U.S. patent application number 10/643431 was filed with the patent office on 2004-03-04 for chemokine uses; compositions; methods.
Invention is credited to Caux, Christophe, Dieu-Nosjean, Marie Caroline, Homey, Bernhard, Oldham, Elizabeth R., Zlotnik, Albert.
Application Number | 20040042998 10/643431 |
Document ID | / |
Family ID | 29406206 |
Filed Date | 2004-03-04 |
United States Patent
Application |
20040042998 |
Kind Code |
A1 |
Oldham, Elizabeth R. ; et
al. |
March 4, 2004 |
Chemokine uses; compositions; methods
Abstract
Agonists or antagonists of MIP-3.alpha., and various methods of
use in dermatological and related applications are provided. In
particular, the method makes use of fact that the MIP-3.alpha.
chemokine is specifically capable of inducing migration of a skin
cell subset.
Inventors: |
Oldham, Elizabeth R.;
(Mountain View, CA) ; Homey, Bernhard; (Palo Alto,
CA) ; Dieu-Nosjean, Marie Caroline; (Rueil-Malmaison,
FR) ; Caux, Christophe; (Bressolles, FR) ;
Zlotnik, Albert; (Palo Alto, CA) |
Correspondence
Address: |
SCHERING-PLOUGH CORPORATION
PATENT DEPARTMENT (K-6-1, 1990)
2000 GALLOPING HILL ROAD
KENILWORTH
NJ
07033-0530
US
|
Family ID: |
29406206 |
Appl. No.: |
10/643431 |
Filed: |
August 19, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10643431 |
Aug 19, 2003 |
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09503219 |
Feb 2, 2000 |
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6645491 |
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60118335 |
Feb 3, 1999 |
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Current U.S.
Class: |
424/85.1 ;
424/145.1 |
Current CPC
Class: |
A61K 2039/505 20130101;
C07K 16/24 20130101 |
Class at
Publication: |
424/085.1 ;
424/145.1 |
International
Class: |
A61K 039/395; A61K
038/19 |
Claims
What is claimed is:
1. A method of modulating migration of a cell within or to the skin
of a mammal, said method comprising administering to said mammal an
effective amount of: a) an antagonist of MIP-3.alpha.; b) an
agonist of MIP-3.alpha.; c) an antagonist of CCR6; or d) an agonist
of CCR6.
2. The method of claim 1, wherein said migration is within said
skin.
3. The method of claim 2, wherein said migration is chemotactic or
chemokinetic.
4. The method of claim 1, wherein said administering is systemic,
local, topical, subcutaneous, intracutaneous, or transdermal.
5. The method of claim 1, wherein said cell is a T cell, B cell,
dendritic cell, or dendritic cell precursor.
6. The method of claim 5, wherein said cell is a T cell.
7. The method of claim 1, wherein said cell migrates into the
dermal and/or epidermal layers of said skin.
8. The method of claim 1, wherein said administering is an
antagonist of MIP-3.alpha..
9. The method of claim 8, wherein said antagonist is selected from:
a) a mutein of natural MIP-3.alpha.; b) an antibody which
neutralizes MIP-3.alpha.; or c) an antibody which binds to
CCR6.
10. The method of claim 8, wherein said mammal is subject to a skin
condition, including one selected from cancer, cancer metastasis,
autoimmunity, inflamation, infection, psoriasis, skin transplant,
or skin graft.
11. The method of claim 8, wherein said antagonist is administered
in combination with an antibiotic, antifungal, antiviral, cancer
therapy, or analgesic.
12. The method of claim 8, wherein said antagonist is administered
in combination with an immune suppressive therapeutic,
anti-inflammatory drug, growth factor, or immune adjuvant.
13. The method of claim 1, wherein said administering is with a
primate MIP-3.alpha..
14. The method of claim 13, wherein said modulating is attracting
said cell.
15. The method of claim 14, wherein said cell is attracted to a
site of cutaneous lesion.
16. The method of claim 13, wherein said primate MIP-3.alpha. is
administered in combination with an antibiotic, antifungal,
antiviral, or analgesic.
17. The method of claim 13, wherein said MIP-3.alpha. is
administered in combination with a vasodilator, growth factor,
cytokine, anti-inflammatory drug, or immune adjuvant.
18. A method of purifying a population of cells, said method
comprising contacting said cells with MIP-3.alpha., thereby
resulting in the identification of cells expressing a receptor for
said MIP-3.alpha..
19. The method of claim 18, wherein: a) said receptor is CCR6; or
b) said contacting results in specific migration of said cells to a
site for purification.
20. The method of claim 18, wherein said migration is through pores
of a membrane.
Description
[0001] The present filing is a conversion to U.S. utility patent
application from provisional U.S. Ser. No. 60/118,335, filed Feb.
3, 1999, which is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The invention relates generally to methods of using various
chemokine related compositions, more particularly, to methods of
treating skin diseases or conditions associated with misregulation
of the chemokine MIP-3.alpha., a ligand for the CCR6 chemokine
receptor.
BACKGROUND
[0003] The immune system consists of a wide range of distinct cell
types, each with important roles to play. See Paul (ed. 1997)
Fundamental Immunology 4th ed., Raven Press, New York. The
lymphocytes occupy central stage because they are the cells that
determine the specificity of immunity, and it is their response
that orchestrates the effector limbs of the immune system. Two
broad classes of lymphocytes are recognized: the B lymphocytes,
which are precursors of antibody secreting cells, and the T
(thymus-dependent) lymphocytes. T lymphocytes express important
regulatory functions, such as the ability to help or inhibit the
development of specific types of immune response, including
antibody production and increased microbicidal activity of
macrophages. Other T lymphocytes are involved in direct effector
functions, such as the lysis of virus infected-cells or certain
neoplastic cells.
[0004] The chemokines are a large and diverse superfamily of
proteins. The superfamily is subdivided into two classical
branches, based upon whether the first two cysteines in the
chemokine motif are adjacent (termed the "C--C" branch), or spaced
by an intervening residue ("C--X--C"). A more recently identified
branch of chemokines lacks two cysteines in the corresponding
motif, and is represented by the chemokines known as lymphotactins.
Another recently identified branch has three intervening residues
between the two cysteines, e.g., CX3C chemokines. See, e.g., Schall
and Bacon (1994) Current Opinion in Immunology 6:865-873; and Bacon
and Schall (1996) Int. Arch. Allergy & Immunol. 109:97-109.
[0005] Many factors have been identified which influence the
differentiation process of precursor cells, or regulate the
physiology or migration properties of specific cell types. These
observations indicate that other factors exist whose functions in
immune function were heretofore unrecognized. These factors provide
for biological activities whose spectra of effects may be distinct
from known differentiation or activation factors. The absence of
knowledge about the structural, biological, and physiological
properties of the regulatory factors which regulate cell physiology
in vivo prevents the modulation of the effects of such factors.
Thus, medical conditions where regulation of the development or
physiology of relevant cells is required remain unmanageable.
SUMMARY OF THE INVENTION
[0006] The present invention is based, in part, upon the surprising
discovery that the MIP-3( chemokine is expressed in inflamed skin
cells. The chemokine is the ligand for the CCR6 receptor. See
Greaves, et al. (1997) J. Expt'l Med. 186:837-844. Both the ligand
and receptor are expressed at essentially undetectable levels in
normal skin, while both are highly upregulated in inflamed
skin.
[0007] The present invention provides methods of modulating
migration of a cell within or to the skin of a mammal comprising
administering to the mammal an effective amount of: an antagonist
of MIP-3.alpha.; an agonist of MIP-3.alpha., and antagonist of
CCR6; or an agonist of CCR6. Typically, the migration is within the
skin; or may be chemotactic or chemokinetic. In preferred
embodiments, the administering is systemic, local, topical,
subcutaneous, intracutaneous, or transdermal. Often, the cell is a
T cell, B cell, dendritic cell, or dendritic cell precursor. In
other embodiments, the cell is a T cell, or moves into the dermal
and/or epidermal layers of the skin.
[0008] In other embodiments, the administering is of an antagonist
of MIP-3.alpha.. Generally, the antagonist is selected from: a
mutein of natural MIP-3.alpha.; an antibody which neutralizes
MIP-3.alpha.; or an antibody which binds to CCR6. In various
embodiments, the mammal is subject to a skin disease or condition,
including one selected from cancer, cancer metastasis, skin
transplant, or skin graft. Often, the antagonist is administered in
combination with an antibiotic, antifungal, antiviral, or
analgesic; or may be with an immune suppressive therapeutic,
anti-inflammatory drug, growth factor, or immune adjuvant.
[0009] In another embodiment, the administering is with a primate
MIP-3.alpha.. Often, the modulating is attracting the cell, e.g.,
to a site of cutaneous lesion. The primate MIP-3.alpha. may be
administered in combination with an antibiotic, antifungal,
antiviral, or analgesic; or with a vasodilator, growth factor,
cytokine, anti-inflammatory drug, or immune adjuvant.
[0010] Alternatively, the invention provides a method of purifying
a population of cells, the method comprising contacting the cells
with MIP-3.alpha., thereby resulting in the identification of cells
expressing a receptor for MIP-3.alpha.. In certain embodiments, the
receptor is CCR6, or the contacting results in specific migration
of the cells to a site for purification, e.g., through pores of a
membrane.
DETAILED DESCRIPTION OF THE INVENTION
[0011] Outline
[0012] I. General
[0013] II. Chemokine Agonists and Antagonists
[0014] A. MIP-3.alpha. and Variants
[0015] B. Antibodies
[0016] C. Other Molecules
[0017] III. Immunoassays
[0018] IV. Uses
[0019] I. General
[0020] The invention is based, in part, on the surprising discovery
that the chemokine MIP-3.alpha. has been implicated in roles in
skin-immunity. In particular, MIP-3.alpha. has been identified as a
ligand for the chemokine receptor designated CCR6. Both
MIP-3.alpha. and CCR6 expression are undetectable in normal skin,
while both are highly upregulated in inflamed skin samples.
[0021] The skin consists of a surface layer of epithelium called
the epidermis and an underlying layer of connective tissue called
the dermis. Under the dermis is a layer which contains large
amounts of adipose tissue, the hypodermis. The skin serves a
variety of functions, and variations in the character of the dermis
and epidermis occur according to functional demands. The appendages
of the skin, hair, nails, and sweat and sebaceous glands, are such
local specializations of the epidermis. Together, the skin and its
appendages form the integument. See, e.g., Fitzpatrick, et al.
(eds. 1993) Dermatology in General Medicine 4th ed., McGraw-Hill,
NY; Bos (ed. 1989) Skin Immune System CRC Press, Boca Raton, Fla.;
Callen (1996) General Practice Dermatology Appleton and Lange;
Rook, et al. (eds. 1998) Textbook of Dermatology Blackwell; Habifor
and Habie (1995) Clinical Dermatology: A Color Guide to Diagnosis
and Therapy Mosby; and Grob (ed. 1997) Epidemiology, Causes and
Prevention of Skin Diseases Blackwell.
[0022] The epidermis consists of many different cell types in
various proportions. The most prevalent cell type is keratinocytes,
which make up some 95% of the cells. Cells in the 1-2% range
include melanocytes and Langerhans cells. The Langerhans cells are
particularly important because they trap antigens that have
penetrated the skin, and transport antigens to regional lymph
nodes. A small population of .gamma..delta. T cells can also reside
in the epidermis.
[0023] The dermis varies in thickness in different regions of the
body. It is tough, flexible, and highly elastic, and consists of a
feltwork of collagen fibers with abundant elastic fibers. The
connective tissue is arranged into deep reticular and superficial
papillary layers.
[0024] The chemokines are a sub-family of chemoattractant cytokines
that were classically characterized by their ability to mediate
leukocyte trafficking or migration by binding to specific
G-protein-linked seven transmembrane spanning receptors, or GPCRs.
Chemokines are divided into four groups based on the primary
sequence of the first two cysteines: the CXC, CC, C, and CX3C
families. The CXC and C families are effective predominantly on
neutrophils and lymphocytes, respectively. The CC chemokines are
preferentially effective on macrophages, lymphocytes, and
eosinophils.
[0025] The chemokine MIP-3.alpha., from human, mouse, and rat, has
been described earlier. See, e.g., human, GenBank HSU77035; mouse,
GenBank AF099052; rat, GenBank U90447; Li and Adams, WO 94-US9484;
and Wilde, et al. WO 9616979; each of which is incorporated herein
by reference for all purposes. The sequences are provided in Table
1.
1TABLE 1 A primate, human, MIP-3.alpha., nucleic acid sequence (SEQ
ID NO: 1) and amino acid sequence (SEQ ID NO: 2). Coding sequence
begins at about nucleotide 1 and ends at about 288; CC motif at
amino acid residues 6-7. A signal sequence is indicated, but based
upon related genes; slightly different processing may occur in
different cell types. atg tgc tgt acc aag agt ttg ctc ctg gct gct
ttg atg tca gtg ctg 48 Met Cys Cys Thr Lys Ser Leu Leu Leu Ala Ala
Leu Met Ser Val Leu -25 -20 -15 cta ctc cac ctc tgc ggc gaa tca gaa
gca gca agc aac ttt gac tgc 96 Leu Leu His Leu Cys Gly Glu Ser Glu
Ala Ala Ser Asn Phe Asp Cys -10 -5 -1 1 5 tgt ctt gga tac aca gac
cgt att ctt cat cct aaa ttt att gtg ggc 144 Cys Leu Gly Tyr Thr Asp
Arg Ile Leu His Pro Lys Phe Ile Val Gly 10 15 20 ttc aca cgg cag
ctg gcc aat gaa ggc tgt gac atc aat gct atc atc 192 Phe Thr Arg Gln
Leu Ala Asn Glu Gly Cys Asp Ile Asn Ala Ile Ile 25 30 35 ttt cac
aca aag aaa aag ttg tct gtg tgc gca aat cca aaa cag act 240 Phe His
Thr Lys Lys Lys Leu Ser Val Cys Ala Asn Pro Lys Gln Thr 40 45 50
tgg gtg aaa tat att gtg cgt ctc ctc agt aaa aaa gtc aag aac atg 288
Trp Val Lys Tyr Ile Val Arg Leu Leu Ser Lys Lys Val Lys Asn Met 55
60 65 70 taa 291 Table 1 (continued): A murine, mouse, MIP-3.alpha.
chemokine nucleic acid sequence (SEQ ID NO: 3) and corresponding
amino acid sequence (SEQ ID NO: 4). SignalP software predicts a
cleavage between A1a(-1) and Ser1; but the actual cleavage may be
on either side by a residue or so. atg gcc tgc ggt ggc aag cgt ctg
ctc ttc ctt gct ttg gca tgg gta 48 Met Ala Cys Gly Gly Lys Arg Leu
Leu Phe Leu Ala Leu Ala Trp Val 25 -20 -15 ctg ctg gct cac ctc tgc
agc cag gca gaa gca agc aac tac gac tgt 96 Leu Leu Ala His Leu Cys
Ser Gln Ala Glu Ala Ser Asn Tyr Asp Cys -10 -5 -1 1 5 tgc ctc tcg
tac ata cag acg cca ctt cct tcc aga gct att gtg ggt 144 Cys Leu Ser
Tyr Ile Gln Thr Pro Leu Pro Ser Arg Ala Ile Val Gly 10 15 20 ttc
aca aga cag atg gcc gat gaa gct tgt gac att aat gct atc atc 192 Phe
Thr Arg Gln Met Ala Asp Glu Ala Cys Asp Ile Asn Ala Ile Ile 25 30
35 ttt cac acg aag aaa aga aaa tct gtg tgc gct gat cca aag cag aac
240 Phe His Thr Lys Lys Arg Lys Ser Val Cys Ala Asp Pro Lys Gln Asn
40 45 50 tgg gtg aaa agg gct gtg aac ctc ctc agc cta aga gtc aag
aag atg 288 Trp Val Lys Arg Ala Val Asn Leu Leu Ser Leu Arg Val Lys
Lys Met 55 60 65 taa 291 Table 1 (continued): A murine, rat,
MIP-3.alpha. chemokine nucleic acid sequence (SEQ ID NO: 5) and
corresponding amino acid sequence (SEQ ID NO: 6). SignalP software
predicts a cleavage between Ala(-1) and Ala1; but the actual
cleavage may be on either side by a residue or so. atg gcc tgc aag
cat ctg ccc ttc ctg gct ttg gcg ggg gta ctg ctg 48 Met Ala Cys Lys
His Leu Pro Phe Leu Ala Leu Ala Gly Val Leu Leu -25 20 -15 -10 gct
tac ctc tgc agc cag tca gaa gca gca agc aac ttt gac tgc tgc 96 Ala
Tyr Leu Cys Ser Gln Ser Glu Ala Ala Ser Asn Phe Asp Cys Cys -5 -1 1
5 ctc acg tac aca aag aac gtg tat cat cat gcg aga aat ttt gtg ggt
144 Leu Thr Tyr Thr Lys Asn Val Tyr His His Ala Arg Asn Phe Val Gly
10 15 20 ttc aca aca cag atg gcc gac gaa gct tgt gac att aat gct
atc atc 192 Phe Thr Thr Gln Met Ala Asp Glu Ala Cys Asp Ile Asn Ala
Ile Ile 25 30 35 ttt cac ctg aag tcg aaa aga tcc gtg tgc gct gac
cca aag cag atc 240 Phe His Leu Lys Ser Lys Arg Ser Val Cys Ala Asp
Pro Lys Gln Ile 40 45 50 55 tgg gtg aaa agg att ttg cac ctc ctc agc
cta aga acc aag aag atg 288 Trp Val Lys Arg Ile Leu His Leu Leu Ser
Leu Arg Thr Lys Lys Met 60 65 70 taa 291
[0026]
2TABLE 2 Nucleotide sequence (5' to 3') of primate, human,
chemokine receptor, CCR6, and the corresponding amino acid sequence
(amino to carboxy), see SEQ ID NO: 7 and 8. Nucleotide 579 may be
A, C, G, or T, and the codon may code for His or Gln. atg ttt tcg
act cca gtg aag att att ttg tgt cag tca ata ctt cat 48 Met Phe Ser
Thr Pro Val Lys Ile Ile Leu Cys Gln Ser Ile Leu His 1 5 10 15 att
act cag ttg att ctg aga tgt tac tgt gct cct tgc agg agg tca 96 Ile
Thr Gln Leu Ile Leu Arg Cys Tyr Cys Ala Pro Cys Arg Arg Ser 20 25
30 ggc agt tct cca ggc tat ttg tac cga att gcc tac tcc ttg atc tgt
144 Gly Ser Ser Pro Gly Tyr Leu Tyr Arg Ile Ala Tyr Ser Leu Ile Cys
35 40 45 gtt ctt ggc ctc ctg ggg aat att ctg gtg gtg atc acc ttt
gct ttt 192 Val Leu Gly Leu Leu Gly Asn Ile Leu Val Val Ile Thr Phe
Ala Phe 50 55 60 tat aag aag gcc agg tct atg aca gac gtc tat ctc
ttg aac atg gcc 240 Tyr Lys Lys Ala Arg Ser Met Thr Asp Val Tyr Leu
Leu Asn Met Ala 65 70 75 80 att gca gac atc ctc ttt gtt ctt act ctc
cca ttc tgg gca gtg agt 288 Ile Ala Asp Ile Leu Phe Val Leu Thr Leu
Pro Phe Trp Ala Val Ser 85 90 95 cat gcc act ggt gcg tgg gtt ttc
agc aat gcc acg tgc aag ttg cta 336 His Ala Thr Gly Ala Trp Val Phe
Ser Asn Ala Thr Cys Lys Leu Leu 100 105 110 aaa ggc atc tat gcc atc
aac ttt aac tgc ggg atg ctg ctc ctg act 384 Lys Gly Ile Tyr Ala Ile
Asn Phe Asn Cys Gly Met Leu Leu Leu Thr 115 120 125 tgc att agc atg
gac cgg tac atc gcc att gta cag gcg act aag tca 432 Cys Ile Ser Met
Asp Arg Tyr Ile Ala Ile Val Gln Ala Thr Lys Ser 130 135 140 ttc cgg
ctc cga tcc aga aca cta ccg cgc agc aaa atc atc tgc ctt 480 Phe Arg
Leu Arg Ser Arg Thr Leu Pro Arg Ser Lys Ile Ile Cys Leu 145 150 155
160 gtt gtg tgg ggg ctg tca gtc atc atc tcc agc tca act ttt gtc ttc
528 Val Val Trp Gly Leu Ser Val Ile Ile Ser Ser Ser Thr Phe Val Phe
165 170 175 aac caa aaa tac aac acc caa ggc agc gat gtc tgt gaa ccc
aag tac 576 Asn Gln Lys Tyr Asn Thr Gln Gly Ser Asp Val Cys Glu Pro
Lys Tyr 180 185 190 can act gtc tcg gag ccc atc agg tgg aag ctg ctg
atg ttg ggg ctt 624 Xaa Thr Val Ser Glu Pro Ile Arg Trp Lys Leu Leu
Met Leu Gly Leu 195 200 205 gag cta ctc ttt ggt ttc ttt atc cct ttg
atg ttc atg ata ttt tgt 672 Glu Leu Leu Phe Gly Phe Phe Ile Pro Leu
Met Phe Met Ile Phe Cys 210 215 220 tac acg ttc att gtc aaa acc ttg
gtg caa gct cag aat tct aaa agg 720 Tyr Thr Phe Ile Val Lys Thr Leu
Val Gln Ala Gln Asn Ser Lys Arg 225 230 235 240 cac aaa gcc atc cgt
gta atc ata gct gtg gtg ctt gtg ttt ctg gct 768 His Lys Ala Ile Arg
Val Ile Ile Ala Val Val Leu Val Phe Leu Ala 245 250 255 tgt cag att
cct cat aac atg gtc ctg ctt gtg acg gct gct aat ttg 816 Cys Gln Ile
Pro His Asn Met Val Leu Leu Val Thr Ala Ala Asn Leu 260 265 270 ggt
aaa atg aac cga tcc tgc cag agc gaa aag cta att ggc tat acg 864 Gly
Lys Met Asn Arg Ser Cys Gln Ser Glu Lys Leu Ile Gly Tyr Thr 275 280
285 aaa act gtc aca gaa gtc ctg gct ttc ctg cac tgc tgc ctg aac cct
912 Lys Thr Val Thr Glu Val Leu Ala Phe Leu His Cys Cys Leu Asn Pro
290 295 300 gtg ctc tac gct ttt att ggg cag aag ttc aga aac tac ttt
ctg aag 960 Val Leu Tyr Ala Phe Ile Gly Gln Lys Phe Arg Asn Tyr Phe
Leu Lys 305 310 315 320 atc ttg aag gac ctg tgg tgt gtg aga agg aag
tac aag tcc tca ggc 1008 Ile Leu Lys Asp Leu Trp Cys Val Arg Arg
Lys Tyr Lys Ser Ser Gly 325 330 335 ttc tcc tgt gcc ggg agg tac tca
gaa aac att tct cgg cag acc agt 1056 Phe Ser Cys Ala Gly Arg Tyr
Ser Glu Asn Ile Ser Arg Gln Thr Ser 340 345 350 gag acc gca gat aac
gac aat gcg tcg tcc ttc act atg tga 1098 Glu Thr Ala Asp Asn Asp
Asn Ala Ser Ser Phe Thr Met 355 360 365
[0027] In contrast to naive lymphocytes, memory/effector
lymphocytes can access non-lymphoid effector sites and display
restricted, often tissue-selective, migration behavior. This
results in the presence of such lymphocytes in the peripheral
tissues, e.g., outside of the lymphatic and blood volume.
[0028] Both human and mouse MIP-3.alpha. are detected in lymph
nodes, appendix, PBL, fetal liver, fetal lung, and various cell
lines. See, e.g., Rossi, et al. (1997) J. Immunol. 158:1033-1036;
Hieshima, et al. (1997) J. Biol. Chem. 272:5846-5853; Baba, et al.
(1997) J. Biol. Chem. 272:14893-14898; and Imai, et al. (1997) J.
Biol. Chem. 272:1503.6-15042. The expression in the Langerhans
islets suggests a role in skin functions. The data is consistent
with MIP-3.alpha. as a product of activated monocytes, and is
preferentially expressed in inflamed tissue. This distribution
would suggest that MIP-3.alpha. may have a role in attracting
memory T cells, and skin dendritic cells (Langerhans cells) and
their precursors. These results suggest an important role for
MIP-3.alpha. in recruitment of T cells and dendritic cells to
peripheral cutaneous sites.
[0029] Chemokine receptors are members of the G protein coupled
receptor family. See, e.g., Yoshie, et al. (1997) J. Leukoc. Biol.
62:634-644. CCR6 expression has been reported in Greaves, et al.
(1997) J. Expt'l Med. 186:837-844; and Liao, et al. (1999) J.
Immunol. 162:186-194. Northern blot data showed expression
predominantly in the spleen, with lesser amounts in thymus, testis,
small intestine, and peripheral blood. Additional transcripts were
detected in spleen. Transcripts were not detected in the TF-1,
Jurkat, MRC5, JY, and U937 cell lines. Message seems not to be
abundantly expressed in the lymphoid lineage, particularly in,
e.g., libraries made from cells made from dendritic cell cultures
derived from cells selected on the basis of CD1a expression.
Expression is lower in DC generated from monocytes.
[0030] Another study showed CCR6 was expressed on memory T cells,
including most .alpha.4.beta.7 memory cells and cutaneous
lymphocyte-associated antigen expressing cells, and on B cells.
Chemotaxis of T cells to MIP-3.alpha. was limited to memory cells.
See Liao, et al. (1998) J. Immunol. 162:186-194. Antiserum detected
CCR6 on CD34+ bone marrow derived dendritic cells.
[0031] Having identified the MIP-3.alpha. as a skin related
chemokine, it will find use in affecting medical abnormalities of
the skin. Common skin disorders involving the immune system include
psoriasis, skin cancers, carcinomas, inflammation, allergies
dermatitis, wound healing, infections (both microbial and
parasitic), and many others. See, e.g., The Merck Manual,
particularly the chapter on dermatologic disorders. These
therapeutics may have useful effects on growth or health of
appendages of the skin, including, e.g., hair, nails, and sweat and
sebaceous glands.
[0032] Psoriasis is a chronic inflammatory skin disease that is
associated with hyperplastic epidermal keratinocytes and
infiltrating mononuclear cells, including T cells, neutrophils and
macrophages. Because of this highly mixed inflammatory picture and
the resulting complex interrelationships between these different
cells, it has been very difficult to dissect the mechanisms that
underlie the induction and progression of the disease.
[0033] This view of psoriasis also implies that although dormant
autoreactive T cells may pre-exist in susceptible individuals, an
environmental stimulus is necessary to trigger disease induction.
Others believe that the immune system plays only a minor modulatory
role in the disease process and that hyperproliferation of
keratinocytes is in fact the initiating event in a genetically
susceptible host. Research into the pathogenesis of psoriasis has
long been hindered by the lack of suitable animal models.
[0034] There is growing data indicating that T cells and not
keratinocytes are the primary pathogenic component in the disease.
The observations herein provide evidence to support the concept
that psoriasis-like conditions can indeed result from unregulated T
cell responses.
[0035] Skin cancers such as basal cell and squamous cell carcinoma
are among the most common malignancies. See, e.g., Miller and
Maloney (eds. 1997) Cutaneous Oncology: Pathophysiology, Diagnosis,
and Management Blackwell; Emmett and Orourke (1991) Malignant Skin
Tumours Churchill Livingstone; Friedman (1990) Cancer of the Skin
Saunders. Most of those tumors arise in sun exposed areas of the
skin. Immune regulation or clearance of such tumors may depend upon
function Qf the skin immune system. Cells which effect such may be
compromised by local misregulation or suppression. The MIP-3.alpha.
or antagonists may break a temporary homeostasis which suppresses
normal immune response, thereby leading to activation of proper
regulatory and immune pathways.
[0036] Dermatitis is a superficial inflammation of the skin,
characterized by vesicles (when acute), redness, edema, oozing,
crusting, scaling, and/or itching. See, e.g., Lepoittevin (ed.
1998) Allergic Contact Dermatitis: The Molecular Basis
Springer-Verlag; Rietschel and Fowler (eds. 1995) Fisher's Contact
Dermatitis Lippincott; and Rycroft, et al. (eds. 1994) Textbook of
Contact Dermatitis Springer-Verlag. The term eczematous dermatitis
is often used to refer to a vesicular dermatitis. Dermatitis may
accompany various immune deficiency conditions or diseases, inborn
metabolic disorders, or nutritional deficiency diseases. Certain of
the symptoms of such conditions may be treated using the present
invention.
[0037] Pruritus is a sensation that the patient attempts to relieve
by scratching. See, e.g., Fleischer and Fleischer (1998) The
Clinical Management of Itching: Therapeutic Protocols for Pruritus
Parthenon. Many parasitic or infectious conditions may result in
those symptoms, which conditions may be cleared by proper
reactivation or suppression of immune functions in the skin.
Likewise with various allergic or other immune reactions to
exposure to various allergic or inflammatory antigens.
[0038] II. Chemokine Agonists and Antagonists
[0039] Mammalian MIP-3.alpha. chemokines were described previously
in U.S. Ser. No. 08/887,977, which describes various migratory
assays. Various agonists and antagonists of the natural ligands can
be produced. The migration assays may-take advantage of the
movement of cells through pores in membranes. Chemotaxis may be
measured thereby. Alternatively, chemokinetic assays may be
developed, which measure the induction of kinetic movement, not
necessarily relative to a gradient, per se.
[0040] A. MIP-3.alpha. and Variants
[0041] MIP-3.alpha. agonists will exhibit some or all of the
signaling functions of MIP-3.alpha. e.g., binding, inducing a Ca++
flux, and chemoattracting appropriate receptor bearing cells.
Various mammalian MIP-3.alpha. sequences may be evaluated to
determine what residues are conserved across species, suggesting
what residues may be changed without dramatic effects on biological
activity. Alternatively, conservative substitutions are likely to
retain biological activity, thus leading to variant forms of the
chemokine which will retain agonist activity. Standard methods for
screening mutant or variant MIP-3.alpha. polypeptides will
determine what sequences will be useful therapeutic agonists.
[0042] In addition, certain nucleic acid expression methods may be
applied. For example, in skin graft contexts, it may be useful to
transfect the grafts with nucleic acids which will be expressed, as
appropriate. Various promoters may be operably linked to the gene,
thereby allowing for regulated expression. Antisense constructs may
prevent expression of the ligand or receptor.
[0043] Alternatively, antagonist activity may be tested or screened
for. Tests for ability to antagonize chemoattractant activity can
be developed using assays as described below. Various ligand
homologs can be created which retain receptor binding capacity, but
lack signaling capability, thus serving as competitive binding
molecules. Small molecules may also be screened for ability to
antagonize MIP-3.alpha. function, e.g., chemoattraction, receptor
binding, Ca++ flux, and other effects mediated by MIP-3.alpha.. See
generally Gilman, et al. (eds. 1990) Goodman and Gilman's: The
Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press;
Remington's Pharmaceutical Sciences, 17th ed. (1990), Mack
Publishing Co., Easton, Pa., each of which is incorporated herein
by reference.
[0044] B. Antibodies
[0045] The present invention provides for the use of an antibody or
binding composition which specifically binds to MIP-3.alpha.,
preferably mammalian, e.g., primate, human, cat, dog, rat, or
mouse, and neutralizes the ability of the chemokine to mediate its
signal. Antibodies can be raised to various MIP-3.alpha. proteins,
including individual, polymorphic, allelic, strain, or species
variants, and fragments thereof, either in their naturally
occurring (full-length) forms or in their recombinant forms.
Additionally, antibodies can be raised to MIP-3.alpha. or
polypeptides in both their native (or active) forms or in their
inactive, e.g., denatured, forms, which may neutralize ligand
capacity to mediate its signal. Antibodies may block the
interaction of the ligand with its receptor.
[0046] Alternatively, receptor antagonists may be produced by
making antibodies which bind to the receptor and block ligand
binding. With the identification of the CCR6 as a receptor for the
cytokine, antibodies to the receptor may be selected for those
which block the binding of, or signaling induced by, ligand.
[0047] A number of immunogens may be selected to produce antibodies
specifically reactive, or selective for binding, with MIP-3.alpha.
or CCR6 proteins. Recombinant protein is a preferred immunogen for
the production of monoclonal or polyclonal antibodies. Naturally
occurring protein, from appropriate sources, e.g., primate, rodent,
etc., may also be used either in pure or impure form. Synthetic
peptides, made using the MIP-3.alpha. or CCR6 protein sequences
described herein, may also be used as an immunogen for the
production of antibodies. Recombinant protein can be expressed and
purified in eukaryotic or prokaryotic cells as described, e.g.,. in
Coligan, et al. (eds. 1995 and periodic supplements) Current
Protocols in Protein Science John Wiley & Sons, New York, N.Y.;
and Ausubel, et al (eds. 1987 and periodic supplements) Current
Protocols in Molecular Biologvy, Greene/Wiley, New York, N.Y.
Naturally folded or denatured material, perhaps expressed on cell
surfaces, can be used, as appropriate, for producing antibodies.
Either monoclonal or polyclonal antibodies may be generated, e.g.,
for subsequent use in immunoassays to measure the protein, or for
immunopurification methods.
[0048] Methods of producing polyclonal antibodies are well known to
those of skill in the art. Typically, an immunogen, preferably a
purified protein, is mixed with an adjuvant and animals are
immunized with the mixture. The animalls immune response to the
immunogen preparation is monitored by taking test bleeds and
determining the titer of reactivity to, e.g., the MIP-3.alpha.
protein or polypeptide of interest. For example, when appropriately
high titers of antibody to the immunogen are obtained, usually
after repeated immunizations, blood is collected from the animal
and antisera are prepared. Further fractionation of the antisera to
enrich for antibodies reactive to the protein can be performed, if
desired. See, e.g., Harlow and Lane Antibodies, A Laboratory
Manual; or Coligan (ed.) Current Protocols in Immunology.
Immunization can also be performed through other methods, e.g., DNA
vector immunization. See, e.g., Wang, et al. (1997) Viroloqy
228:278-284. Affinity purification, or absorptions, can be used to
select for desired specificity of binding.
[0049] Monoclonal antibodies may be obtained by various techniques
familiar to those skilled in the art. Typically, spleen cells from
an animal immunized with a desired antigen are immortalized,
commonly by fusion with a myeloma cell. See, Kohler and Milstein
(1976) Eur. J. Immunol. 6:511-519. Alternative methods of
immortalization include transformation with Epstein Barr Virus,
oncogenes, or retroviruses, or other methods known in the art. See,
e.g., Doyle, et al. (eds. 1994 and periodic supplements) Cell and
Tissue Culture: Laboratory Procedures, John Wiley and Sons, New
York, NY. Colonies arising from single immortalized cells are
screened for production of antibodies of the desired specificity
and affinity for the antigen, and yield of the monoclonal
antibodies produced by such cells may be enhanced by various
techniques, including injection into the peritoneal cavity of a
vertebrate host. Alternatively, one may isolate DNA sequences which
encode a monoclonal antibody or a binding fragment thereof by
screening a DNA library from human B cells according, e.g., to the
general protocol outlined by Huse, et al. (1989) Science
246:1275-1281.
[0050] Antibodies or binding compositions, including binding
fragments and single chain versions, against predetermined
fragments of MIP-3.alpha. or CCR6 polypeptides can be raised by
immunization of animals with conjugates of the fragments with
carrier proteins as described above. Monoclonal antibodies are
prepared from cells secreting the desired antibody. These
antibodies can be screened for binding to normal or defective
MIP-3.alpha. protein, or screened for capacity to block cell
MIP-3.alpha. mediated chemoattraction or chemokinetic activity.
These monoclonal antibodies will usually bind with at least a KD of
about 1 mM, more usually at least about 300 .mu.M, typically at
least about 10 .mu.M, more typically at least about 30 .mu.M,
preferably at least about 10 .mu.M, and more preferably at least
about 3 .mu.M or better.
[0051] In some instances, it is desirable to prepare monoclonal
antibodies (mAbs) from various mammalian hosts, such as mice,
rodents, primates, humans, etc. Description of techniques for
preparing such monoclonal antibodies may be found in, e.g., Stites,
et al. (eds.) Basic and Clinical Immunology (4th ed.) Lange Medical
Publications, Los Altos, Calif., and references cited therein;
Harlow and Lane (1988) Antibodies: A Laboratory Manual CSH Press;
Goding (1986) Monoclonal Antibodies: Principles and Practice (2d
ed.) Academic Press, New York, N.Y.; and particularly in Kohler and
Milstein (1975) Nature 256:495-497, which discusses one method of
generating monoclonal antibodies. Summarized briefly, this method
involves injecting an animal with an immunogen. The animal is then
sacrificed and cells taken from its spleen, which are then fused
with myeloma cells. The result is a hybrid cell or "hybridoma" that
is capable of reproducing in vitro. The population of hybridomas is
then screened to isolate individual clones, each of which secrete a
single antibody species to the immunogen. In this manner, the
individual antibody species obtained are the products of
immortalized and cloned single B cells from the immune animal
generated in response to a specific site recognized on the
immunogenic substance.
[0052] Other suitable techniques involve selection of libraries of
antibodies in phage or similar vectors. See, e.g., Huse, et al.
(1989) "Generation of a Large Combinatorial Library of the
Immunoglobulin Repertoire in Phage Lambda," Science 246:1275-1281;
and Ward, et al. (1989) Nature 341:544-546. The polypeptides and
antibodies of the present invention may be used with or without
modification, including chimeric or humanized antibodies.
Frequently, the polypeptides and antibodies will be labeled by
joining, either covalently or non-covalently, a substance which
provides for a detectable signal. A wide variety of labels and
conjugation techniques are known and are reported extensively in
both the scientific and patent literature. Suitable labels include
radionuclides, enzymes, substrates, cofactors, inhibitors,
fluorescent moieties, chemiluminescent moieties, magnetic
particles, and the like. Patents teaching the use of such labels
include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;
4,277,437; 4,275,149; and 4,366,241. Also, recombinant
immunoglobulins may be produced, see, Cabilly, U.S. Pat. No.
4,816,567; and Queen, et al. (1989) Proc. Nat'l Acad. Sci. USA
86:10029-10033; or made in transgenic mice, see Mendez, et al.
(1997) Nature Genetics 15:146-156.
[0053] Antibody binding compounds, including binding fragments, of
this invention can have significant diagnostic or therapeutic
value. They can be useful as non-neutralizing binding compounds and
can be coupled to toxins or radionuclides so that when the binding
compound binds to the antigen, a cell expressing it, e.g., on its
surface, is killed. Further, these binding compounds can be
conjugated to drugs or other therapeutic agents, either directly or
indirectly by means of a linker, and may effect drug targeting.
[0054] C. Other Molecules
[0055] Antibodies are merely one form of specific binding
compositions. Other binding compositions, which will often have
similar uses, include molecules that bind with specificity to
MIP-3.alpha. receptor, e.g., CCR6, in a binding partner-binding
partner fashion, an antibody-antigen interaction, or in a natural
physiologically relevant protein-protein interaction, either
covalent or non-covalent, e.g., proteins which specifically
associate with MIP-3.alpha. receptor protein. The molecule may be a
polymer, or chemical reagent. A functional analog may be a protein
with structural modifications, or may be a structurally unrelated
molecule, e.g., which has a molecular shape which interacts with
the appropriate binding determinants. Application of, e.g.,
Systematic Evolution of Ligand by Exponential Enrichment (SELEX)
technology, methods are available to select specific binding
constructs for desired targets. See, e.g., Colas, et al. (1996)
Nature 380:548-550; Cohen, et al. (1998) Proc. Nat'l Acad. Sci. USA
95:14272-14277; Kolonin, et al. (1998) Proc. Nat'l Acad. Sci. USA
95:14266-14271; Famulok, et al. (1998) Curr. Opin. Chem. Biol.
2:320-327; and Eaton, et al. (1997) Bioorg. Med. Chem.
5:1087-1096.
[0056] Drug screening using antibodies or MIP-3.alpha. or fragments
thereof can be performed to identify compounds having binding
affinity to MIP-3.alpha., or can block or simulate the natural
interaction with ligand. Subsequent biological assays can then be
utilized to determine if the compound has intrinsic blocking
activity and is therefore an antagonist. Likewise, a compound
having intrinsic stimulating activity can signal to the cells via
the MIP-3.alpha. pathway and is thus an agonist in that it
simulates the activity of a ligand. Mutein antagonists may be
developed which maintain receptor binding but lack signaling.
[0057] Structural studies of the ligands will lead to design of new
variants, particularly analogs exhibiting agonist or antagonist
properties on the receptor. This can be combined with previously
described screening methods to isolate muteins exhibiting desired
spectra of activities.
[0058] As receptor specific binding molecules are provided, also
included are small molecules identified by screening procedures. In
particular, it is well known in the art how to screen for small
molecules which interfere, e.g., with ligand binding to the
receptor, often by specific binding to the receptor and blocking of
binding by natural ligand. See, e.g., meetings on High Throughput
Screening, International Business Communications, Southborough,
Mass. 01772-1749. Such molecules may compete with natural ligands,
and selectively bind to the MIP-3.alpha. or CCR6
[0059] III. Immunoassays
[0060] Immunoassays are valuable in diagnosing a disease or
disorder associated with MIP-3.alpha. imbalance or pathology.
Qualitative or quantitative measurement of a particular protein can
be performed by a variety of immunoassay methods. For a review of
immunological and immunoassay procedures in general, see Stites and
Terr (eds. 1991) Basic and Clinical Immunology (7th ed.). Moreover,
the immunoassays of the present invention can be performed in many
configurations, which are reviewed extensively in, e.g., Maggio
(ed. 1980) Enzyme Immunoassay CRC Press, Boca Raton, Fla.; Tijan
(1985) "Practice and Theory of Enzyme Immunoassays," Laboratory
Techniques in Biochemistry and Molecular Biology, Elsevier Science
Publishers B.V., Amsterdam; Harlow and Lane Antibodies: A
Laboratory Manual, supra;.Chan (ed. 1987) Immunoassay: A Practical
Guide Academic Press, Orlando, Fla.; Price and Newman (eds. 1991)
Principles and Practice of Immunoassays Stockton Press, NY; and Ngo
(ed. 1988) Non-isotopic Immunoassays Plenum Press, NY.
[0061] In particular, the present invention provides various skin
related diseases as conditions susceptible to analysis or diagnosis
by evaluating MIP-3.alpha. and/or CCR6. For example, the likelihood
of skin rejection in a graft situation would be evaluated by the
numbers or types of MIP-3.alpha. or CCR6 bearing cells present.
Prophylactic downregulation may be useful to prevent the
recruitment of dermal T or NK cells. Response to various skin
tumors may be evaluated by the presence or absence of MIP-3.alpha.
and/or CCR6 bearing cells.
[0062] Immunoassays for measurement of MIP-3.alpha. proteins or
peptides can be performed by a variety of methods known to those
skilled in the art. In brief, immunoassays to measure the protein
can be either competitive or noncompetitive binding assays. In
competitive binding assays, the sample to be analyzed competes with
a labeled analyte for specific binding sites on a capture agent
bound to a solid surface. Preferably the capture agent is an
antibody specifically reactive with MIP-3.alpha. proteins produced
as described above. The concentration of labeled analyte bound to
the capture agent is inversely proportional to the amount of free
analyte present in the sample.
[0063] In a competitive binding immunoassay, typically the
MIP-3.alpha. protein present in the sample competes with labeled
protein for binding to a specific binding agent, e.g., an antibody
specifically reactive with the MIP-3.alpha. protein. The binding
agent may be bound to a solid substrate or surface to effect
separation of bound labeled protein from the unbound labeled
protein. Alternately, the competitive binding assay may be
conducted in liquid phase and a variety of techniques known in the
art may be used to separate the bound labeled protein from the
unbound labeled protein. Following separation, the amount of bound
labeled protein is determined. The amount of protein present in the
sample is inversely proportional to the amount of labeled protein
binding.
[0064] Alternatively, a homogeneous immunoassay may be performed in
which a separation step is not needed. In these immunoassays, the
label on the protein is altered by the binding of the protein to
its specific binding agent. This alteration in the labeled protein
results in a decrease or increase in the signal emitted by label,
so that measurement of the label at the end of the immunoassay
allows for detection or quantitation of the protein.
[0065] MIP-3.alpha. proteins may also be determined by a variety of
noncompetitive immunoassay methods. For example, a two-site, solid
phase sandwich immunoassay may be used. In this type of assay, a
binding agent for the protein, e.g., an antibody, is attached to a
solid support. A second protein binding agent, which may also be an
antibody, and which binds the protein at a different site, is
labeled. After binding at both sites on the protein has occurred,
the unbound labeled binding agent is removed and the amount of
labeled binding agent bound to the solid phase is measured. The
amount of labeled binding agent bound is directly proportional to
the amount of protein in the sample.
[0066] Western blot analysis can be used to determine the presence
of MIP-3.alpha. or CCR6 proteins in a sample. Electrophoresis is
carried out, for example, on a tissue sample suspected of
containing the protein. Following electrophoresis to separate the
proteins, and transfer of the proteins to a suitable solid support,
e.g., a nitrocellulose filter, the solid support is incubated with
an antibody reactive with the protein. This antibody may be
labeled, or alternatively may be detected by subsequent incubation
with a second labeled antibody that binds the primary antibody.
[0067] The immunoassay formats described above may employ labeled
assay components. The label may be coupled directly or indirectly
to the desired component of the assay according to methods well
known in the art. A wide variety of labels and methods may be used.
Traditionally, a radioactive label incorporating .sup.3H,
.sup.125I, .sup.35S, .sup.14C, or .sup.32P was used.
Non-radioactive labels include ligands which bind to labeled
antibodies, fluorophores, chemiluminescent agents, enzymes, and
antibodies which can serve as specific binding pair members for a
labeled ligand. The choice of label depends on sensitivity
required, ease of conjugation with the compound, stability
requirements, and available instrumentation. For a review of
various labeling or signal producing systems which may be used, see
U.S. Pat. No. 4,391,904.
[0068] Antibodies reactive with a particular protein can also be
measured by a variety of immunoassay methods. Thus modifications of
the above procedures may be used to determine the amounts or
affinities of various MIP-3.alpha. or CCR6 antibodies or antibody
preparations. For a review of immunological and immunoassay
procedures applicable to the measurement of antibodies by
immunoassay techniques, see, e.g., Stites and Terr (eds.) Basic and
Clinical Immunology (7th ed.) supra; Maggio (ed.) Enzyme
Immunoassay, supra; and Harlow and Lane Antibodies, A Laboratory
Manual, supra.
[0069] Screens to evaluate the binding and activity of mAbs and
binding compositions encompass a variety of methods. Binding can be
assayed by detectably labeling the antibody or binding composition
as described above. Cells responsive to MIP-3.alpha. can be used to
assay antibody or binding composition.
[0070] To evaluate MIP-3.alpha. chemoattraction or chemokinetic
ability, experimental animals, e.g., mice, are preferably used.
Skin, e.g., Langerhans, cell counts are made prior to and at
various time points after administration of a bolus of the
candidate agonist or antagonist. Levels are analyzed in various
samples, e.g., blood, serum, nasal or pulmonary lavages, or tissue
biopsy staining. A successful depleting mAb or binding composition
will significantly lower the level of CCR6 bearing cells. Such may
be at least about 10%, preferably at least about 20%, 30%, 50%,
70%, or more.
[0071] Evaluation of antibodies can be performed in other animals,
e.g., humans using various methods. For example, blood samples are
withdrawn from patients suffering from a skin related disease or
disorder before and after treatment with a candidate mAb.
[0072] IV. Uses
[0073] The exquisite tissue-selective homing of lymphocytes has
long been appreciated as central for the control of systemic immune
responses. Recent advances in the field support a model in which
leukocyte homing is achieved by sequential engagement of
differentially expressed and independently regulated vascular and
leukocyte adhesion molecules, and signaling receptors and their
ligands. Butcher and Picker (1996) Science 272:60-66. The
observation that chemokines, a superfamily of small secreted
proteins with G protein-coupled receptors (Baggiolini (1998) Nature
392:565-568) can attract leukocytes led to the hypothesis that
chemokines provide key signals directing recruitment of T
lymphocyte subsets into lymphoid and extra-lymphoid immune effector
sites. The inflamed skin-specific expression of MIP-3.alpha. and
OCR6 suggests that such skin-specific chemokines selectively
attract functional subsets of lymphocytes into the skin.
[0074] As such, the present invention provides means to purify
desired skin cell subsets. The chemoattractive or chemokinetic
effects on those cells can be the basis of purification methods.
Methods exist for selective migration and recovery of cells to or
from the chemokine, e.g., through porous membrane, or to various
locations in a culture. Other methods exist to selectively separate
cells of particular shapes from others. Alternatively, labeling can
be used to FACS sort cells which specifically bind the chemokine.
Populations of substantially homogeneous Langerhans or skin derived
cells will have important utility in research or therapeutic
environments.
[0075] While MIP-3.alpha. is likely to have functional effects on
CCR6 bearing subsets of cells, e.g., T and B cells-and precursors,
other cells which may also be responsive include dendritic cells or
granulocytes, e.g., neutrophils and/or eosinophils, or their
precursors. Effects on various cell types may be indirect, as well
as direct. A statistically significant change in the numbers of
cells will typically be at least about 10%, preferably 20%, 30%,
50%, 70%, 90%, or more. Effects of greater than 100%, e.g., 130%,
150%, 2.times., 3.times., 5.times., etc., will often be desired.
The effects may be specific in causing chemotaxis to specific
points, or may be chemokinetic, in inducing general movement of
cells, but not necessarily in a specific direction, e.g., of
concentration gradient.
[0076] The present invention will be useful in the treatment of
medical conditions or diseases associated with immunological
conditions of the skin. See, e.g., Bos (ed. 1990) Skin Immune
System CRC Press, Boca Raton, Fla.; Fitzpatrick, et al. (eds. 1993)
Dermatology in General Medicine (4th ed.) McGraw-Hill, NY; Rook, et
al. (eds. 1998) Textbook of Dermatology Blackwell; Habifor and
Habie (1995) Clinical Dermatology: A Color Guide to Diagnosis and
Therapy Mosby; Grob (ed. 1997) Epidemiology, Causes and Prevention
of Skin Diseases Blackwell; Frank, et al. (eds. 1995) Samter's
Immunologic Diseases, 5th Ed., vols. I-II, Little, Brown and Co.,
Boston, Mass.; Coffman, et al (1989) Science 245:308-310; and
Frick, et al. (1988) J. Allergy Clin. Immunol. 82:199-225. The
agonists or antagonists described may be combined with other
treatments of the medical conditions described herein, e.g., an
antibiotic, antifungal, antiviral, immune suppressive therapeutic,
immune adjuvant, analgesic, anti-inflammatory drug, growth factor,
cytokine, vasodilator, or vasoconstrictor. See, e.g, the
Physician's Desk Reference, both prescription and non-prescription
compendiums.
[0077] The CCR6 receptor appears to be preferentially expressed on
CD4+ memory T cells. Its ligand, MIP-3.alpha., is an inflammatory
chemokine expressed by cellular constituents of the skin, whose
expression is inducible after stimulation with T cell-derived
proinflammatory meidators such as IFN-.gamma. and IL-17. Thus, CD4+
memory T cell mediated skin conditions are therapeutic targets of
the antagonists, e.g., psoriasis, atopic dermatitis, contact
dermatitis, SLE, and lichen ruber planus.
[0078] Preferred combination therapies include the MIP-3.alpha.
reagent with various anti-inflammatory agents, such as topical,
transdermal, or systemic steroids or corticosteroids. Systemic,
topical, transdermal, or systemic retinoid or retinoid-like
compounds, or vitamin D analogs, may be administered with the
MIP-3.alpha. therapeutics. Alternatively, various forms of UV light
may be used in combination with these therapeutics, e.g.,
ultraviolet A, ultraviolet B, or narrow bands of UVB.
[0079] For example, the MIP-3.alpha. ligands would be expected to
signal specifically to the cell types expressing their receptor.
Thus, it will be possible to block signaling, e.g., to the T cell
or B cell subsets, by reagents which block receptor signaling,
e.g., antibodies to ligand, and small drug antagonists.
[0080] Standard immunological techniques are described, e.g., in
Hertzenberg, et al. (eds. 1996) Weir's Handbook of Experimental
Immunology vols. 1-4, Blackwell Science; Coligan (1991) Current
Protocols in Immunology Wiley/Greene, NY; and Methods in Enzymology
volumes 70, 73, 74, 84, 92, 93, 108, 116, 121, 132, 150, 162, and
163. These will allow use of the reagents for purifying cell
subpopulations, etc.
[0081] To prepare pharmaceutical or sterile compositions including,
e.g., MIP-3.alpha., the material is admixed with a pharmaceutically
acceptable carrier or excipient which is preferably inert.
Preparation of such pharmaceutical compositions is known in the
art, see, e.g., Remington's Pharmaceutical Sciences and U.S.
Pharmacopeia: National Formulary, Mack Publishing Company, Easton,
Pa. (1984). Typically, therapeutic compositions are sterile.
Alternatively, MIP-3.alpha. antagonist compositions can be
prepared.
[0082] Agonists, e.g., natural ligand, or antagonists, e.g.,
antibodies or binding compositions, are normally administered
parenterally, preferably intravenously. Since such protein or
peptide antagonists may be immunogenic they are preferably
administered slowly, either by a conventional IV administration set
or from a subcutaneous depot, e.g. as taught by Tomasi, et al.,
U.S. Pat. No. 4,732,863. However, as a skin target, the
administration may be topical, transdermal, intradermal,
subcutaneous, or even systemic.
[0083] When administered parenterally the therapeutics will be
formulated in a unit dosage injectable form (solution, suspension,
emulsion) in association with a pharmaceutically acceptable
parenteral vehicle. Such vehicles are inherently nontoxic and
nontherapeutic. The antagonist may be administered in aqueous
vehicles such as water, saline, or buffered vehicles with or
without various additives and/or diluting agents. Alternatively, a
suspension, such as a zinc suspension, can be prepared to include
the peptide. Such a suspension can be useful for subcutaneous (SQ),
intradermal (ID), or intramuscular (IM) injection. The proportion
of therapeutic entity and additive can be varied over a broad range
so long as both are present in effective amounts. The therapeutic
is preferably formulated in purified form substantially free of
aggregates, other proteins, endotoxins, and the like, at
concentrations of about 5 to 30 mg/ml, preferably 10 to 20 mg/ml.
Preferably, the endotoxin levels are less than 2.5 EU/ml. See,
e.g., Avis, et al. (eds. 1993) Pharmaceutical Dosage Forms:
Parenteral Medications 2d ed., Dekker, NY; Lieberman, et al. (eds.
1990) Pharmaceutical Dosage Forms: Tablets 2d ed., Dekker, NY;
Lieberman, et al. (eds. 1990) Pharmaceutical Dosage Forms: Disperse
Systems Dekker, NY; Fodor, et al. (1991) Science 251:767-773;
Coligan (ed.) Current Protocols in Immunology; Hood, et al.
Immunology Benjamin/Cummings; Paul (ed. 1997) Fundamental
Immunology 4th ed., Academic Press; Parce, et al. (1989) Science
246:243-247; Owicki, et al. (1990) Proc. Nat'l Acad. Sci. USA
87:4007-4011; and Blundell and Johnson (1976) Protein
Crystallography, Academic Press, New York. Local, e.g., topical or
transdermal, administration will often be particularly useful.
[0084] Selecting an administration regimen for a therapeutic
agonist or antagonist depends on several factors, including the
serum or tissue turnover rate of the therapeutic, the
immunogenicity of the therapeutic, or the accessibility of the
target cells. Preferably, an administration regimen maximizes the
amount of therapeutic delivered to the patient consistent with an
acceptable level of side effects. Accordingly, the amount of
therapeutic delivered depends in part on the particular agonist or
antagonist and the severity of the condition being treated.
Guidance in selecting appropriate doses of antibodies is found in
the literature on therapeutic uses, e.g. Bach et al., chapter 22,
in Ferrone, et al. (eds. 1985) Handbook of Monoclonal Antibodies
Noges Publications, Park Ridge, N.J.; and Russell, pgs. 303-357,
and Smith et al., pgs. 365-389, in Haber, et al. (eds. 1977)
Antibodies in Human Diagnosis and Therapy Raven Press, New York,
N.Y.
[0085] Determination of the appropriate dose is made by the
clinician, e.g., using parameters or factors known in the art to
affect treatment or predicted to affect treatment. Generally, the
dose begins with an amount somewhat less than the optimum dose and
it is increased by small increments thereafter until the desired or
optimum effect is achieved relative to any negative side effects.
Numbers of CCR6 bearing cells in defined samples might be important
indicators of when an effective dose is reached. Preferably, an
antibody or binding composition thereof that will be used is
derived from the same species as the animal targeted for treatment,
thereby minimizing a humoral response to the reagent.
[0086] The total weekly dose ranges for antibodies or fragments
thereof, which specifically bind to MIP-3a, range generally from
about 1 ng, more generally from about 10 ng, typically from about
100 ng; more typically from about 1 .mu.g, more typically from
about 10 .mu.g, preferably from about 100 pg, and more preferably
from about 1 mg per kilogram body weight. Although higher amounts
may be more efficacious, the lower doses typically will have fewer
adverse effects. Generally the range will be less than 100 mg,
preferably less than about 50 mg, and more preferably less than
about 25 mg per kilogram body weight.
[0087] The weekly dose ranges for antagonists, e.g., antibody,
binding fragments, range from about 10 .mu.g, preferably at least
about 50 .mu.g, and more preferably at least about 100 .mu.g per
kilogram of body weight. Generally, the range will be less than
about 1000 .mu.g, preferably less than about 500 .mu.g, and more
preferably less than about 100 .mu.g per kilogram of body weight.
Dosages are on a schedule which effects the desired treatment and
can be periodic over shorter or longer term. In general, ranges
will -be from at least about 10 .mu.g to about 50 mg, preferably
about 100 .mu.g to about 10 mg per kilogram body weight.
[0088] Other antagonists of the ligands, e.g., muteins, are also
contemplated. Hourly dose ranges for muteins range from at least
about 10 .mu.g, generally at least about 50 .mu.g, typically at
least about 100 .mu.g, and preferably at least 500 .mu.g per hour.
Generally the dosage will be less than about 100 mg, typically less
than about 30 mg, preferably less than about 10 mg, and more
preferably less than about 6 mg per hour. General ranges will be
from at least about 1 .mu.g to about 1000 .mu.g, preferably about
10 .mu.g to about 500 .mu.g per hour.
[0089] In particular contexts, e.g., transplant or skin grafts, may
involve the administration of the therapeutics in different forms.
For example, in a skin graft, the tissue may be immersed in a
sterile medium containing the therapeutic resulting in a
prophylactic effect on cell migration soon after the graft is
applied.
[0090] The present invention also provides for administration of
MIP-3.alpha. antibodies or binding compositions in combination with
known therapies, e.g., steroids, particularly glucocorticoids,
which alleviate the symptoms associated with excessive inflammatory
responses. Daily dosages for glucocorticoids will range from at
least about 1 mg, generally at least about 2 mg, and preferably at
least about 5 mg per day. Generally, the dosage will be less than
about 100 mg, typically less than about 50 mg, preferably less than
about 20 mg, and more preferably at least about 10 mg per day. In
general, the ranges will be from at least about 1 mg to about 100
mg, preferably from about 2 mg to 50 mg per day.
[0091] The phrase "effective amount" means an amount sufficient to
effect a desired response, or to ameliorate a symptom or sign of
the skin condition. Typical mammalian hosts will include mice,
rats, cats, dogs, and primates, including humans. An effective
amount for a particular patient may vary depending on factors such
as the condition being treated, the overall health of the patient,
the method, route, and dose of administration and the severity of
side affects. Preferably, the effect will result in a change in
quantitation of at least about 10%, preferably at least 20%, 30%,
50%, 70%, or even 90% or more. When in combination, an effective
amount is in ratio to a combination of components and the effect is
not limited to individual components alone.
[0092] An effective amount of therapeutic will modulate the
symptoms typically by at least about 10%; usually by at least about
20%; preferably at least about 30%; or more preferably at least
about 50%. Alternatively, modulation of migration will mean that
the migration or trafficking of various cell types is affected.
Such will result in, e.g., statistically significant and
quantifiable changes in the numbers of cells being affected. This
may be an increase or decrease in the numbers of target cells being
attracted within a time period or target area.
[0093] The present invention provides reagents which will find use
in therapeutic applications as described elsewhere herein, e.g., in
the general description for treating disorders associated with skin
conditions. See, e.g., Berkow (ed.) The Merck Manual of Diagnosis
and Therapy, Merck & Co., Rahway, N.J.; Thorn, et al.
Harrison's Principles of Internal Medicine, McGraw-Hill, NY;
Gilman, et al. (eds. 1990) Goodman and Gilman's: The
Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press;
Remington's Pharmaceutical Sciences, 17th ed. (1990), Mack
Publishing Co., Easton, Penn; Langer (1990) Science 249:1527-1533;
and Merck Index, Merck & Co., Rahway, N.J.
[0094] Antibodies to MIP-3.alpha. proteins may be used for the
identification or sorting of cell populations expressing
MIP-3.alpha. protein, e.g., fibroblasts or Langerhans cells.
Methods to sort such populations are well known in the art, see,
e.g., Melamed, et al. (1990) Flow Cytometry and Sorting Wiley-Liss,
Inc., New York, N.Y.; Shapiro (1988) Practical Flow Cytometry Liss,
New York, N.Y.; and Robinson, et al. (1993) Handbook of Flow
Cytometry Methods Wiley-Liss, New York, N.Y. Populations of cells
expressing the MIP-3.alpha. receptor, e.g., CCR6, can also be
purified, e.g., using magnetic beads as described, e.g., in Bieva,
et al. (1989) Exp. Hematol. 17:914-920; Hernebtub, et al. (1990)
Bioconj. Chem. 1:411-418; Vaccaro (1990) Am. Biotechnol. Lab.
3:30.
[0095] Moreover, antisense nucleic acids may be used. For example,
antisense polynucleotides against the ligand encoding nucleic acids
may function in a manner like ligand antagonists, and antisense
against the receptor may function like receptor antagonists. Thus,
it may be possible to block the signaling through the pathway with
antisense nucleic acids. Conversely, nucleic acids for the receptor
may serve as agonists, increasing the numbers of receptor on the
cell, thereby increasing cell sensitivity to ligand, and perhaps
blocking the normal apoptotic signal described.
[0096] Using the assay methods described above, the antibodies or
binding compositions are useful in diagnosing diseases states which
result in skin disorders. Antibodies raised against a MIP-3.alpha.
or CCR6 protein will also be useful to raise anti-idiotypic
antibodies. These will be useful in detecting or diagnosing various
immunological conditions related to expression of the respective
antigens. Combinations of these signals may be also pursued.
[0097] The broad scope of this invention is best understood with
reference to the following examples, which are not intended to
limit the inventions to the specific embodiments.
EXAMPLES
[0098] I. General Methods
[0099] Some of the standard methods are described or referenced,
e.g., in Maniatis, et al. (1982) Molecular Cloning, A Laboratory
Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor Press;
Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual, (2d
ed.), vols. 1-3, CSH Press, NY; Ausubel, et al., Biology, Greene
Publishing Associates, Brooklyn, N.Y.; or Ausubel, et al. (1987 and
Supplements) Current Protocols in Molecular Bioloqy, Greene/Wiley,
New York; Innis, et al. (eds.)(1990) PCR Protocols: A Guide to
Methods and Applications Academic Press, N.Y. Methods for protein
purification include such methods as ammonium sulfate
precipitation, column chromatography, electrophoresis,
centrifugation, crystallization, and others. See, e.g., Ausubel, et
al. (1987 and periodic supplements); Deutscher (1990) "Guide to
Protein Purification" in Methods in Enzymology, vol. 182, and other
volumes in this series; manufacturer's literature on use of protein
purification products, e.g., Pharmacia, Piscataway, N.J., or
Bio-Rad, Richmond, Calif.; and Coligan, et al. (eds.) (1995 and
periodic supplements) Current Protocols in Protein Science, John
Wiley & Sons, New York, NY. Combination with recombinant
techniques allow fusion to appropriate segments, e.g., to a FLAG
sequence or an equivalent which can be fused via a
protease-removable sequence. See, e.g., Hochuli (1989) Chemische
Industrie 12:69-70; Hochuli (1990) "Purification of Recombinant
Proteins with Metal Chelate Absorbent" in Setlow (ed.) Genetic
Engineering, Principle and Methods 12:87-98, Plenum Press, N.Y.;
and Crowe, et al. (1992) QIAexpress: The Hiqh Level Expression
& Protein Purification System QIAGEN, Inc., Chatsworth,
Calif.
[0100] Standard immunological techniques are described, e.g., in
Hertzenberg, et al. (eds. 1996) Weir's Handbook of Experimental
Immunology vols. 1-4, Blackwell Science; Coligan (1991) Current
Protocols in Immunology Wiley/Greene, NY; and Methods in Enzvmology
volumes. 70, 73, 74, 84, 92, 93, 108, 116, 121, 132, 150, 162, and
163.
[0101] Lymphocyte migration assays are performed as previously
described, e.g., in Bacon, et al. (1988) Br. J. Pharmacol.
95:966-974. Other trafficking assays are also available. See, e.g.,
Quidling-Jrbrink, et al. (1995) Eur. J. Immunol. 25:322-327; Koch,
et al. (1994) J. Clinical Investigation 93:921-928; and Antony, et
al. (1993). J. Immunol. 151:7216-7223.
[0102] Alternatively, an activation assay or attraction assay is
used. An appropriate cell type is selected, e.g., hematopoietic
cells, myeloid (macrophages, neutrophils, polymorphonuclear cells,
etc.) or lymphoid (T cell, B cell, or NK cells), neural cells
(neurons, neuroglia, oligodendrocytes, astrocytes, etc.), or stem
cells, e.g., progenitor cells which differentiate to other cell
types, e.g., gut crypt cells and undifferentiated cell types.
[0103] Chemokines may also be assayed for activity in hemopoietic
assays as described, e.g., by H. Broxmeyer. See Bellido, et al.
(1995) J. Clinical Investigation 95:2886-2895; and Jilka, et al.
(1995) Expt'l Hematology 23:500-506. They may be assayed for
angiogenic activities as described, e.g., by Streiter, et al.
(1992) Am. J. Pathol. 141:1279-1284. Or for a role in inflammation.
See, e.g., Wakefield, et al. (1996) J. Surgical Res. 64:26-31.
[0104] Other assays will include those which have been demonstrated
with other chemokines. See, e.g., Schall and Bacon (1994) Current
Opinion in Immunology 6:865-873; and Bacon and Schall (1996) Int.
Arch. Allergy & Immunol. 109:97-109. Ca2+ flux upon chemokine
stimulation is measured according to the published procedure
described in Bacon, et al. (1995) J. Immunol. 154:3654-3666.
[0105] FACS analyses are described in Melamed, et al. (1990) Flow
Cytometry and Sorting Wiley-Liss, Inc., New York, N.Y.; Shapiro
(1988) Practical Flow Cytometry Liss, New York, N.Y.; and Robinson,
et al. (1993) Handbook of Flow Cytometry Methods Wiley-Liss, New
York, N.Y.
[0106] II. Cell Culture and Tissue Samples
[0107] Adult human primary cells including keratinocytes,
melanocytes, and dermal fibroblasts are obtained from Clonetics and
cultured according to the suppliers instructions. For cytokine
treatment, cells are cultured with 10 ng/ml hTNF-.alpha. plus 3
ng/ml hIL-1.beta. (R&D Systems) in culture medium. Human T
cells are purified from PBMCs using a T-cell enrichment column
(R&D Systems) according to the manufacturers instructions.
[0108] III. Isolation of Encoding Sequences
[0109] The human, mouse, or rat MIP-3.alpha. sequence is readily
available. See Table 1 and GenBank. Appropriate PCR primers or
hybridization probes can be selected.
[0110] Similarly, the human CCR6, or others, can be readily
isolated. See Table 2 and GenBank.
[0111] IV. Distribution Analysis
[0112] For Southern blotting, 5 .mu.g of each cDNA library is
digested with the appropriate restriction enzymes to release the
insert, subjected to gel electrophoresis, and transferred to
Hybond-N.sup.+ membrane. For Northern blotting all RNAs are
isolated using RNAzol B (TEL-TEST, Inc.) and analyzed by
electrophoresis on a 1% formaldehyde-agarose gel and transferred to
Hybond-N.sup.+ membrane. Northern and Southern blots are hybridized
for 16 hr at 650 C with .sup.32P-labeled probes obtained by
randomly priming (Prime-it; Stratagene) the full length inserts
from mouse or human MIP-3.alpha. or CCR6 clones. After
hybridization, blots are washed at high-stringency and exposed to
film.
[0113] The MIP-3.alpha. was identified from a cDNA library made
from human monocytes activated with LPS and IFN-.gamma., in the
presence of anti-IL-10. See, Rossi, et al. (1997) J. Immunology
158:1033-1036. Message of the chemokine has also been detected in
pancreatic islet cells, fetal lung, and hepatic HEPG2 cells,
suggesting a physiological role in inflammation or medical
conditions in such organs/tissues.
[0114] The gene is expressed in HL-60 (promyelocytic leukemia); S3
(HeLa cell); K562 (chronic myelogenous leukemia); MOLT-4
(lymphblastic leukemia); Raji (Burkitt's lymphoma); SW480
(colorectal adenocarcinoma); A549 (lung carcinoma); and G361
(melanoma) cell lines, as determined by probing on a tissue blot
from CLONTECH. Tissue expression gave a positive signal in lymph
node, appendix, peripheral blood lymphocytes, fetal liver, and
fetal lung, suggesting a physiological role in inflammation or
medical conditions in such organs/tissues; but no detectable signal
in spleen, bone marrow, brain, and kidney.
[0115] The main transcript appears to be about 1.2 kb, with two
additional transcript sizes in fetal lung RNA. Among the various
tissues, transcript sizes of 1.8, 2.7, and 4.2 kb were
detected.
[0116] Positive signals were also detected in the following cDNA
libraries: dendritic cells activated with LPS, but not when
activated with GM-CSF and IL-4; monocytes treated with LPS,
IFN-.gamma., and anti-IL-10, but not when treated with LPS,
IFN-.gamma., and IL-10; and activated PBMC.
[0117] These expression data implicate this chemokine in
inflammatory responses upon cell activation. The lymph nodes,
appendix, and PBL are sites where inflammatory processes take
place. The MIP-3.alpha. may exert its effects on monocytes and
cells involved in inflammatory events. Other structural features
implicate this chemokine in eosinophil and lung physiology, e.g.,
asthma indications. Thus, an antagonist of the chemokine, e.g., an
antibody, may be important for treatment of asthmatic conditions.
Also, IL-10 appears to inhibit MIP-3.alpha. expression.
[0118] The human MIP-3.alpha. is a ligand for the CCR6. Thus, a
positive control exists for the Ca++ flux assay for that receptor.
This allows for the further screening of agonist ligands for the
CCR6. Moreover, the CCR6 was isolated from eosinophil cDNA, and
observations have been made that eosinophils migrate to
MIP-3.alpha. in vitro. See, e.g., Greaves, et al. (1997) J. Exp.
Med. 186:837-844; Liao, et al. (1997) Biochem. Biophys. Res.
Commun. 236:212-217; and Liao, et al. (1998) J. Immunol.
162:186-194. These suggest that the MIP-3.alpha. interaction with
the CCR6 is important in recruitment of eosinophils, as occurs with
the eotaxin ligand and the CCR3. As such, antagonists of the
MIP-3.alpha. interaction with the CCR6 will likely be useful in
inhibiting eosinophilia, particularly in the lung, or lung
inflammation. These may accompany asthmatic or other pulmonary
conditions. The specific upregulation of the pair in inflamed skin
suggests a role in skin immunity.
[0119] The CCR6 was isolated from a cDNA library made from a
dendritic cell cDNA library. It appears to be expressed in certain
T cells, spleen cell subsets, NK cells, and other cell populations
enriched in dendritic cells, including CD1a.sup.+, CD14.sup.+, and
CD1Aa.sup.+ cells. It did not give a detectable signal in TF1,
Jurkat, MRC5, JY, or U937 cell lines.
[0120] Quantitative PCR methods have been applied, e.g., TAQMAN.TM.
. High levels of CCR6 cDNA was detected in libraries made from
peripheral blood mononuclear cells, resting; T cell, THO clone Mot
72, resting; T cell, TH1 clone HY06, anergic; T cell clones,
pooled, resting; T cell .gamma..delta. clones, resting;
Splenocytes, resting; Splenocytes, activated; B cell EBV lines,
resting; NK 20 clones pooled, resting; NK cell clone, NKA6; NK
cytotoxic clone, resting; NK cell clone, NK non cytotox; monocytes,
LPS, .gamma.IFN, anti-IL-10, 4+16 hr; monocytes, LPS, .gamma.IFN,
IL-10, 4+16 hr; DC 70% CD1a+, ex CD34+ GM-CSF, TNF.alpha.,
activated 1 hr; DC 70% CD1a+, ex CD34+ GM-CSF, TNF.alpha.,
activated 6 hr; DC 95% CD1a+, ex CD34+ GM-CSF, TNF.alpha.,
activated 1+6 hr; DC 95% CD14+, ex CD34+ GM-CSF, TNF.alpha.,
activated 1+6 hr; DC CD1a+ CD86+, ex CD34+ GM-CSF, TNF.alpha.,
activated 1+6 hr; DC resting CD34 derived; DC CD4lo activated mo
derived; DC resting activated mo derived; DC TGF and TGFb CD34
derived; lung fetal; gall bladder fetal; small intestine fetal;
ovary fetal; spleen fetal; normal human colon; normal human
thyroid; tonsil inflammed; pool of three heavy smoker human lung
samples; allergic lung sample; Hashimoto's thyroiditis thyroid
sample; and Psoriasis patient skin sample. Intermediate levels were
detected in libraries derived from peripheral blood mononuclear
cells, activated; T cell, THO clone Mot 72, activated; T cell, THO
clone Mot 81, resting; T cell, THO clone Mot 81, Activated; T cell,
TH1 clone HY06, resting; T cell, TH1 clone HY06, activated; B cell
line JY, activated; NK 20 clones pooled, activated; NK cell clone,
NKB1, pSPORT; NK cell clone, NKB1; DC ex monocytes GM-CSF, IL-4,
resting; DC ex monocytes GM-CSF, IL-4, monokine activated 4+16 hr;
eosinophils; testes fetal; placenta 28 wk; pool of two normal human
lung samples; ulcerative colitis human colon sample; pool of
rheumatoid arthritis samples, human; and normal w.t. monkey colon.
Low or undetectable levels were detected in libraries from T cell,
THO clone Mot 72, anergic; T cell, TH2 clone HY935, resting; T
cell, TH2 clone HY935, activated; T cell, TH1 clone TA20-23,
resting; T cell, TH1 clone TA20-23, activated; T cell, THO clone
B21, resting; T cell, THO clone B21, Activated; T cells CD4+, TH2
polarized, activated; T cell lines Jurkat and Hut78, resting; U937
premonocytic line, resting; U937 premonocytic line, activated;
monocytes, LPS, .gamma.IFN, anti-IL-10; monocytes, LPS, .gamma.IFN,
IL-10; monocytes, LPS, 1 hr; monocytes, LPS, 6 hr; DC 70% CD1a+, ex
CD34+ GM-CSF, TNF.alpha., resting; DC ex monocytes GM-CSF, IL-4,
resting; DC ex monocytes GM-CSF, IL-4, LPS activated 4+16 hr;
kidney fetal; liver fetal; heart fetal; brain fetal; Allergic lung
#19; adipose tissue fetal; uterus fetal; normal human skin;
Pneumocystic carnii pneumonia lung sample; normal w.t. monkey lung;
Ascaris-challenged monkey lung, 24 hr.; and Ascaris-challenged
monkey lung, 4 hr.
[0121] Being found on dendritic cells, its ligand, including the
MIP-3.alpha., may be important in attracting appropriate cells for
the initiation of an immune response. MIP-3.alpha. has been shown
to be a very potent chemoattractant for dendritic cells.
Significant roles of the ligand and receptor in skin physiology are
suggested. The receptor may be also present in other cells
important in such responses.
[0122] V. Chemotaxis.
[0123] Recombinant mouse MIP-3.alpha. is produced in E. coli and
purified, e.g., as previously described for other chemokines.
Hedrick, et al. (1998) Blood 91:4242-4-247. Total human T cells in
DMEM, pH 6.9, 1% bovine serum albumin, were added to the top
chamber of 3 pm pore polycarbonate Transwell culture insert
(Costar) and incubated with the indicated concentrations of
purified chemokine in the bottom chamber for 3 h. The number of
migrating cells of each subtype is determined by multi-parameter
flow cytometry using fluorochrome conjugated antibodies. A known
number of 15 .mu.m microsphere beads (Bangs Laboratories, Fishers,
Ind.) is added to each sample before analysis in order to determine
the absolute number of migrating cells.
[0124] Chemotaxis assays are performed with purified human
peripheral-blood T cells and/or skin-homing T cells. Other cell
types express the CCR6, e.g., T cells, B cells, DC cells, and
granulocyte cells, e.g., neutrophils and/or eosinophils.
Recombinant murine MIP-3.alpha. should have effects on the cell
types expressing CCR6.
[0125] The MIP-3.alpha. and CCR6 expression levels are very low in
normal skin samples, but are highly upregulated in inflamed skin
tissues.
[0126] VI. Antibody Production Appropriate mammals are immunized
with appropriate amounts, e.g., of MIP-3.alpha. or MIP-3.alpha.
gene transfected cells, e.g., intraperitoneally every 2 weeks for 8
weeks. Similar methods may be used to produce antibodies which bind
to CCR6, e.g., purified CCR6, polypeptides, or transfected cells
expressing the receptor may be used. Typically, rodents are used,
though other species should accommodate production of selective and
specific antibodies. The final immunization is given intravenously
(IV) through the tail vein.
[0127] Generic polyclonal antibodies may be collected.
Alternatively, monoclonal antibodies can be produced. For example,
four days after the IV injection, the spleen is removed and fused
to SP2/0 and NS1 cells. HAT resistant hybridomas are selected,
e.g., using a protocol designed by Stem Cell Technologies
(Vancouver, BC). After 10 days of HAT selection, resistant foci are
transferred to 96 well plates and expanded for 3 days. Antibody
containing supernatants are analyzed, e.g., by FACS for binding to
NIH3T3/surface MIP-3.alpha. transfectantsM Many different
MIP-3.alpha. mAbs are typically produced. Those antibodies may be
isolated and modified, e.g., by labeling or other means as is
standard in the art. See, e.g., Harlow and Lane (1988) Antibodies:
A Laboratory Manual CSH Press; Goding (1986) Monoclonal Antibodies:
Principles and Practice (2d ed.) Academic Press, New York, N.Y.
Methods to conjugate magnetic reagents, toxic entities, labels,
attach the antibodies to solid substrates, to sterile filter, etc.,
are known in the art.
[0128] VII. Purification of Cells
[0129] MIP-3.alpha. responsive cells may be identified using the
reagents described herein. For example, cells which are
chemoattracted towards MIP-3.alpha. may be purified from other
cells by collecting those cells which traverse towards
MIP-3.alpha.. Such chemotaxis may be to a source of chemokine, or
may be across a porous membrane or other substrate. See above, in
the microchemotaxis assay.
[0130] Alternatively, responsive cells may be identified by
expression of the receptor, e.g., CCR6, as provided herein. Thus,
antibodies which recognize CCR6 may be used as a positive marker
for sorting cells likely to respond to MIP-3.alpha.. Conversely,
the marker may be used to deplete CCR6 bearing cells, e.g., by
magnetic depletion or toxic conjugates.
[0131] Analysis of human samples can be evaluated in a similar
manner. A biological sample, e.g., blood, tissue biopsy sample,
lung or nasal lavage, skin punch, is obtained from an individual
suffering from a skin related disorder. MIP-3.alpha. responsive
cell analysis is performed, e.g., by FACS analysis, or similar
means.
[0132] VIII. MIP-3.alpha. Antagonists
[0133] Various antagonists of MIP-3.alpha. are made available. For
example, antibodies against the chemokine itself may block the
binding of ligand to its receptor, thereby serving as a direct
receptor antagonist. Other antagonists may function by blocking the
binding of ligand to receptor, e.g., by binding to the receptor in
a way to preclude the possibility of binding of ligand. Other
antagonists, e.g., mutein antagonists or aptamers, may bind to the
receptor without signaling, thereby blocking a true agonist from
binding. Many of these may serve to block the signal transmitted to
target cells, specifically MIP-3.alpha.-responsive cells. These may
be skin cells, including Langerhans, fibroblasts, or
keratinocytes.
[0134] Information on the criticality of particular residues is
determined using standard procedures and analysis. Standard
mutagenesis analysis is performed, e.g., by generating many
different variants at determined positions, e.g., at the positions
identified above, and evaluating biological activities of the
variants. This may be performed to the extent of determining
positions which modify activity, or to focus on specific positions
to determine the residues which can be substituted to either
retain, block, or modulate biological activity.
[0135] Alternatively, analysis of natural variants can indicate
what positions tolerate natural mutations. This may result from
populational analysis of variation among individuals, or across
strains or species. Samples from selected individuals are analyzed,
e.g., by PCR analysis and sequencing. This allows evaluation of
population polymorphisms.
[0136] IX. Psoriasis Studies
[0137] Psoriasis is a common chronic inflammatory skin disease
characterized by a marked inflammatory infiltrate and
hyperproliferation of keratinocytes. The infiltrate is composed of
skin-infiltrating T cells, predominantly of the memory phenotype,
neutrophils, lining macrophages and increased numbers of dendritic
cells. Ortonne (1996) Br. J. Dermatol. 135:Suppl 49:1-5; Prens, et
al. (1995) Clin. Dermatol. 13:115-129; and Elder, et al. (1994) J.
Invest. Dermatol. 102:24S-27S. There is evidence that T cells play
a crucial role in the immunopathogenesis of this disease. See,
e.g., Gottlieb, et al. (1995) Nature Med. 1:442-447; Nicolas, et
al. (1991) Lancet 338:321; Cooper, et al. (1990) J. Am. Acad.
Dermatol. 23:1318-1326; Ellis, et al. (1986) JAMA 256:3110-3116;
Krueger, et al. (1995) J. Exp. Med. 182:2057-2068; Schon, et al.
(1997) Nature Med. 3:183-188; Schon (1999) J. Invest. Dermatol.
112:405-410; Wrone-Smith and Nickoloff (1996) [see comments] J.
Clin. Invest. 98:1878-1887; Uyemura, et al. (1993) J. Invest.
Dermatol. 101:701-705; (1996) Arch. Dermatol. 132:419-423; and
Barker (1998) Hosp. Med. 59:530-533. An early cellular event in the
development of psoriatic lesions is the infiltration of target
sites by activated T cells which in turn produce inflammatory
mediators, such as IFN-.alpha., induce epidermal hyperplasia and
may act with keratinocytes and dermal macrophages to sustain a
cycle of inflammation which finally leads to a psoriatic phenotype.
Bata-Csorgo, et al. (1995) J. Invest. Dermatol. 105:89S-94S.
[0138] A CC chemokine designated macrophage inflammatory
protein-3.alpha. (MIP-3.alpha.; also known as LARC) was previously
cloned and characterized, and CCR6 identified as its specific
receptor. See, e.g., Rossi, et al. (1997) J. Immunol.
158:1033-1036; and Greaves, et al. (1997) J. Exp. Med. 186:837-844.
MIP-3.alpha. is known to attract both T and dendritic cells. See
Dieu, et al. (1998) J. Exp. Med. 188:373-386; and Power, et al.
(1997) J. Exp. Med. 186:825-835. Among dendritic cells,
MIP-3.alpha. is a highly potent chemokine for the chemoattraction
of epithelial Langerhans type dendritic cells derived from CD34+
hematopoetic progenitor cells. Dieu, et al. (1998) J. Exp. Med.
188:373-386. Recently, MIP-3.alpha. has been shown to
preferentially attract the memory subset of T cells. Liao, et al.
(1999) J. Immunol. 162:186-194; and Campbell, et al. (1998) Science
279:381-384.
[0139] This study sought to identify chemokines and chemokine
receptors involved in autoimmune diseases. To this end, an analysis
was made of the expression of mRNA of various chemokines and
receptors in samples of inflammatory skin diseases using real time
quantitative PCR. Here is reported that the expression of the CC
chemokine MIP-3.alpha. and its receptor CCR6 are significantly
upregulated in psoriasis. Within psoriatic lesions,
MIP-3.alpha.-expressing keratinocytes co-localize with
skin-infiltrating T lymphocytes. Furthermore, CCR6 is expressed at
high levels on the skin-homing CLA+ subset of memory T cells.
Finally, biologically active MIP-3.alpha. is induced in cellular
constituents of the skin by proinflammatory cytokines and T
cell-derived inflammatory mediators. Taken together, these
observations strongly suggest that this ligand/receptor pair may
represent a unique role in the development of psoriasis.
[0140] Patients: 6 mm punch biopsies were taken, after obtaining
informed consent, from either lesional (n=10) and non-lesional
(n=5) skin of psoriatic patients or from normal (n=5) healthy
individuals. Skin samples were immediately frozen in liquid
nitrogen and stored at -80.degree. C. In addition, 50 ml
heparinized blood was drawn from either psoriatic patients (n=15)
in lesional phases of the disease or healthy donors (n=3) and
peripheral blood mononuclear cells were prepared using standard
protocols. The psoriatic patients used in this study had been
untreated for at least three weeks.
[0141] Real Time Quantitative PCR (TaqMan.RTM.) Analysis of
MIP-3.alpha. and CCR6 mRNA Expression: Skin biopsies were
homogenized in liquid nitrogen using a Mikro-Dismembrator U (Braun
Biotech, San Diego, Calif.) and RNA was extracted with RNAzol
according to the manufacturer's protocol (Tel-Test, Friedensburg,
Tex.). 4 .mu.g of RNA were treated with DNase I (Boehringer,
Mannheim, Germany) and reverse transcribed with oligo dT.sub.14-18
(Gibco BRL, Gaithersburg, Md.) and random hexamer primers (Promega,
Madison, Wis.) using standard protocols. cDNA was diluted to a
final concentration of 5 ng/.mu.l. 10 .mu.l of cDNA were amplified
in the presence of 12.5 .mu.l of TaqMan.RTM. universal master mix
(Perkin Elmer, Foster City, Calif.), 0.625 .mu.l of gene-specific
TaqMan.RTM. probe, 0.5 .mu.l of gene-specific forward and reverse
primers, and 0.5 .mu.l of water. As an internal positive control,
0.125 .mu.l of 18S RNA-specific TaqMan.RTM. probe and 0.125 .mu.l
of 18S RNA-specific forward and reverse primers were added to each
reaction. Specific primers and probes for MIP-3.alpha., CCR6, and
the other chemokine receptors measured were obtained from Perkin
Elmer (Foster City, Calif.). Gene-specific probes used FAM as
reporter whereas probes for the internal positive control (18S RNA)
were associated with either the JOE or VIC reporters. Samples
underwent the following stages: stage 1, 50.degree. C. for 2
minutes; stage 2, 95.degree. C. for 10 minutes; and stage 3,
95.degree. C. for 15 seconds followed by 60.degree. C. for 1
minute. Stage 3 was repeated 40 times. Gene-specific PCR products
were measured by means of an ABI PRISM.RTM. 7700 Sequence Detection
System (Perkin Elmer, Foster City, Calif.) continuously during 40
cycles. Specificity of primer probe combination was confirmed in
crossreactivity studies performed against plasmids of all known
chemokine receptors (CCR1-CCR9, CXCR1-CXCR5, XCR1, CX3CR1) and the
following panel of chemokines: MIP-1.alpha., MIP-1.beta.,
MIP-1.delta., MIP-3.beta., 6Ckine, IP-10, Mig, TCA-3, I-309, I-TAC,
HCC-1, HCC-4, Gro-.alpha./.beta., ENA78, eotaxin, eotaxin-2,
DC-CK1, BCA-1, fractalkine, SDF-1.alpha., RANTES, PF4, PBP, MDC,
lymphotactin, IL-8, TARC, MDC, TECK, and MCP-1-MCP-4. Target gene
expression was normalized between different samples based on the
values of the expression of the internal positive control. Human
cDNA libraries used in this study were generated as described
previously. See, e.g., Rossi, et al. (1997) J. Immunol.
158:1033-1036; Bolin, et al. (1997) J. Neurosci. 17:5493-5502; and
Vicari, et al. (1997) Immunity 7:291-301.
[0142] Cell Culture: Human primary epidermal keratinocytes, dermal
fibroblasts, melanocytes, and dermal microvascular endothelial
cells were purchased from Clonetics (San Diego, Calif.) and
cultured in keratinocyte (KGM), fibroblast (FGM), melanocyte (MGM),
or endothelial cell (EGM-2) growth medium (Clonetics, San Diego,
Calif.) as described in Detmar, et al. (1994) J. Exp. Med.
180:1141-1146. Cells were treated with TNF-.alpha. (10 ng/ml) /
IL-1.beta. (5 ng/ml), IFN-.gamma. (20 ng/ml), IL-4 (50 ng/ml),
IL-17 (100 ng/ml) (R&D Systems Inc., Mineapolis, Minn.), or
left untreated. The epidermal .gamma..delta. T cell line, 7-17, was
kindly provided by Richard Boismenu (The Scripps Institute, La
Jolla, Calif.) and cultured as described previously. See Boismenu,
et al. (1996) J. Immunol. 157:985-992. Epidermal .gamma..delta. T
cells were cultured with Con A, TNF-.alpha. (10 ng/ml) /
IL-1.gamma. (5 ng/ml), or medium alone. Supernatants as well as
cells were harvested after 6 or 18 h. Generation of dendritic cells
either from cord blood CD34+ hematopoetic progenitor cells or from
peripheral blood monocytes was performed as described in Dieu, et
al. (1998) J. Exp. Med. 188:373-386. Immature dendritic cells from
CD34+ hematopoetic progenitor cells or monocyte-derived dendritic
cells were activated for 3-24 h in the presence of CD40L
transfected L cells (one per five dendritic cells) as described.
See Caux, et al. (1994) J. Exp. Med. 180:1263-1272. Cells and
supernatants were harvested 3, 12, 24, 48 hours after CD40L
stimulation. RNA was extracted from cells as described above.
[0143] Flow Cytometry and Cell Sorting: In order to analyze
chemokine receptor expression of skin-homing T cell subsets,
CLA.sup.+/CD45RO.sup.+/CD4.sup.+ cells were sorted from PBMCs
isolated from 2 different donor pool buffy coats (70 ml) from 3
individual donors each using a FACS Vantage (Becton Dickinson, San
Jose, Calif.) and the following monoclonal antibodies (mAb)
(Pharmingen, San Diego, Calif.): FITC-conjugated anti-CLA
(HECA4522) mAb, PE-conjugated anti-CD45RO (UCHL1), APC-conjugated
anti-CD4 (RPA-T4). The purity of the cells was detected as
>99.5%. Subsequently, RNA was extracted and reverse transcribed
as described above.
[0144] In other experiments, CCR6 expression was analyzed on memory
T cell subsets using the following antibodies: FITC-conjugated
anti-CLA (HECA4522) mAb (Pharmingen, San Diego, Calif.),
FITC-conjugated anti-CD45RO (UCHL1) mAb (Pharmingen, San Diego,
Calif.), APC-conjugated afiti-CD8 (RPA-T8) mAb (Pharmingen, San
Diego, Calif.), Cy-chrome-conjugated anti-CD4 (RPA-T4) mAb
(Pharmingen, San Diego, Calif.), anti-CCR6-PE conjugated
(53103.111) mAb (R&D Systems, Mineapolis, Minn.), mouse
IgG.sub.2b-PE-conjugated (R&D Systems, Mineapolis, Minn.).
Briefly, 10.sup.6 PBMCs were stained with anti-CD4, anti-CD8,
anti-CLA, anti-CCR6 mAb or isotype and analyzed using a FACSCalibur
and CELLQuest software (Becton Dickinson, San Jose, Calif.).
[0145] In Situ Hybridization: In situ hybridization was performed
as described. Dieu, et al. (1998) J. Exp. Med. 188:373-386. Coupled
primers were used for amplifying by RT-PCR the majority of the open
reading frame of the MIP-3.alpha. gene. +77/MIP-3.alpha. forward
primer and -425/MIP-3.alpha. were used with an annealing
temperature of 62.degree. C. Then, PCR products were cloned into
pCRII TA cloning vector (Invitogen, Leek, The Netherlands), RNA
probes were synthesized using Sp6 and T7 RNA polymerases
(Boehringer Mannheim, Germany) and radiolabled with .sup.35S-UTP
(Amersham Corp., United Kingdom). Sense and anti-sense
.sup.35S-labeled probes of MIP-3.alpha. were obtained by run-off
transcription of the 367 bp fragment and then partially degraded by
alkaline hydrolysis for 20 min at 60.degree. C. 6 .mu.m cryostat
sections were prepared on charged electrostatic slides
(SuperFrost/Plus, Polylabo, Strasbourg, France) and fixed with cold
acetone for 20 min, with 4% paraformaldehyde for 20 min followed by
0.1 M triethanolamine/0.25% acetic anhydride. The sections were
hybridized overnight at 50.degree. C. (2 to 3.times.10.sup.6
cpm/slide), RNAse A treated, and exposed for 40 days. After
development, the sections were stained with hematoxylin.
[0146] Immunohistochemistry: Frozen 6 .mu.m tissue sections were
fixed in acetone and in paraformaldehyde before the immunostaining.
To block the non-specific binding.of avidin, biotin system
components, or endogenous peroxidase activity, sections were
pre-treated with avidin D and biotin solutions (Blocking kit,
Vector, Biosys SA, Compiegne, France) for 10 min each step and with
PBS containing 0.3% hydrogen peroxide (Sigma, France) for 15 min at
room temperature. After brief washing in PBS, the sections were
incubated with blocking serum (2% normal rabbit serum) for at least
30 min before adding both primary antibodies. Sections were double
stained simultanously with anti-hMIP-3.alpha. goat polyclonal
antibody (IgG isotype, R&D Systems Inc., Minesota, Minn.) and
anti-hCD3 mouse monoclonal antibody (Leu-4, IgG1 isotype,
Becton-Dickinson, Moutain View, Calif.) for 1 hour at room
temperature in a humid atmosphere. The binding of goat IgG was
detected using biotinylated rabbit anti-goat IgG followed by
streptavidin-peroxidase (both included in the Vectastain ABC kit:
Goat IgG PK-4005, Vector) and the binding of mouse IgGl was
detected by rabbit alkaline phosphatase-labeled anti-mouse IgG
(D0314, Dako, Glostrup, Denmark) at the same time at room
temperature in a humid atmosphere. The peroxidase and alkaline
phosphatase activities were revealed using 3-amino-9-ethylcarbazole
(AEC) substrate (SK-4200, Vector) and alkaline phosphatase
substrate III (SK-5300, Vector) for 5 to 10 min at room
temperature, respectively. Negative controls were established by
adding non-specific isotype controls as primary antibodies.
[0147] Generation of Mouse mAbs Against hMIP-3.alpha. and
Development of an hMIP-3.alpha. ELISA: Inbred BALB/c mice were
immunized with three successive intraperitoneal injections of
complete Freund's adjuvant (Sigma Chemical Co., St. Louis, Mo.),
incomplete Freund's adjuvant, or without Freund's adjuvant,
respectively, with 50 ng of purified hMIP-3.alpha. obtained from
supernatants of hMIP-3.alpha. transient transfected COP5 cells.
Spleens were removed for fusion 3 days after a final i.v. injection
of hMIP-3.alpha.. Hybridization was carried out using the
non-secreting myeloma cell line SP2/0-Ag8 with polyethylene glycol
1000 (Sigma Chemical Co, St. Louis, Mo.). hMIP-3.alpha. transient
transfected COP5 cells were cultured for 2 days in 96 well plates
and fixed in acetone. Then, hybridoma supernatants were harvested
after 6 days, were incubated for 30 min on fixed hMIP-3.alpha.
transient transfected COP5 cells. Antibody binding was then
revealed with peroxidase-conjugated sheep anti-mouse IgG (Biosys,
Compiegne, France) at a 1:200 dilution in PBS for 30 min at
37.degree. C. Positive hybridomas were cloned by limiting dilution
and expanded using a high density culture system (Integra cell line
CL1000, Integra Biosciences, France). After sodium sulphate
precipitation, the mAbs were purified by anion-exchange
chromatography on a Hyper-D column and peroxidase labeled
(Sepracor, Villeneuve, France). An ELISA was set up using one of
the anti-hMIP-3.alpha. mAbs, 319F6, as a capture mAb and a
peroxidase-coupled mouse anti-hMIP-3.alpha. mAb to reveal the
captured hMIP-3.alpha.. The assay proved to be specific for
MIP-3.alpha. with a sensitivity of 0.2 ng/ml.
[0148] Analysis of hMIP-3.alpha. bioactivity by calcium
mobilization assay: A cell line expressing the human CCR6 chemokine
receptor was kindly provided by Chuan Chu Chou (SPRI, Kennilworth,
N.J.). Briefly, the CCR6 open reading frame was cloned into the
pME18sneo eukaryotic expression vector and transfected into the
murine B-cell line, BAF/3 by electroporation. Stable transfectants
were isolated by selection in medium containing 1 mg/ml G418. CCR6
expression was confirmed using calcium signaling, ligand binding
analysis with recombinant human MIP-3.alpha. (R&D Systems,
Minneapolis, Minn.), and immunohistochemistry with anti-CCR6
(53103.111) mAb (R&D Systems, Minneapolis, Minn.). The average
number of binding sites per cell was estimated to be 220,000.
[0149] To measure the biological activity of the MIP-3.alpha.
produced by keratinocytes fibroblast or endothelial cells,
supernatants from these cell cultures were concentrated 20-fold
using Centriplus concentrators with a cut off of 3 kD (Amicon,
Beverly, Mass.). The calcium response to supernatants from these
resting or activated cells was measured using standard protocols.
See, e.g., Greaves, et al. (1997) J. Exp. Med. 186:837-844.
Briefly, the BAF/3 parental and CCR6 transfectant were loaded for
60 min at 37.degree. C. with 3 .mu.M INDO-1A (Molecular-Probes,
Eugene, Oreg.). Cells were washed and resuspended in Hank's
balanced salt solution (HBSS) (Gibco/BRL, Grand Island, N.Y.) to a
final concentration of 10.sup.7 cells/ml. Calcium mobilization was
measured using a Photon, Technology International spectrophotometer
with excitation at 350 nm and dual simultaneous recording of
fluorescence emission at 400 nm and 490 nm. Relative intracellular
calcium levels are expressed as the 400 nm/490 nm emission ratio.
Experiments were performed at 37.degree. C. with constant mixing in
cuvettes containing 10.sup.6 cells in 2 ml of HBSS with 1 mM
CaCl.sub.2. In order to demonstrate specificity of
MIP-3.alpha.-induced calcium mobilization, neutralization studies
were performed using a blocking mouse anti-human MIP-3.alpha.
(IgG.sub.1) (67310.111) mAb (R&D Sytems, Minneapolis, Mo.) or
isotype control (IgG.sub.1) (Sigma, St. Louis Mo.).
[0150] MIP-3.alpha. and its Specific Receptor CCR6 Are
Significantly Upregulated in Lesional Psoriatic Versus Non-Lesional
or Normal Skin: After cloning and initial characterization of mouse
and human MIP-3.alpha. and identification of its receptor, CCR6,
the potential role of this ligand-receptor pair was investigated in
human diseases. See Rossi, et al. (1997) J. Immunol. 158:1033-1036;
and Greaves, et al. (1997) J. Exp. Med. 186:837-844. To this end, a
systematic screening of human tissue cDNA libraries with
MIP-3.alpha.- and CCR6-specific TaqMan.RTM. probe and primers was
undertaken. The cDNA library panel included various libraries
derived from human autoimmune disease samples. This initial
screening showed that MIP-3.alpha. was expressed more than
100-times higher in a cDNA library derived from lesional psoriatic
skin (568000 fg/50 ng cDNA) when compared to normal skin (5530
fg/50 ng cDNA). Moreover, TaqMan.RTM. analyses showed abundant CCR6
message (864 fg/50 ng cDNA) in the cDNA library generated from
psoriatic skin. In contrast, CCR6 was undetectable in a cDNA
library derived from normal skin. Other chemokine receptors that
have been reported to be upregulated in psoriasis were investigated
in the cDNA libraries from normal and psoriatic skin. To this end,
the expression of the IL-8 receptors was studied, and confirmed
previous reports. See Kulke, et al. (1998) J. Invest. Dermatol.
110:90-94; and Schulz, et al. (1993) J. Immunol. 151:4399-4406.
CXCR1 and CXCR2 were constitutively expressed in the cDNA library
derived from normal skin and markedly upregulated in the psoriatic
skin cDNA library. Thus, the cDNA libraries derived from normal or
psoriatic skin provided representative tools to study gene
expression. These initial observations prompted investigation of a
possible role for MIP-3.alpha. and CCR6 in the pathogenesis of
psoriasis in more detail. These led to efforts to validate these
observations in more patient samples. Consecutive quantitative real
time PCR analysis of cDNA derived from either lesional (n=10),
non-lesional (n=5) psoriatic or normal (n=5) skin confirmed that
both MIP-3.alpha. and CCR6 were significantly upregulated in
lesional psoriatic versus non-lesional or normal skin (P<0.005).
An average 7- and 4-fold induction of MIP-3.alpha. and CCR6 could
be detected, respectively. To look more precisely at the
distribution of MIP-3.alpha., in situ hybridization and
immunohistochemistry were performed using specific probes and
polyclonal antibodies directed against MIP-3.alpha.. In situ
hybridization with a MIP-3.alpha. specific probe showed selective
expression of this chemokine in the suprabasal layers of the
epidermis of lesional psoriatic skin. These showed specific
hybridization within the stratum granulosum of lesional psoriatic
skin. In addition, MIP-3.alpha. mRNA expression could be detected
within the stratum spinosum of psoriatic epidermis. Sense (vs
antisense) controls as well as in situ hybridization with normal
and non-lesional psoriatic skin confirmed the specific detection of
MIP-3.alpha. hybridization in lesional psoriatic skin. Furthermore,
irmunohistochemical staining of lesional psoriatic skin confirmed
the focal upregulation of MIP-3.alpha. protein within suprabasal
layers of the epidermis. In contrast, non-lesional psoriatic and
normal skin showed no specific staining for MIP-3.alpha.. Staining
with isotype controls showed the specificity of MIP-3.alpha.
detection. Attempts to detect CCR6by immunohistochemistry for CCR6
were inconclusive due to the low sensitivity of the antibody.
Therefore, double stainings were performed for MIP-3.alpha. and CD3
to study localization of MIP-3(-expressing cells and T cells.
Immunohistochemisty of lesional psoriatic skin showed that focal
accumulation of T cells in the papillary dermis of lesional
psoriatic skin was directly adjacent to foci of
MIP-3.alpha.-expressing epidermal cells. Moreover,
MIP-3.alpha.-expressing keratinocytes within the epidermis
co-localized with intraepidermal CD3.sup.+ T cells.
[0151] Peripheral Blood Mononuclear Cells From Psoriasis Patients
Express Significantly Higher Levels of CCR6 When Compared With
Those of Healthy Donors: Clinically, it is well known that
infections may trigger psoriatic episodes and recently it has been
suggested that superantigens may play a role in T cell activation
during the pathogenesis of psoriasis. See Valdimarsson, et al.
(1997) Clin. Exp. Immunol. 107 Suppl 1:21; Nickoloff, et al. (1993)
J. Immunol. 150:2148-2159; and Nickoloff, et al. (1993) J.
Dermatol. Sci. 6:127-133. However, very little is known about
chemokine receptor expression on PBMCs of normal healthy donors
versus psoriatic patients. Interestingly, CCR6 was also
significantly upregulated in PBMCs derived from psoriasis patients
(n=10) versus PBMC from healthy donors (n=5) (P<0.001). PBMCs
from psoriatic patients expressed on average 4-fold higher levels
of CCR6 mRNA when compared with PBMCs from healthy donors. The CLA+
T cell subset represents a skin-associated population of memory T
cells that migrates preferentially to normal and chronically
inflamed cutaneous sites. See Picker, et al. (1990) J. Immunol.
145:3247-3255.
[0152] Subsequent experiments focused on the chemokine receptor
profile of pathologically relevant skin-homing CLA.sup.+ memory T
cells. Flow cytometric analyses revealed that CCR6 was expressed at
high levels on the surface of skin-homing CLA.sup.+ T cells of
normal donors. Moreover, CCR6 was predominantly expressed on the
CD4.sup.+ subset of CLA.sup.+ T cells. The latter observation may
account for the therapeutic effect of anti-human CD4 antibodies in
the treatment of psoriasis. See Isaacs, et al. (1997) Clin. Exp.
Immunol. 110:158-166; Thivolet and Nicolas (1994) Int. J. Dermatol.
33:327-332; and Morel, et al. (1992) J. Autoimmun. 5:465-477.
[0153] In agreement with flow cytometric analyses, TaqMan.RTM.
analyses on sorted CLA+ memory T cells from normal donors indicated
that they express CCR6 at high levels. In fact, the expression of
CCR6 was the highest one detected among chemokine receptors,
especially when compared to receptors of other chemokines (IL-8,
Gro-.alpha., IP-10, Mig, MCP-1, RANTES) that have already been
associated with psoriasis. See Schulz, et al. (1993) J. Immunol.
151:4399-4406; Gottlieb, et al. (1988) J. Exp. Med. 168:941-948;
Lemster, et al. (1995) Clin. Exp. Immunol. 99:148-154; Gillitzer,
et al. (1993) J. Invest. Dermatol. 101:127-131; Fukuoka, et al.
(1998) Br. J. Dermatol. 138:63-70; and Goebeler, et al. (1998) J.
Pathol. 184:89-95. CCR6 expression was 100 to more than 1000-times
higher than CXCR1, CXCR2, CXCR3, CCR2, CCR3 and CCR5 on this
skin-homing subset of memory T cells.
[0154] TNF-.alpha., IL-1.beta., IFN-.gamma., IL-17, and CD40L
Regulate the Expression of MIP-3.alpha. in Cellular Constituents of
the Skin: The pattern of MIP-3.alpha. expression within the
epidermis suggested that keratinocytes may be a major source for
MIP-3.alpha. in the skin. To further investigate the cellular
origin of MIP-3.alpha. within the skin and to get insights into its
regulation, human primary keratinocytes, melanocytes, and dermal
fibroblasts were cultured with TNF-.alpha./IL-1.beta., IFN-.gamma.,
IL-4, IL-17, or medium alone as control. Furthermore, it was tested
whether cultured epidermal .gamma..delta. T cells resting or
stimulated with either TNF-.alpha./IL-1.beta. or Con A may express
MIP-3.alpha.. Human primary dermal microvascular endothelial cells
were also cultured in the presence or absence of
TNF-.alpha./IL-1.beta.. TNF-.alpha. and IL-1.beta. were used for
stimulation since these proinflammatory cytokines are known to be
upregulated during inflammatory conditions and in psoriatic
lesional skin. See Terajima, et al. (1998) Arch. Dermatol. Res.
290:246-252; Mizutani, et al. (1997) J. Dermatol. Sci. 14:145-153;
Debets, et al. (1995) Eur. J. Immunol. 25:1624-1630; Nickoloff, et
al. (1991) Am. J. Pathol. 138:129-140; and Gomi, et al. (1991) [see
comments] Arch. Dermatol. 127:827-830. Furthermore, the effects of
T helper cell-derived cytokines such as IL-4, IFN-.gamma., and
IL-17 were tested on cellular constituents of the skin. TaqMan.RTM.
analyses showed that only keratinocytes and dermal microvascular
endothelial cells constitutively express low levels of MIP-3.alpha.
and that TNF-.alpha./IL-1.beta. can induce MIP-3.alpha. mRNA
expression in both keratinocytes and dermal microvascular
endothelial cells. In addition, TNF-.alpha./IL-1.beta. stimulation
of dermal microvascular endothelial cells induced strong
upregulation of MIP-3.alpha. expression. Activation of both
CD34+hematopoetic progenitor cell- or monocyte-derived dendritic
cells with CD40L also induced MIP-3.alpha.-specific transcripts.
Interestingly, melanocytes also showed significant expression of
MIP-3.alpha. following TNF-.alpha./IL-1.beta. stimulation.
Moreover, MIP-3.alpha. expression could be markedly induced in
these cells by IFN-.gamma. or IL-4 stimulation. Keratinocytes
showed a weak (2-4 fold) upregulation of MIP-3.alpha. mRNA after
activation with either IFN-.gamma. or IL-4. In contrast, resting or
stimulated epidermal .gamma..delta. cells failed to express
MIP-3.alpha. mRNA under any conditions. Similar results were
observed in keratinocytes (n=4), melanocytes (n=2), CD34+
hematopoietic progenitor cell-derived dendritic cells (n=2),
monocyte-derived dendritic cells (n=2), dermal microvascular
endothelial cells (n=2) and dermal fibroblasts (n=2) of different
donors. Interestingly, keratinocytes, fibroblasts, melanocytes, or
epidermal .gamma..delta. T cells were never observed to express
significant levels of CCR6 mRNA.
[0155] Analyses of MIP-3.alpha. protein expression by ELISA
confirmed that activated keratinocytes, dermal microvascular
endothelial cells, dermal fibroblasts, and monocyte-derived
dendritic cells are potent producers of this chemokine.
Supernatants from cultured primary human keratinocytes, dermal
microvascular endothelial cells, and dermal fibroblasts activated
with TNF-.alpha. and IL-1.beta. showed a marked induction of
MIP-3.alpha. protein while resting cells showed either little or no
production of MIP-3.alpha.. These levels of hMIP-3.alpha.
production are in the range of biological activity reported for
prokaryote-derived recombinant hMIP-3.alpha.. Low levels of
MIP-3.alpha. protein were also detected after stimulation of
keratinocytes with IFN-.gamma. or IL-17, however, additional
TNF-.alpha. stimulation showed synergistic effects and markedly
enhanced MIP-3.alpha. protein production.
[0156] Supernatants of monocyte-derived dendritic cells showed
significant production of MIP-3.alpha. protein (1.14-8.76 ng/ml) 12
to 48 h following CD40L stimulation. MIP-3.alpha. protein
expression generally confirmed the data obtained using real time
quantitative PCR (TaqMan.sup.+). However, supernatants from CD34+
hematopoetic progenitor cells stimulated with CD40L did not contain
detectable MIP-3.alpha. protein despite a marked induction of
MIP-3.alpha. mRNA expression. This may be due to the high level of
CCR6 expression by these cells suggesting that they may be binding
and internalizing MIP-3.alpha..
[0157] It was then sought to determine if the MIP-3.alpha. protein
detected in these supernatants was biologically active. To this
end, supernatants were tested against keratinocytes, fibrobasts, or
endothelial cells, either resting or following stimulation with
TNF-.alpha./IL-1.beta., IFN-.gamma., IL-4, in a calcium signaling
assay using CCR6-transfected BAF/3 cells. These cells are known to
express endogenous CXCR4. Therefore, to obtain a CCR6-specific
assay, the endogenous CXCR4 was blocked with human SDF-1.alpha.
prior to testing the supernatants for MIP-3.alpha. activity. In
agreement with the ELISA data, supernatants from keratinocytes,
dermal fibroblasts, and dermal microvascular endothelial cells
stimulated with TNF-.alpha./IL-1.beta. induced significant calcium
mobilization responses in CCR6-transfected BAF/3 cells but not in
the parental untransfected BAF/3 cells. However, the parental BAF/3
cell line showed the expected calcium mobilization response due to
the triggering of endogenous CXCR4 by SDF-1.alpha.. Furthermore,
treatment with anti-MIP-3.alpha. mAb completely neutralized
supernatant-induced calcium mobilization in CCR6-transfected BAF/3
cells, however, isotype control showed no effect. Concentrated
medium with or without cytokine (TNF-.alpha./IL-1.beta., IL-4,
IFN-.gamma.) addition did not produce any intracellular Ca++
mobilization in parental or CCR6-transfected cells. These results
confirm the production of bioactive MIP-3.alpha. protein by
keratinocytes, dermal fibroblasts, and dermal microvascular
endothelial cells initially detected at the mRNA level by
quantitative PCR.
[0158] This study demonstrates that the CC chemokine MIP-3.alpha.
and its receptor CCR6 are significantly upregulated in psoriasis.
Furthermore, clusters of skin-infiltrating T cells in the papillary
dermis of lesional psoriatic skin are directly adjacent to foci of
MIP-3.alpha.-expressing epidermal cells. Moreover,
MIP-3.alpha.-expressing keratinocytes within the epidermis
co-localize with intraepidermal CD3.sup.+ T cells. These findings
together with the observation of Liao, et al. (1999) J. Immunol.
162:186-194 that MIP-3.alpha. specifically attracts the memory
subset of T cells in vitro strongly suggests that MIP-3.alpha.
plays an important role in T cell recruitment to lesional psoriatic
skin. The significantly increased expression of CCR6 in PBMCs
derived from psoriatic donors and the high expression of CCR6 on
skin-homing CLA T cells further supports this concept. In addition
to memory T cells, peripheral blood B cells also express CCR6,
however, they are not present in psoriatic skin lesions
(Bata-Csorgo, et al. (1995) J. Invest. Dermatol. 105:89S-94S; Liao,
et al. (1999) J. Immunol. 162:186-194; Bos, et al. (1983) Arch.
Dermatol. Res. 275:181-189; and Bos, et al. (1989) Arch. Dermatol.
Res. 281:24-30) suggesting that there may be further necessary
requirements such as E-selectin or CLA expression for effective
skin homing. The CLA+ T cell subset represents a skin-associated
population of memory T cells that preferentially extravasates to
normal and chronically inflamed cutaneous sites. See Picker, et al.
(1990) J. Immunol. 145:3247-3255. Comparison of CCR6 expression in
CLA+ T cells with those of receptors for chemokines (IL-8;
Gro-.alpha., IP-10, MIG, MCP-1, RANTES) reported to be associated
with psoriasis underscore the relevance of this specific
chemokine/receptor pair in psoriasis. Schulz, et al. (1993) J.
Immunol. 151:4399-4406; Gottlieb, et al. (1988) J. Exp. Med.
168:941-948; Lemster, et al. (1995) Clin. Exp. Immunol. 99:148-154;
Gillitzer, et al. (1993) J. Invest. Dermatol. 101:127-131; Fukuoka,
et al. (1998) Br. J. Dermatol. 138:63-70; and Goebeler, et al.
(1998) J. Pathol. 184:89-95. Furthermore, the predominance of CCR6
expression on the CD4 subset of skin-homing CLA+ T cells suggests a
link with the effective treatment of psoriasis using anti-human CD4
antibodies. See Isaacs, et al. (1997) Clin. Exp. Immunol.
110:158-166; Thivolet and Nicolas (1994) Int. J. Dermatol.
33:327-332; and Morel, et al. (1992) J. Autoimmun. 5:465-477. These
findings support previous observations by Campbell, et al. (1998)
Science 279:381-384 showing that MIP-3.alpha. induces rapid
adhesion to ICAM-1 only in memory but not in naive CD4+ T cells.
Furthermore, immunohistological studies have shown that the
inflammatory infiltrate in psoriasis is mainly composed of CD4+
memory T cells. See Prens, et al. (1995) Clin. Dermatol.
13:115-129; and Bata-Csorgo, et al. (1995) J. Invest. Dermatol.
105:89S-94S.
[0159] Interestingly, TaqMan.RTM. analyses of cDNA libraries show
that CCR6 is predominantly expressed in cDNA libraries derived from
human Th.sub.0 or Th.sub.1 clones. However, cDNA libraries derived
from either activated Th.sub.2 polarized cells or resting or
activated Th.sub.2 (HY935) clones show little or no CCR6
expression. This expression pattern supports previous observations
indicating that lesional psoriatic T cells predominantly display a
Th.sub.1 phenotype. See Uyemura, et al. (1993) J. Invest. Dermatol.
101:701-705; Barker (1998) Hosp. Med. 59:530-533; Nestle, et al.
(1994) J. Clin. Invest. 94:202-209; and Schlaak, et al. (1994) J.
Invest. Dermatol. 102:145-149. In addition, preliminary experiments
show that activated T cells are capable of inducing high levels of
MIP-3.alpha. production in epithelial cells.
[0160] Keratinocytes are potent producers of MIP-3.alpha. in
lesional psoriatic skin. Here it is shown that TNF-.alpha. and
IL-1, both proinflammatory cytokines known to be upregulated in
psoriasis, (see Terajima, et al. (1998) Arch. Dermatol. Res.
290:246-252; Mizutani, et al. (1997) J. Dermatol. Sci. 14:145-153;
Debets, et al. (1995) Eur. J. Immunol. 25:1624-1630; Nickoloff, et
al. (1991) Am. J. Pathol. 138:129-140; and Gomi, et al. (1991) [see
comments] Arch. Dermatol. 127:827-830) as well as CD40L are potent
inducers of bioactive MIP-3.alpha. protein in keratinocytes,
melanocytes, dermal microvascular endothelial cells, dermal
fibroblast, and dendritic cells in vitro. Furthermore, T helper
cell-derived mediators (e.g., IFN-.gamma., IL-17, CD40L) regulate
MIP-3.alpha. production in cellular constituents of the skin. IL-17
is known to be upregulated in lesional psoriatic skin, suggesting
that it may play a role in the amplification and the development of
cutaneous inflammation. See Nestle, et al. (1994) J. Clin. Invest.
94:202-209; and Teunissen, et al. (1998) J. Invest. Dermatol.
111:645-649. Here it is shown that it is another inducer of
MIP-3.alpha. protein production by primary keratinocytes.
[0161] In vitro epithelial Langerhans-type dendritic cells can be
generated from CD34+ hematopoietic progenitor cells, however,
monocyte-derived dendritic cells share phenotypic characteristics
with dermal dendritic cells. It has been reported previously that
CCR6 is highly expressed on dendritic cells derived from CD34+
hematopoetic progenitor cells and that MIP-3.alpha. selectively
induces migratory responses in CD34+ hematopoetic progenitor
cell--but not in monocyte-derived dendritic cells. See Greaves, et
al. (1997) J. Exp. Med. 186:837-844; and Dieu, et al. (1998) J.
Exp. Med. 188:373-386. In lesional psoriatic skin, large numbers of
dermal dendritic cells are present and show potent stimulatory
functions. Nestle, et al. (1994) J. Clin. Invest. 94:202-209.
Activation of dendritic cells via CD40 triggering resulted in a
marked upregulation of MIP-3.alpha. suggesting that dendritic
cell-T lymphocyte interactions may amplify inflammatory-processes
in psoriasis.
[0162] In summary, these data suggest that T cell-derived
mediators, such as CD40L, IFN-.gamma., and IL-17, may amplify
chemokine, particularly MIP-3.alpha., production in lesional
psoriatic skin and may contribute to the development and/or
chronicity of psoriatic lesions. Thus, blockage of effect may be
therapeutically effective, e.g., by use of antagonists, receptor
antagonists, or receptor desensitization compounds.
[0163] Along with its expression in intestinal epithelial cells,
cutaneous MIP-3.alpha. expression supports the hypothesis that this
inflammatory chemokine plays an important role in the interface
between the organism and the environment. Tanaka, et al. (1999)
Eur. J. Immunol. 29:633-642. Other chemokines have been shown to be
associated with psoriasis, including RANTES which has been reported
to be expressed in psoriatic lesions by activated keratinocytes.
See Fukuoka, et al. (1998) Br. J. Dermatol. 138:63-70; and
Raychaudhuri, et al. (1999) Acta Derm. Venereol. 79:9-11. However,
peak levels of RANTES expression (2.072 ng/ml) in activated
keratinocytes were 10-50 times lower than those detected for
MIP-3.alpha. in the present study. Fukuoka, et al. (1998) Br. J.
Dermatol. 138:63-70. Furthermore, there is divergent data regarding
RANTES expression in lesional psoriatic skin. Fukuoka, et al. (n=3)
as well as Raychaudhuri, et al. (n=8) reported increased RANTES
expression in lesional psoriatic skin using immunohistochemistry in
a total of 11 psoriatic patients, whereas Goebler, et al. reported
no detectable RANTES expression in either lesional psoriatic
(n=11), non-lesional psoriatic (n=11), or normal skin (n=5).
Goebeler, et al. (1998) J. Pathol. 184:89-95. The present study
observed markedly lower levels of RANTES expression in a cDNA
library derived from psoriatic skin when compared with one derived
from normal skin using RANTES-specific primers and probes for real
time quantitative PCR (TaqMan.RTM.) analyses. In contrast to
RANTES, Goebler, et al. detect strong selective expression of Mig
in the upper lesional dermis with pronounced clustering in the tips
of the papillae, whereas expression in normal or non-lesional
psoriatic or normal skin was quiescent. Co-localization studies
have suggested that highly activated dermal macrophages and dermal
microvascular endothelial cells are major sources of Mig in
lesional psoriatic skin. Goebeler, et al. (1998) J. Pathol.
184:89-95. The pro-inflammatory CC chemokines, IP-10 and Mig, are
able to attract activated T cells and are mainly regulated by T
cell-derived cytokines, such as IFN-.gamma.. Thus,
skin-infiltrating activated T cells release inflammatory mediators
which, in turn, induce Mig, IP-10, and MIP-3.alpha., contributing
to the amplification of inflammatory responses and to the
chronicity of psoriatic lesions. Moreover, MCP-1 expression of
keratinocytes in the stratum basale of lesional psoriatic skin is
associated with chemoattraction of dermal macrophages to lesional
sites. See Gillitzer, et al. (1993) J. Invest. Dermatol.
101:127-131; Goebeler, et al. (1998) J. Pathol. 184:89-95; and
Deleuran, et al. (1996) J. Dermatol. Sci. 13:228-236.
[0164] Given the cumulating evidence that psoriasis is a T-cell
mediated disease, MIP-3.alpha./CCR6 is the first ligand/receptor
pair identified in this disease which is directly associated with
memory T cell recruitment to lesional psoriatic skin. The only
other chemokine/receptor pairs reported in psoriasis, such as IL-8
and GRO-.alpha. with their receptors CXCR1 and CXCR2, are mainly
involved in the recruitment of neutrophils to lesional psoriatic
skin. See Gillitzer, et al. (1996) J. Invest. Dermatol.
107:778-782; Gillitzer, et al. (1991) J. Invest. Dermatol.
97:73-79; and Kulke, et al. (1996) J. Invest. Dermatol.
106:526-530. In addition, the expression pattern of those CXC
chemokines did not fully coincide with the pattern of T cell
accumulation. See Gillitzer, et al. (1996) J. Invest. Dermatol.
107:778-782; Gillitzer, et al. (1991) J. Invest. Dermatol.
97:73-79; and Kulke, et al. (1996) J. Invest. Dermatol.
106:526-530.
[0165] Recently, Campbell, et al. suggested that the interaction of
the CC chemokine TARC with its receptor CCR4 plays an important
role in the homing of memory T cells to the skin. Campbell, et al.
(1999) Nature 400:776-780. TARC expression was shown to be
exclusively expressed in endothelial cells of both normal and
inflamed human skin and this CC chemokine could induce firm
adhesion and chemotaxis of skin-homing memory T cells. Taken
together, these findings suggest that TARC and CCR4 may play a role
in the interaction of skin-homing T cells with the dermal
endothelium and transendothelial migration, however, other
chemokines (e.g., MIP-3.alpha., IP-10, Mig) expressed by
keratinocytes, fibroblasts, or dendritic cells may mediate the
localization of skin-homing T cells to the dermis and epidermis.
MIP-3.alpha. has also been shown to mediate firm adhesion of CD4+
memory T cells suggesting that chemokines may also act in an
orchestrated fashion at this particular step of leukocyte
trafficking.
[0166] Most recently, a non-chemokine ligand for CCR6 has been
identified. Yang, et al. showed that human .beta.-defensin-2 is
able to bind CCR6 transfected cells and to induce chemotaxis,
however, its chemotactic activity was considerably lower than that
of MIP-3.alpha.. Yang, et al. (1999) [In Process Citation] Science
286:525-528. Interestingly, like MIP-3.alpha., human
.beta.-defensin-2 is upregulated by proinflammatory mediators, such
as TNF-.alpha. and IL-1.beta.. Most importantly, bacterial and
viral infections markedly induce the production of human
.beta.-defensin-2. Since both MIP-3.alpha. and human
.beta.-defensin-2 are expressed in psoriatic skin, they could both
contribute to the recruitment of memory T cells to lesional sites,
however, these studies found no evidence for a role for human
.beta.-defensin-2 since the intracellular Ca.sup.2.sup..sub.+
mobilization induced by supernatants of
TNF-.alpha./IL-1.beta.-stimulated primary keratinocytes in CCR6
transfectants was completely blocked by an anti-MIP-3.alpha.
antibody.
[0167] These findings suggest the following model for the
involvement of MIP-3.alpha. and CCR6 in the pathogenesis of
psoriasis: MIP-3.alpha. may be induced in keratinocytes and/or
dermal microvascular endothelial cells at sites of physical injury
or infection due to the release of proinflammatory cytokines, such
as TNF-.alpha. and IL-1. In turn, TARC and MIP-3.alpha. may induce
adhesion (Campbell, et al. (1998) Science 279:381-384; and
Campbell, et al. (1999) Nature 400:776-780) and chemotaxis of
skin-homing memory T cells (Liao, et al. (1999) J. Immunol.
162:186-194) through the endothelium into the skin. Subsequently,
the skin-homing CLA+ T cells may encounter their specific antigen
presented by dendritic cells, get activated, and produce
inflammatory mediators, such as IFN-.gamma., IL-17, or CD40L,
which, in turn, induce additional MIP-3.alpha., IP-10, and Mig
production by activated keratinocytes, dendritic cells, and dermal
macrophages. Gottlieb, et al. (1988) J. Exp. Med. 168:941-948;
Goebeler, et al. (1998) J. Pathol. 184:89-95; and Boorsma, et al.
(1998) Arch. Dermatol. Res. 290:335-341. This "second wave" of
chemokine production may complete a self-sustaining cycle of
inflammation which leads to the development of a psoriatic
phenotype.
[0168] In conclusion, this study shows the potential that a highly
specific and sensitive real time quantitative PCR technique
(TaqMan.RTM. ) offers to identify novel disease associations with
the expression of specific genes. This technology led to the
identification of MIP-3.alpha./CCR6 as a new ligand/receptor pair
potentially involved in the pathogenesis of psoriasis.
[0169] All citations herein are incorporated herein by reference to
the same extent as if each individual publication or patent
application was specifically and individually indicated to be
incorporated by reference.
[0170] Many modifications and variations of this invention can be
made without departing from its spirit and scope, as will be
apparent to those skilled in the art. The specific embodiments
described herein are offered by way of example only, and the
invention is to be limited by the terms of the appended claims,
along with the full scope of equivalents to which such claims are
entitled; and the invention is not to be limited by the specific
embodiments that have been presented herein by way of example.
Sequence CWU 1
1
8 1 291 DNA Homo sapiens CDS (1)..(288) 1 atg tgc tgt acc aag agt
ttg ctc ctg gct gct ttg atg tca gtg ctg 48 Met Cys Cys Thr Lys Ser
Leu Leu Leu Ala Ala Leu Met Ser Val Leu -25 -20 -15 cta ctc cac ctc
tgc ggc gaa tca gaa gca gca agc aac ttt gac tgc 96 Leu Leu His Leu
Cys Gly Glu Ser Glu Ala Ala Ser Asn Phe Asp Cys -10 -5 -1 1 5 tgt
ctt gga tac aca gac cgt att ctt cat cct aaa ttt att gtg ggc 144 Cys
Leu Gly Tyr Thr Asp Arg Ile Leu His Pro Lys Phe Ile Val Gly 10 15
20 ttc aca cgg cag ctg gcc aat gaa ggc tgt gac atc aat gct atc atc
192 Phe Thr Arg Gln Leu Ala Asn Glu Gly Cys Asp Ile Asn Ala Ile Ile
25 30 35 ttt cac aca aag aaa aag ttg tct gtg tgc gca aat cca aaa
cag act 240 Phe His Thr Lys Lys Lys Leu Ser Val Cys Ala Asn Pro Lys
Gln Thr 40 45 50 tgg gtg aaa tat att gtg cgt ctc ctc agt aaa aaa
gtc aag aac atg 288 Trp Val Lys Tyr Ile Val Arg Leu Leu Ser Lys Lys
Val Lys Asn Met 55 60 65 70 taa 291 2 96 PRT Homo sapiens 2 Met Cys
Cys Thr Lys Ser Leu Leu Leu Ala Ala Leu Met Ser Val Leu -25 -20 -15
Leu Leu His Leu Cys Gly Glu Ser Glu Ala Ala Ser Asn Phe Asp Cys -10
-5 -1 1 5 Cys Leu Gly Tyr Thr Asp Arg Ile Leu His Pro Lys Phe Ile
Val Gly 10 15 20 Phe Thr Arg Gln Leu Ala Asn Glu Gly Cys Asp Ile
Asn Ala Ile Ile 25 30 35 Phe His Thr Lys Lys Lys Leu Ser Val Cys
Ala Asn Pro Lys Gln Thr 40 45 50 Trp Val Lys Tyr Ile Val Arg Leu
Leu Ser Lys Lys Val Lys Asn Met 55 60 65 70 3 291 DNA Mus musculus
CDS (1)..(288) 3 atg gcc tgc ggt ggc aag cgt ctg ctc ttc ctt gct
ttg gca tgg gta 48 Met Ala Cys Gly Gly Lys Arg Leu Leu Phe Leu Ala
Leu Ala Trp Val -25 -20 -15 ctg ctg gct cac ctc tgc agc cag gca gaa
gca agc aac tac gac tgt 96 Leu Leu Ala His Leu Cys Ser Gln Ala Glu
Ala Ser Asn Tyr Asp Cys -10 -5 -1 1 5 tgc ctc tcg tac ata cag acg
cca ctt cct tcc aga gct att gtg ggt 144 Cys Leu Ser Tyr Ile Gln Thr
Pro Leu Pro Ser Arg Ala Ile Val Gly 10 15 20 ttc aca aga cag atg
gcc gat gaa gct tgt gac att aat gct atc atc 192 Phe Thr Arg Gln Met
Ala Asp Glu Ala Cys Asp Ile Asn Ala Ile Ile 25 30 35 ttt cac acg
aag aaa aga aaa tct gtg tgc gct gat cca aag cag aac 240 Phe His Thr
Lys Lys Arg Lys Ser Val Cys Ala Asp Pro Lys Gln Asn 40 45 50 tgg
gtg aaa agg gct gtg aac ctc ctc agc cta aga gtc aag aag atg 288 Trp
Val Lys Arg Ala Val Asn Leu Leu Ser Leu Arg Val Lys Lys Met 55 60
65 taa 291 4 96 PRT Mus musculus 4 Met Ala Cys Gly Gly Lys Arg Leu
Leu Phe Leu Ala Leu Ala Trp Val -25 -20 -15 Leu Leu Ala His Leu Cys
Ser Gln Ala Glu Ala Ser Asn Tyr Asp Cys -10 -5 -1 1 5 Cys Leu Ser
Tyr Ile Gln Thr Pro Leu Pro Ser Arg Ala Ile Val Gly 10 15 20 Phe
Thr Arg Gln Met Ala Asp Glu Ala Cys Asp Ile Asn Ala Ile Ile 25 30
35 Phe His Thr Lys Lys Arg Lys Ser Val Cys Ala Asp Pro Lys Gln Asn
40 45 50 Trp Val Lys Arg Ala Val Asn Leu Leu Ser Leu Arg Val Lys
Lys Met 55 60 65 5 291 DNA Rattus sp. CDS (1)..(288) 5 atg gcc tgc
aag cat ctg ccc ttc ctg gct ttg gcg ggg gta ctg ctg 48 Met Ala Cys
Lys His Leu Pro Phe Leu Ala Leu Ala Gly Val Leu Leu -25 -20 -15 -10
gct tac ctc tgc agc cag tca gaa gca gca agc aac ttt gac tgc tgc 96
Ala Tyr Leu Cys Ser Gln Ser Glu Ala Ala Ser Asn Phe Asp Cys Cys -5
-1 1 5 ctc acg tac aca aag aac gtg tat cat cat gcg aga aat ttt gtg
ggt 144 Leu Thr Tyr Thr Lys Asn Val Tyr His His Ala Arg Asn Phe Val
Gly 10 15 20 ttc aca aca cag atg gcc gac gaa gct tgt gac att aat
gct atc atc 192 Phe Thr Thr Gln Met Ala Asp Glu Ala Cys Asp Ile Asn
Ala Ile Ile 25 30 35 ttt cac ctg aag tcg aaa aga tcc gtg tgc gct
gac cca aag cag atc 240 Phe His Leu Lys Ser Lys Arg Ser Val Cys Ala
Asp Pro Lys Gln Ile 40 45 50 55 tgg gtg aaa agg att ttg cac ctc ctc
agc cta aga acc aag aag atg 288 Trp Val Lys Arg Ile Leu His Leu Leu
Ser Leu Arg Thr Lys Lys Met 60 65 70 taa 291 6 96 PRT Rattus sp. 6
Met Ala Cys Lys His Leu Pro Phe Leu Ala Leu Ala Gly Val Leu Leu -25
-20 -15 -10 Ala Tyr Leu Cys Ser Gln Ser Glu Ala Ala Ser Asn Phe Asp
Cys Cys -5 -1 1 5 Leu Thr Tyr Thr Lys Asn Val Tyr His His Ala Arg
Asn Phe Val Gly 10 15 20 Phe Thr Thr Gln Met Ala Asp Glu Ala Cys
Asp Ile Asn Ala Ile Ile 25 30 35 Phe His Leu Lys Ser Lys Arg Ser
Val Cys Ala Asp Pro Lys Gln Ile 40 45 50 55 Trp Val Lys Arg Ile Leu
His Leu Leu Ser Leu Arg Thr Lys Lys Met 60 65 70 7 1098 DNA Homo
sapiens CDS (1)..(1095) 7 atg ttt tcg act cca gtg aag att att ttg
tgt cag tca ata ctt cat 48 Met Phe Ser Thr Pro Val Lys Ile Ile Leu
Cys Gln Ser Ile Leu His 1 5 10 15 att act cag ttg att ctg aga tgt
tac tgt gct cct tgc agg agg tca 96 Ile Thr Gln Leu Ile Leu Arg Cys
Tyr Cys Ala Pro Cys Arg Arg Ser 20 25 30 ggc agt tct cca ggc tat
ttg tac cga att gcc tac tcc ttg atc tgt 144 Gly Ser Ser Pro Gly Tyr
Leu Tyr Arg Ile Ala Tyr Ser Leu Ile Cys 35 40 45 gtt ctt ggc ctc
ctg ggg aat att ctg gtg gtg atc acc ttt gct ttt 192 Val Leu Gly Leu
Leu Gly Asn Ile Leu Val Val Ile Thr Phe Ala Phe 50 55 60 tat aag
aag gcc agg tct atg aca gac gtc tat ctc ttg aac atg gcc 240 Tyr Lys
Lys Ala Arg Ser Met Thr Asp Val Tyr Leu Leu Asn Met Ala 65 70 75 80
att gca gac atc ctc ttt gtt ctt act ctc cca ttc tgg gca gtg agt 288
Ile Ala Asp Ile Leu Phe Val Leu Thr Leu Pro Phe Trp Ala Val Ser 85
90 95 cat gcc act ggt gcg tgg gtt ttc agc aat gcc acg tgc aag ttg
cta 336 His Ala Thr Gly Ala Trp Val Phe Ser Asn Ala Thr Cys Lys Leu
Leu 100 105 110 aaa ggc atc tat gcc atc aac ttt aac tgc ggg atg ctg
ctc ctg act 384 Lys Gly Ile Tyr Ala Ile Asn Phe Asn Cys Gly Met Leu
Leu Leu Thr 115 120 125 tgc att agc atg gac cgg tac atc gcc att gta
cag gcg act aag tca 432 Cys Ile Ser Met Asp Arg Tyr Ile Ala Ile Val
Gln Ala Thr Lys Ser 130 135 140 ttc cgg ctc cga tcc aga aca cta ccg
cgc agc aaa atc atc tgc ctt 480 Phe Arg Leu Arg Ser Arg Thr Leu Pro
Arg Ser Lys Ile Ile Cys Leu 145 150 155 160 gtt gtg tgg ggg ctg tca
gtc atc atc tcc agc tca act ttt gtc ttc 528 Val Val Trp Gly Leu Ser
Val Ile Ile Ser Ser Ser Thr Phe Val Phe 165 170 175 aac caa aaa tac
aac acc caa ggc agc gat gtc tgt gaa ccc aag tac 576 Asn Gln Lys Tyr
Asn Thr Gln Gly Ser Asp Val Cys Glu Pro Lys Tyr 180 185 190 can act
gtc tcg gag ccc atc agg tgg aag ctg ctg atg ttg ggg ctt 624 Thr Thr
Val Ser Glu Pro Ile Arg Trp Lys Leu Leu Met Leu Gly Leu 195 200 205
gag cta ctc ttt ggt ttc ttt atc cct ttg atg ttc atg ata ttt tgt 672
Glu Leu Leu Phe Gly Phe Phe Ile Pro Leu Met Phe Met Ile Phe Cys 210
215 220 tac acg ttc att gtc aaa acc ttg gtg caa gct cag aat tct aaa
agg 720 Tyr Thr Phe Ile Val Lys Thr Leu Val Gln Ala Gln Asn Ser Lys
Arg 225 230 235 240 cac aaa gcc atc cgt gta atc ata gct gtg gtg ctt
gtg ttt ctg gct 768 His Lys Ala Ile Arg Val Ile Ile Ala Val Val Leu
Val Phe Leu Ala 245 250 255 tgt cag att cct cat aac atg gtc ctg ctt
gtg acg gct gct aat ttg 816 Cys Gln Ile Pro His Asn Met Val Leu Leu
Val Thr Ala Ala Asn Leu 260 265 270 ggt aaa atg aac cga tcc tgc cag
agc gaa aag cta att ggc tat acg 864 Gly Lys Met Asn Arg Ser Cys Gln
Ser Glu Lys Leu Ile Gly Tyr Thr 275 280 285 aaa act gtc aca gaa gtc
ctg gct ttc ctg cac tgc tgc ctg aac cct 912 Lys Thr Val Thr Glu Val
Leu Ala Phe Leu His Cys Cys Leu Asn Pro 290 295 300 gtg ctc tac gct
ttt att ggg cag aag ttc aga aac tac ttt ctg aag 960 Val Leu Tyr Ala
Phe Ile Gly Gln Lys Phe Arg Asn Tyr Phe Leu Lys 305 310 315 320 atc
ttg aag gac ctg tgg tgt gtg aga agg aag tac aag tcc tca ggc 1008
Ile Leu Lys Asp Leu Trp Cys Val Arg Arg Lys Tyr Lys Ser Ser Gly 325
330 335 ttc tcc tgt gcc ggg agg tac tca gaa aac att tct cgg cag acc
agt 1056 Phe Ser Cys Ala Gly Arg Tyr Ser Glu Asn Ile Ser Arg Gln
Thr Ser 340 345 350 gag acc gca gat aac gac aat gcg tcg tcc ttc act
atg tga 1098 Glu Thr Ala Asp Asn Asp Asn Ala Ser Ser Phe Thr Met
355 360 365 8 365 PRT Homo sapiens misc_feature (579)..(579) n may
be a, c, g, or t 8 Met Phe Ser Thr Pro Val Lys Ile Ile Leu Cys Gln
Ser Ile Leu His 1 5 10 15 Ile Thr Gln Leu Ile Leu Arg Cys Tyr Cys
Ala Pro Cys Arg Arg Ser 20 25 30 Gly Ser Ser Pro Gly Tyr Leu Tyr
Arg Ile Ala Tyr Ser Leu Ile Cys 35 40 45 Val Leu Gly Leu Leu Gly
Asn Ile Leu Val Val Ile Thr Phe Ala Phe 50 55 60 Tyr Lys Lys Ala
Arg Ser Met Thr Asp Val Tyr Leu Leu Asn Met Ala 65 70 75 80 Ile Ala
Asp Ile Leu Phe Val Leu Thr Leu Pro Phe Trp Ala Val Ser 85 90 95
His Ala Thr Gly Ala Trp Val Phe Ser Asn Ala Thr Cys Lys Leu Leu 100
105 110 Lys Gly Ile Tyr Ala Ile Asn Phe Asn Cys Gly Met Leu Leu Leu
Thr 115 120 125 Cys Ile Ser Met Asp Arg Tyr Ile Ala Ile Val Gln Ala
Thr Lys Ser 130 135 140 Phe Arg Leu Arg Ser Arg Thr Leu Pro Arg Ser
Lys Ile Ile Cys Leu 145 150 155 160 Val Val Trp Gly Leu Ser Val Ile
Ile Ser Ser Ser Thr Phe Val Phe 165 170 175 Asn Gln Lys Tyr Asn Thr
Gln Gly Ser Asp Val Cys Glu Pro Lys Tyr 180 185 190 Thr Thr Val Ser
Glu Pro Ile Arg Trp Lys Leu Leu Met Leu Gly Leu 195 200 205 Glu Leu
Leu Phe Gly Phe Phe Ile Pro Leu Met Phe Met Ile Phe Cys 210 215 220
Tyr Thr Phe Ile Val Lys Thr Leu Val Gln Ala Gln Asn Ser Lys Arg 225
230 235 240 His Lys Ala Ile Arg Val Ile Ile Ala Val Val Leu Val Phe
Leu Ala 245 250 255 Cys Gln Ile Pro His Asn Met Val Leu Leu Val Thr
Ala Ala Asn Leu 260 265 270 Gly Lys Met Asn Arg Ser Cys Gln Ser Glu
Lys Leu Ile Gly Tyr Thr 275 280 285 Lys Thr Val Thr Glu Val Leu Ala
Phe Leu His Cys Cys Leu Asn Pro 290 295 300 Val Leu Tyr Ala Phe Ile
Gly Gln Lys Phe Arg Asn Tyr Phe Leu Lys 305 310 315 320 Ile Leu Lys
Asp Leu Trp Cys Val Arg Arg Lys Tyr Lys Ser Ser Gly 325 330 335 Phe
Ser Cys Ala Gly Arg Tyr Ser Glu Asn Ile Ser Arg Gln Thr Ser 340 345
350 Glu Thr Ala Asp Asn Asp Asn Ala Ser Ser Phe Thr Met 355 360
365
* * * * *