U.S. patent application number 10/666286 was filed with the patent office on 2004-04-01 for reversal of proinflammatory response by ligating the macrophage fcgammari receptor.
This patent application is currently assigned to Temple University - Of The Commonwealth System of Higher Education. Invention is credited to Mosser, David M., Sutterwala, Fayyaz.
Application Number | 20040062763 10/666286 |
Document ID | / |
Family ID | 22184635 |
Filed Date | 2004-04-01 |
United States Patent
Application |
20040062763 |
Kind Code |
A1 |
Mosser, David M. ; et
al. |
April 1, 2004 |
Reversal of proinflammatory response by ligating the macrophage
FcgammaRI receptor
Abstract
Ligation of the Fc.gamma. receptor type I (Fc.gamma.RI) on
IL-10-producing cells leads to a selective upregulation of IL-10
production, which in turn induces a marked suppression of IL-12
biosynthesis by IL-12-producing cells, particularly macrophages.
The ligation of the Fc.gamma.RI receptor thus downmodulates IL-12
production via a mechanism that is dependent on macrophage-derived
IL-10. Agents for ligating Fc.gamma.RI comprise, for example,
multivalent antibodies which bind the Fc.gamma.RI receptor, immune
complexes comprising antibodies which contain the Fc region of IgG,
and IgG multimers, preferably IgG dimers and trimers. The ligating
agent may be administered to therapeutically inhibit
proinflammatory immune responses. In particular, the ligating agent
may be administered to treat or prevent endotoxic shock associated
with bacterial endotoxemia, and to treating autoimmune
disorders.
Inventors: |
Mosser, David M.;
(Hyattsville, MD) ; Sutterwala, Fayyaz;
(Northford, CT) |
Correspondence
Address: |
DRINKER BIDDLE & REATH
ONE LOGAN SQUARE
18TH AND CHERRY STREETS
PHILADELPHIA
PA
19103-6996
US
|
Assignee: |
Temple University - Of The
Commonwealth System of Higher Education
Philadelphia
PA
|
Family ID: |
22184635 |
Appl. No.: |
10/666286 |
Filed: |
September 19, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10666286 |
Sep 19, 2003 |
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09692586 |
Oct 19, 2000 |
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6660266 |
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09692586 |
Oct 19, 2000 |
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PCT/US99/09269 |
Apr 29, 1999 |
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60084385 |
May 6, 1998 |
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Current U.S.
Class: |
424/131.1 ;
424/144.1 |
Current CPC
Class: |
A61P 37/02 20180101;
A61K 2039/505 20130101; C07K 16/1203 20130101; C07K 16/2806
20130101; A61K 38/00 20130101; C07K 2317/52 20130101; C07K 16/28
20130101; C07K 16/1242 20130101 |
Class at
Publication: |
424/131.1 ;
424/144.1 |
International
Class: |
A61K 039/395 |
Goverment Interests
[0002] The invention described herein was supported in part by
National Institutes of Health grant AI24313. The Federal government
has certain rights in the invention.
Claims
1. A method of inhibiting a proinflammatory immune response in a
mammal comprising administering an effective amount of a ligating
agent which causes ligation of Fc.gamma.RI receptors on cells of
the mammal.
2. A method according to claim 1 wherein the ligating agent
comprises a multivalent antibody which binds to the Fc.gamma.RI
receptor.
3. A method according to claim 1 wherein the ligating agent
comprises an immune complex containing at least two antibody
molecules or fragments thereof which contain the Fc region of
IgG.
4. A method according to claim 1 wherein the ligating agent
comprises an antibody multimer containing at least two antibody
molecules or fragments thereof which contain the Fc region of
IgG.
5. A method according to claim 4 wherein the ligating agent
comprises an IgG preparation comprising IgG dimers, IgG trimers, or
a combination thereof.
6. A method according to. Claim 5 wherein the IgG content of the
IgG preparation comprises, on a weight percent basis, at least
about 50% IgG dimers, IgG trimers, or a mixture thereof.
7. A method according to claim 1 wherein the ligating agent does
not cause ligation of Fc.gamma.RIII receptors.
8. A method of inhibiting a proinflammatory immune response in a
mammal comprising administering an IgG antibody which binds to
antigen in the mammal to form an immune complex capable of ligating
Fc.gamma.RI receptors present on host cells.
9. A method according to claim 8 wherein the immune complex does
not cause ligation of Fc.gamma.RIII receptors.
10. A method for treating or preventing shock associated with
bacterial endotoxemia comprising administering to a mammal in need
of such treatment an effective amount of ligating agent which
causes ligation of Fc.gamma.RI receptors on cells of the
mammal.
11. A method according to claim 10 wherein the ligating agent
comprises an immune complex containing at least two antibody
molecules or fragments thereof which contain the Fc region of
IgG.
12. A method according to claim 10 wherein the ligating agent
comprises a multivalent antibody which binds to the Fc.gamma.RI
receptor.
13. A method according to claim 10 wherein the ligating agent
comprises an antibody multimer containing at least two antibody
molecules or fragments thereof which contain the Fc region of
IgG.
14. A method according to claim 13 wherein the ligating agent
comprises an IgG preparation comprising IgG dimers, IgG trimers, or
a combination thereof.
15. A method according to claim 14 wherein the IgG content of the
IgG preparation comprises, on a weight percent basis, at least
about 50% IgG dimers, IgG trimers, or a mixture thereof.
16. A method according to claim 10 wherein the ligating agents does
not cause ligation of Fc.gamma.RIII receptors.
17. A method of treating or preventing shock associated with
bacterial endotoxemia in a mammalian host comprising administering
an IgG antibody which binds to antigen in the host to form an
immune complex capable of ligating Fc.gamma.RI receptors present on
host cells.
18. A method for treating an autoimmune disorder comprising
administering to an individual in need of such treatment an
effective amount of a ligating agent which causes ligation of
Fc.gamma.RI receptors on cells of the individual.
19. A method according to claim 18 wherein the ligating agent
comprises a multivalent antibody which binds to the Fc.gamma.RI
receptor.
20. A method according to claim 18 wherein the ligating agent
comprises an immune complex containing at least two antibody
molecules or fragments thereof which contain the Fc region of
IgG.
21. A method according to claim 18 wherein the ligating agent
comprises an antibody multimer containing at least two antibody
molecules or fragments thereof which contain the Fc region of
IgG.
22. A method according to claim 21 wherein the ligating agent
comprises an IgG preparation comprising IgG dimers, IgG trimers, or
a combination thereof.
23. A method according to claim 22 wherein the IgG content of the
IgG preparation comprises, on a weight percent basis, at least
about 50% IgG dimers, IgG trimers, or a mixture thereof.
24. A method according to claim 18 wherein the ligating agent does
not cause ligation of Fc.gamma.RIII receptors.
25. A method according to claim 18 wherein the autoimmune disease
is selected from the group consisting of Kawasaki Disease, systemic
lupus erythematosus, rheumatoid arthritis, inflammatory bowel
disease, Sydenham's chorea, and autoimmune hemolytic anemia.
26. A method according to claim 25, wherein the autoimmune disease
is systemic lupus erythematosus.
27. A method for treating an autoimmune disorder in a mammalian
host comprising administering an IgG antibody which binds to
antigen in the host to form an immune complex capable of ligating
Fc.gamma.RI receptors present on host cells.
28. The method of claim 1, wherein the at least two IgG Fc regions
are joined by a covalent bond.
29. The method of claim 1, wherein the at least two IgG Fc regions
are joined by a homobifunctional or heterobifunctional
cross-linking reagent.
30. The method of claim 29, wherein the homobifunctional
cross-linking agent is selected from the group consisting of
disuccinimidyl tartrate; disuccinimidyl suberate; ethylene
glycolbis(succinimidyl succinate); 1,5-difluoro-2,4-dinitrobenzene;
4,4'-diisothiocyano-2,2'-disulfonic acid stilbene; and
bismaleimidohexane.
31. The method of claim 29, wherein the heterobifunctional
cross-linking agent is selected from the group consisting of
N-succinimidyl-3-(2-pyridy- ldithio) propionate; sulfo
succinimidyl-2-(p-azidosalicylamido)ethyl-1-3'-- dithiopropionate;
N-maleimidobenzoyl-N-hydroxy-succinimidyl ester;
m-maleimidobenzoylsulfosuccinimide ester;
N-succinimidyl(4-iodoacetyl) aminobenzoate; succinimidyl
4-(N-maleimidomethyl)-cyclohexane-1-carboxyla- te;
succinimidyl-4-(p-maleimidophenyl) butyrate;
sulfosuccinimidyl(4-iodoa- cetyl) aminobenzoate; sulfosuccinimidyl
4-(N-maleimidomethyl) cyclohexane-1-carboxylate; sulfosuccinimidyl
4-(p-maleimidophenyl)-butyra- te;
bromoacetyl-p-aminobenzoyl-N-hydroxy-succinimidyl ester; and
iodoacetyl-N-hydroxysuccinimidyl ester.
32. The method of claim 1, wherein the ligating agent comprises a
synthetic or recombinant peptide.
33. The method of claim 10, wherein the at least two IgG Fc regions
are joined by a covalent bond.
34. The method of claim 10, wherein the at least two IgG Fc regions
are joined by a homobifunctional or heterobifunctional
cross-linking reagent.
35. The method of claim 34, wherein the homobifunctional
cross-linking agent is selected from the group consisting of
disuccinimidyl tartrate; disuccinimidyl suberate; ethylene
glycolbis(succinimidyl succinate); 1,5-difluoro-2,4-dinitrobenzene;
4,4'-diisothiocyano-2,2'-disulfonic acid stilbene; and
bismaleimidohexane.
36. The method of claim 34, wherein the heterobifunctional
cross-linking agent is selected from the group consisting of
N-succinimidyl-3-(2-pyridy- ldithio)propionate;
sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1-3'-di-
thiopropionate; N-maleimidobenzoyl-N-hydroxy-succinimidyl ester;
m-maleimidobenzoylsulfosuccinimide ester;
N-succinimidyl(4-iodoacetyl) aminobenzoate; succinimidyl
4-(N-maleimidomethyl)-cyclohexane-1-carboxyla- te;
succinimidyl-4-(p-maleimidophenyl)butyrate;
sulfosuccinimidyl(4-iodoac- etyl)aminobenzoate; sulfosuccinimidyl
4-(N-maleimidomethyl) cyclohexane-1-carboxylate; sulfosuccinimidyl
4-(p-maleimidophenyl)-butyra- te;
bromoacetyl-p-aminobenzoyl-N-hydroxy-succinimidyl ester; and
iodoacetyl-N-hydroxysuccinimidyl ester.
37. The method of claim 10, wherein the ligating agent comprises a
synthetic or recombinant peptide.
38. The method of claim 18, wherein the at least two IgG Fc regions
are joined by a covalent bond.
39. The method of claim 18, wherein the at least two IgG Fc regions
are joined by a homobifunctional or heterobifunctional
cross-linking reagent.
40. The method of claim 39, wherein the homobifunctional
cross-linking agent is selected from the group consisting of
disuccinimidyl tartrate; disuccinimidyl suberate; ethylene
glycolbis(succinimidyl succinate); 1,5-difluoro-2,4-dinitrobenzene;
4,4'-diisothiocyano-2,2'-disulfonic acid stilbene; and
bismaleimidohexane.
41. The method of claim 39, wherein the heterobifunctional
cross-linking agent is selected from the group consisting of
N-succinimidyl-3-(2-pyridy- ldithio) propionate;
sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1-3'-d-
ithiopropionate; N-maleimidobenzoyl-N-hydroxy-succinimidyl ester;
m-maleimidobenzoylsulfosuccinimide ester;
N-succinimidyl(4-iodoacetyl) aminobenzoate; succinimidyl
4-(N-maleimidomethyl)-cyclohexane-1-carboxyla- te;
succinimidyl-4-(p-maleimidophenyl)butyrate;
sulfosuccinimidyl(4-iodoac- etyl)aminobenzoate; sulfosuccinimidyl
4-(N-maleimidomethyl) cyclohexane-1-carboxylate; sulfosuccinimidyl
4-(p-maleimidophenyl)-butyra- te;
bromoacetyl-p-aminobenzoyl-N-hydroxy-succinimidyl ester; and
iodoacetyl-N-hydroxysuccinimidyl ester.
42. The method of claim 18, wherein the ligating agent comprises a
synthetic or recombinant peptide.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of co-pending U.S.
application Ser. No. 09/692,586, filed Oct. 19, 2000, which is a
continuation of International Application No. PCT/US99/09269, filed
Apr. 29, 1999, which claims the benefit of U.S. Provisional
Application No. 60/084,385 filed May 6, 1998, the disclosures of
which are incorporated herein by reference.
FIELD OF THE INVENTION
[0003] The invention relates to the therapeutic modulation of
inflammatory response modulation, and in particular, to the
suppression of macrophage proinflammatory responses to infectious
and/or inflammatory stimuli.
BACKGROUND OF THE INVENTION
[0004] Macrophages are prodigious secretory cells which can produce
a number of molecules which can either potentiate or dampen immune
responses (Nathan, J. Clin. Invest. 79:319-322, 1987). In response
to infectious or inflammatory stimuli, macrophages can produce
several proinflammatory molecules, including TNF.alpha., IL-1, 1L-6
and IL-12 (Nathan, J. Clin. Invest. 79:319-322, 1987; Trinchieri et
al., J. Leukocyte Biol. 59:505-511, 1996). These proinflammatory
molecules are important for host defense, because experimentally
infected animals deficient in these cytokines are invariably more
susceptible to acute bacterial infections than are normal animals
(Dalrymple et al., Infect. Immun. 63:2262-2268, 1995; Kincy-Cain et
al., Infect. Immun. 64:1437-1440, 1996).
[0005] IL-12 is a 70 kDa heterodimer consisting of two covalently
linked polypeptide chains, one of 35 kDa (p35) and the other of 40
kDa (p40). IL-12 plays an important role in the development of
T.sub.H1-type immune responses (Trinchieri et al., J. Leukocyte
Biol. 59:505-511, 1996). This cytokine is a potent inducer of
IFN.gamma. from T and NK cells, and it has been shown to play a
crucial role in the development of immunity to intracellular
pathogens (Heinzel et al., J. Exp. Med. 177:1505-1512, 1993; Tripp
et al., Proc. Natl. Acad. Sci. USA 90:3725-3729, 1993).
[0006] IL-12 is a potent inducer of cell-mediated immune responses,
and animals lacking IL-12 are invariably more susceptible to
infections with intracellular pathogens (Mattner et al., Eur. J.
Immunol. 26:1553-1559, 1996). It has been recently demonstrated
that some microbes can influence IL-12 production by macrophages.
Leishmania major, measles virus, and HIV have all been shown to
downregulate the production of IL-12 by macrophages or monocytes
infected with them (Carrera et al., J. Exp. Med 183:515-526, 1996;
Karp et al., Science 273:228-231, 1996; Chehimi et al., J. Exp.
Med. 179:1361-1366, 1994). This downmodulation of IL-12 has the
potential of providing these pathogens with a means of suppressing
the development of cell-mediated immunity.
[0007] The production of proinflammatory cytokines such as IL-12,
however, must be tightly regulated, since their production is also
correlated with many of the pathologies associated with acute
sepsis or with autoimmune diseases. The overproduction of IL-12
during an immune response, however, has the potential to be
detrimental to the host. IL-12 produced during endotoxemia (Wysocka
et al., Eur. J. Immunol. 25:672-676, 1995), and during a number of
autoimmune disorders, including insulin-dependent diabetes mellitus
(Trembleau et al., J. Exp. Med. 181:817-821, 1995), experimental
allergic encephalomyelitis (Leonard et al., J. Exp. Med
181:381-386, 1995), or collagen-induced arthritis (Germann et al.,
Proc. Natl. Acad. Sci. USA 92:4823-4827, 1995), can lead to
exacerbated disease.
[0008] In many instances, macrophages can participate in the
regulation of proinflammatory cytokines by the production of
anti-inflammatory molecules. The secretion of prostaglandins,
TGF.beta., and IL-10 by macrophages has been associated with
anti-inflammatory responses (Tsunawaki et al., Nature 334:260-262,
1988; Bogdan et al., J. Exp. Med 174:1549-1555, 1991; Kunkel et
al., J. Biol. Chem. 263:5380-5384, 1988). These anti-inflammatory
molecules have the potential to ameliorate the potentially
deleterious effects of an overly aggressive immune response. Thus,
the balance between the secretion of pro- and anti-inflammatory
molecules by macrophages is a critical component of the acute phase
response and has the potential to affect the adaptive immune
response that subsequently develops.
[0009] Interleukin-10 (IL-10) is an 18 kDa cytokine produced by the
T.sub.H2 subset of CD4+ helper cells. It is also produced by some
activated B cells, by some T.sub.H1 cells (in humans), by activated
macrophages, and by some non-lymphocytic cell types (e.g.,
keratinocytes). In contrastto IL-12, IL-10 has been associated with
an inhibition of T.sub.H1-type immune responses. IL-10 has been
shown to inhibit the production of T.sub.H1 cytokines and the
proliferation of T.sub.H1 cells to antigen (Malefyt et al., J. Exp.
Med. 174:915-924, 1991; Fiorentino et al., J. Immunol.
146:34443451, 1991). IL-10 inhibits IL-12 production bymacrophages
(D'Andrea et al., J. Exp. Med. 178:1041-1048, 1993), and the
administration of exogenous IL-10 can diminish the toxicity of LPS
(Howard et al., J. Exp. Med. 177:1205-1208, 1993; Berg et al., J.
Clin. Invest. 96:2339-2347, 1995). IL-10 has been considered for
the treatment of autoimmune diseases such as arthritis (Hart et
al., Immunology 84:536-542, 1995) and colitis (Davidson et al., J.
Exp. Med 184:241-251, 1996).
[0010] The Fc.gamma. receptor (Fc.gamma.R) is a receptor for the Fc
region of IgG. B and T lymphocytes, natural killer cells,
polymorphonuclear leukocytes, mononuclear phagocytes, and platelets
contain Fc.gamma. receptor. The three types of Fc.gamma. receptors
include Fc.gamma.RI (CD64), Fc.gamma.RII (CD32) and Fc.gamma.RIII
(CD16). CD16, the Fc.gamma.RIII, is the prototypical
proinflammatory Fc receptor. Ligating Fc.gamma.RIII has been
associated with the production of proinflammatory cytokines
(Cassatella et al., J. Exp. Med. 169:549-567, 1989), and mice
lacking Fc.gamma.RIII undergo diminished Arthus reactions (Hazenbos
et al., Immunity 5:181-188, 1996). CD32, the Fc.gamma.RII, is a
negative regulator of immune complex-triggered immune responses,
and mice lacking Fc.gamma.RII have augmented anaphylactic responses
to IgG (Takai et al., Nature 379:346-349, 1996). Fc.gamma.RI
represents a high-affinity receptor found on mononuclear
phagocytes. In humans, its binds IgG1 and IgG3. Fc.gamma.RII and
Fc.gamma.RIII are low-affinity IgG receptors.
[0011] A mechanism whereby receptor ligation can downmodulate IL-12
production by macrophages has been described (Sutterwala et al, J.
Exp. Med. 185:1977-1985 (1997). However, this previously described
mechanism of IL-12 downregulation did not exhibit specificity with
regard to the macrophage phagocytic receptors that could induce
this downmodulation.
[0012] Lipopolysaccharide endotoxin (LPS) is a complex
macromolecule from the cell walls of certain bacteria, some of
which cause diseases like typhoid fever, dysentery, and urinary
tract infections and from other bacteria which are common
inhabitants of animal and human intestinal tracts but ordinarily do
not cause disease. All of these bacteria have in common the same
type of cell wall and are classified as Gram-negative. LPS induces
the production and release of immunologically active cytokines and
other mediators of the proinflammatory response.
[0013] There are many pathophysiological effects of LPS, one of
which is endotoxemia or septic shock which results from large
amounts of endotoxin in the blood. The majority of the cases of
septic shock are a consequence of Gram-negative bacteremia
(bacteria in the blood). However, the septic shock syndrome can be
induced by other organisms including Gram-positive bacteria and
fungi. A key factor in the development of toxic shock is the
release of LPS from Gram-negative bacteria and the subsequent
effects of the endotoxin on various cells in the body which become
highly activated. As a result, the host is overwhelmed with many
cell substances that lead to circulatory failure, shock and
death.
[0014] There is a need for a therapeutic modality which is capable
of reversing the proinflammatory responses of macrophages to
stimuli such as bacteria and bacterial products, and stimuli
associated with autoimmune disease. There is needed a therapeutic
modality which is capable of inhibiting host proinflammatory immune
responses while at the same time inducing host anti-inflammatory
responses. In particular, what is needed is a modality for
dampening the acute response to inflammatory stimuli, such as LPS
or Gram-negative bacteria. Such therapeutic modalities would be
useful in treating various proinflammatory diseases such as
autoimmune disorders and bacteremia caused by Gram-negative
bacilli.
SUMMARY OF THE INVENTION
[0015] A method for enhancing IL-10 production by Fc.gamma.RI
receptor-expressing cells of a mammal is provided. An agent is
administered to the mammal, which agent either alone or in
combination with one or more substances in the body of the mammal,
causes ligation of the Fc.gamma.RI receptors on those Fc.gamma.RI
receptor-expressing cells. The mammal may be a human being. The
cells most particularly comprise macrophages.
[0016] According to one embodiment, the administered agent is a
ligating agent comprising a multivalent antibody which binds to the
Fc.gamma.RI receptor.
[0017] According to another embodiment, the ligating agent
comprises an immune complex containing at least two antibody
molecules or fragments thereof which contain the Fc region of
IgG.
[0018] According to yet another embodiment of the invention, the
ligating agent comprises an antibody multimer containing at least
two antibody molecules or fragments thereof which contain the Fc
region of IgG. Preferably, the ligating agent comprises a
preparation of IgG comprising IgG dimers, trimers or a mixture
thereof. The IgG content of the preparation preferably comprises,
on a weight percent basis, at least about 50% IgG dimers, trimers,
or a mixture thereof.
[0019] A method of inhibiting a proinflammatory immune response in
a mammal is provided comprising administering an effective amount
of the ligating agent to the mammal to cause ligation of
Fc.gamma.RI receptors on cells of the mammal.
[0020] According to another embodiment of the invention, a method
of inhibiting a proinflammatory immune response in a mammal is
provided. An IgG antibody is administered, which binds to antigen
in the mammal to form an immune complex capable of ligating
Fc.gamma.RI receptors present on host cells.
[0021] According to another embodiment of the invention, a method
for treating or preventing shock associated with bacterial
endotoxemia, or for treating an autoimmune disorder, is provided.
An effective amount of a ligating agent is administered to a mammal
in need of such treatment. The ligating agent causes ligation of
Fc.gamma.RI receptors on cells of the mammal.
[0022] According to another embodiment of the invention, a method
is provided for treating or preventing shock associated with
bacterial endotoxemia in a mammalian host. An IgG antibody is
administered, which binds to antigen in the host to form an immune
complex capable of ligating Fc.gamma.RI receptors present on host
cells.
[0023] According to another embodiment of the invention, a method
for treating an autoimmune disorder in a mammalian host is
provided, comprising administering an IgG antibody which binds to
antigen in the host to form an immune complex capable of ligating
Fc.gamma.RI receptors present on host cells.
DESCRIPTION OF THE FIGURES
[0024] FIG. 1A shows the competitive quantitative reverse
transcription-polymerase chain reaction (RT-PCR) analysis of IL-10
production in murine bone marrow-derived macrophages (BMM.PHI.)
exposed to LPS alone or LPS in combination with either
IgG-opsonized sheep erythrocytes (E-IgG) or complement-opsonized
sheep erythrocytes (E-C3bi). Concentrations of input cDNAs were
first adjusted, using the housekeeping gene hypoxanthine-guanine
phosphoribosyltransferase (HPRT), to yield comparable ratios of
competitor (upper band in each reaction in top panel of FIG. 1A) to
wild-type (lower band in each reaction in top panel of FIG. 1A)
intensities for the amplification reaction for HPRT. The adjusted
input cDNAs were then used in subsequent RT-PCR reactions using
primers for IL-10 (bottom panel in FIG. 1A).
[0025] FIG. 1B shows the competitive quantitative RT-PCR analysis
of IL-10 production in murine BMM.PHI. exposed to LPS alone or LPS
in combination with E-IgG, following normalization for HPRT levels.
Constant volumes of normalized cDNAs were amplified in the presence
of increasing concentration of the multiple cytokine-containing
competitor PQRS, using primers for IL-10.
[0026] FIG. 2 is a graph of the enzyme-linked immunosorbent assay
(ELISA) determination of IL-10 in supernatants of murine BMM.PHI.
exposed to either media, LPS, E-IgG, or E-C3bi (inset), or LPS
alone or LPS in combination with either E-IgG or E-C3bi.
[0027] FIGS. 3A through 3D comprise graphs of ELISA determinations
of IL-10 in supernatants of BMM.PHI. of various mouse strains
exposed to LPS alone or LPS in combination with either E-IgG or
unopsonized erythrocytes (E): 3A, C57BL/6; 3B, FcR.gamma..sup.-/-;
3C, Fc.gamma.RII.sup.-/-; 3D, Fc.gamma.RIII.sup.-/-.
[0028] FIG. 4 is a graph of the ELISA determination of IL-10 in the
supernatant of murine BMM.PHI. exposed to either media,
IgG3-opsonized sheep erythrocytes (E-IgG3), E, or LPS alone, or LPS
in combination with either E-IgG3 or E.
[0029] FIG. 5 is a graph of the ELISA determination of IL-12(p70)
in the supernatant of murine BMM.PHI.. The test supernatant was
generated by (i) priming the test BMM.PHI. with IFN.gamma., (ii)
treating the test BMM.PHI. with another BMM.PHI. supernatant, which
other BMM.PHI. supernatant was generated by exposing BMM.PHI. to
either media alone or LPS in combination with E-IgG, followed by
incubation of the supernatant in the presence or absence of a
neutralizing monoclonal antibody to IL-10 (anti-IL-10), and (iii)
treating the test BMM.PHI. with LPS.
[0030] FIG. 6A is a graph of the ELISA determination of IL-10 in
the supernatant of BMM.PHI. exposed to either media, LPS, or
IgG-LPS.
[0031] FIG. 6B is a graph of the ELISA determination of IL-12(p40)
in the supernatant of BMM.PHI. exposed to either media, LPS, or
IgG-LPS.
[0032] FIG. 7A is a graph of the ELISA determination of IL-10 in
the supernatant of BMM.PHI. incubated with media alone or with
unopsonized or IgG-opsonized H. Influenzae.
[0033] FIG. 7B is a graph of the ELISA determination of IL-12(p40)
in the supernatant of BMM.PHI. incubated with media alone or with
unopsonized or IgG-opsonized H. Influenzae.
[0034] FIG. 8A is a plot of IL-12(p40) serum levels in
RAG-1.sup.-/- mice which received either LPS or IgG-LPS
intravenously at a final LPS dose of 4 .mu.g. Serum levels were
measured at the indicated times post-challenge.
[0035] FIG. 8B is a plot of IL-10 serum levels in RAG-1.sup.-/-
mice which received either LPS or IgG-LPS intravenously at a final
LPS dose of 4 .mu.g. Serum levels were measured at the indicated
times post-challenge.
[0036] FIG. 9A is similar to FIG. 8A, with IL-12(p40) serum levels
sampled at 2, 4 and 8 hours.
[0037] FIG. 9B is similar to FIG. 8B, with IL-10 serum levels
sampled at 2, 4 and 8 hours.
[0038] FIG. 10A is a plot IL-12(p40) serum levels in RAG-1.sup.-/-
mice which received 100 .mu.l of anti-LPS antibody
intraperitoneally 2 or 18 hours before being injected intravenously
with LPS (4 .mu.g, E. coli 0128:B12). Control uninjected mice
received LPS alone. Serum was collected at 2, 4, and 8 hours post
LPS injection and assayed for IL-12(p40) production. Symbols in the
figures represent mean serum cytokine levels from 5 mice.+-.SD.
[0039] FIG. 10B is a graph of IL-10 serum levels in RAG-1.sup.-/-
mice which received anti-LPS and/or LPS according to the same
schedule of FIG. 10A. Serum was sampled two hours post LPS
injection. Symbols in the figures represent mean serum cytokine
levels from 5 mice .+-.SD.
DETAILED DESCRIPTION OF THE INVENTION
[0040] Macrophages can respond to a variety of infectious and/or
inflammatory stimuli by secreting an array of proinflammatory
cytokines, such as IL-1, IL-6, IL-12 and TNF.alpha.. The
overproduction of these proinflammatory cytokines, particularly
IL-12, can result in shock or even death.
[0041] According to the present invention, ligation of the Fcy
receptor type I (Fc.gamma.RI) on Fc.gamma.RI-expressing cells such
as macrophages leads to a selective upregulation of IL-10
production by those cells in response to proinflammatory signals.
This upregulation occurs at the level of gene transcription and
results in a increase in IL-10 protein secretion. The upregulation
of IL-10 production is specific to Fc.gamma.R1 ligation.
[0042] The human Fc.gamma.RI receptor is a monomeric molecule
having three Ig-like domains. The cDNAs for human and murine
Fc.gamma.RI have been identified by Allen and Seed, Science
243:378-80 (1989), and Sears et al., J. Immunol. 144:371-78 (1990),
respectively, the entire disclosures of which are incorporated
herein by reference.
[0043] The upregulation of IL-10 which results from Fc.gamma.RI
ligation occurs in cells exposed to a proinflammatory stimulus. By
"proinflammatory stimulus" is meant an agent or condition, which
acts on Fc.gamma.RI-expressing cells such as macrophages to induce
a proinflammatory response by those cells. Proinflammatory response
is characterized by secretion of one or more proinflammatory
molecules, such as TNFA, IL-1, IL-6 and IL-12. The proinflammatory
stimulus which acts on macrophages to secrete these molecules
typically comprises bacteria or components from bacterial cell
walls, such as lipopolysaccharide (LPS). A macrophage or other
Fc.gamma.RI-expressing cell which has been acted upon by a
proinflammatory stimulus is said to be "stimulated".
[0044] By "ligation" with respect to the Fc.gamma.RI receptor on a
cell is meant the formation of cross-links between a sufficient
number of molecules of such receptor on the cell sufficient to
stimulate IL-10 production by the cell.
[0045] By "ligating agent" is meant any substance which is capable
of carrying out ligation of Fc.gamma.RI receptors on cells.
[0046] By "enhance" or "enhancement" or "upregulation" with respect
to the IL-10 production by Fc.gamma.RI receptor-expressing cells is
meant an increase in IL-10 production of at least two fold over the
IL-10 production level of stimulated cells of the same type which
are not exposed to ligating agent. Increases in IL-10 production of
four fold, five fold and even as high as eight fold are
possible.
[0047] It may be appreciated that any ligating agent must be at
least bifunctional with respect to Fc.gamma.RI binding in order to
achieve cross-linking of the receptor, that is, a single molecule
of ligating agent must be capable of simultaneously binding two or
more receptors.
[0048] Ligation of Fc.gamma.RI on Fc.gamma.RI-expressing cells such
as macrophages stimulates IL-10 production and leads to a marked
suppression of IL-12 biosynthesis by IL-12 producing cells,
particularly macrophages. Macrophage-derived IL-10 is a potent
inhibitor of macrophage IL-12 production. Even IFN.gamma.-primed
macrophages fail to make IL-12 in response to LPS when exposed to
macrophage supernatants containing IL-10. The ligation of the
macrophage Fc.gamma.RI downmodulates IL-12 production via a
mechanism that is dependent on macrophage-derived IL-10.
[0049] The identification of the specific Fc.gamma.R subtype,
Fc.gamma.RI, as the Fc.gamma.RI receptor responsible for IL-10
upregulation, was determined in gene knockout mice. Macrophages
from mice lacking the FcR.gamma. chain, which is required for
assembly and signaling by Fc.gamma.RI and Fc.gamma.RIII, failed to
upregulate IL-10 in response to immune complexes. However, mice
lacking either the Fc.gamma.RII or the Fc.gamma.RIII were fully
capable of upregulating IL-10 production, thus establishing that
Fc.gamma.RI, and not Fc.gamma.RII or Fc.gamma.RIII, is the receptor
responsible for IL-10 upregulation.
[0050] As further proof of the identification of the Fc.gamma.RI
receptor as the receptor responsible for IL-10 upregulation,
Fc.gamma.RI erythrocytes were opsonized with IgG3. IgG3 binds
Fc.gamma.RI exclusively, and does not bind Fc.gamma.RII or
Fc.gamma.RIII (Gavin et al., J. Immunol 160:20-23,1998). The
IgG3-opsonized erythrocytes were observed to enhance IL-10
production, confirming the role of Fc.gamma.RI in this process.
[0051] The biological consequences of Fc.gamma.RI ligation were
determined in both in vitro and in vivo models of inflammation and
sepsis. In all of the models tested the ligation of Fc.gamma.R
promoted the production of IL-10 and inhibited the secretion of
IL-12. This reciprocal alteration in the pattern of macrophage
cytokine production provides a useful therapeutic modality in
suppressing macrophage proinflammatory responses.
[0052] According to the present invention, the Fc.gamma.RI receptor
is ligated by an agent which is capable of ligating that receptor.
Preferably, the ligating agent binds specifically to the
Fc.gamma.RI, and does not also ligate the Fc.gamma.RIII receptor,
or ligates the Fc.gamma.RIII receptor only minimally. The
Fc.gamma.RIII receptor is the prototypical proinflammatory Fc
receptor. Ligating Fc.gamma.RIII has been associated with the
production of proinflammatory cytokines (Cassatella et al., J. Exp.
Med. 169:549-567,1989, and mice lacking Fc.gamma.RIII undergo
diminished Arthus reactions (Hazenbos et al., Immunity 5:181-188,
1996). Ligating the Fc.gamma.RI receptor, without simultaneously
ligating the Fc.gamma.RIII receptor, provides enhancement of the
anti-inflammatory response in treated individuals via upregulation
of macrophage-derived IL-10 production without triggering the
production of the proinflammatory cytokines associated with
Fc.gamma.RIII ligation. At the same time, the method of the present
invention results in the suppression of the proinflammatory
response through the potent inhibition of macrophage IL-12
production by macrophage-derived IL-10.
[0053] While a mechanism whereby receptor ligation can downmodulate
IL-12 production by macrophages was previously described by
Sutterwala et al., J. Exp. Med 185:1977-1985 (1997), that mechanism
of IL-12 downregulation did not exhibit specificity with regard to
the macrophage phagocytic receptors that induced the
downmodulation. Moreover, the IL-12 downregulation following
receptor ligation described by Sutterwala et al. was transient,
Ca.sup.++ dependent and IL-10 independent.
[0054] The present invention arises from a novel mechanism of IL-12
downregulation which is distinct from the previously described
mechanisms in several important ways. The present mechanism does
not involve a direct regulation of IL-12 transcription, but rather
depends on the production of the inhibitory cytokine IL-10, which
acts on IL-12-producing cells. The IL-12 downregulation mediated by
IL-10 is not Ca.sup.++-dependent, and has a markedly longer
duration than the transient downregulation of IL-12 observed by
Sutterwala et al. Moreover, the IL-10-mediated IL-12 downregulation
of the present invention is specific to a single receptor class on
macrophages, the Fc.gamma.RI.
[0055] Fc.gamma.RII has been shown to be a negative regulator of
immune complex-triggered immune responses, and mice lacking
Fc.gamma.RII have augmented anaphylactic responses to IgG (Takai et
al., Nature 379:346-349 (1996)). However, Fc.gamma.RI operates via
a different mechanism than that observed for Fc.gamma.RII. Whereas
Fc.gamma.RII inhibits signaling through the Fc.gamma.R (Muta et
al., Nature 368:70-73, 1994), Fc.gamma.RI actively promotes the
transcription of an inhibitory cytokine, IL-10. Thus, although both
Fc.gamma.RI and Fc.gamma.RII can mediate inhibition of inflammatory
responses to immune complexes, they do so by two distinct
mechanisms.
[0056] Preferably, the ligating agent of the present invention is
completely specific for the Fc.gamma.RI receptor and does not
induce ligation of either Fc.gamma.RII or Fc.gamma.RIII.
[0057] Ligating agents for Fc.gamma.RI may be identified by a
screening assay utilizing appropriate test cells as reagents.
Fibroblasts or eptheliod cells, which do not express Fc.gamma.RI,
are transfected with Fc.gamma.RI cDNA to express Fc.gamma.RI.
Transfection may be carried out utilizing the procedure described
by Sutterwala et al., J. Leukocyte Biol. 59:883 (1996)
(incorporated herein by reference) for obtaining stable cell
surface expression of complement receptor type I (CRI) and
complement receptor type 3 (CR3) in Chinese hamster ovary (CHO)
cells. Briefly, cells are cotransfected with pRSVneo and a plasmid
containing the complete cDNA of the human Fc.gamma.RI receptor
(Allen and Seed, Science 243:378-80, 1989) (incorporated herein by
reference) cloned into the ApPM8 expression vector. Transfected
cells are selected in medium containing G418 sulfate. The cell
surface expression of Fc.gamma.RI is confirmed by flow cytometry,
according to known techniques. As a negative control screening
reagent, transfectants expressing the human Fc.gamma.RIII are
prepared according to the same procedure.
[0058] One of two assays may be employed in utilizing the
transfectants to detect ligating agents that specifically bind to
the human Fc.gamma.RI, According to a direct binding assay, the
candidate ligating agent is labeled with fluorescein isothiocyanate
(FITC) according to standard techniques. FITC-labeled ligating
agent is then added to parallel wells of transfected cells
expressing either the Fc.gamma.RI or the Fc.gamma.RIII for thirty
minutes at 4.degree. C. The cells are washed and then fixed in 4%
paraformaldehyde and analyzed by flow cytometry. Agents which bind
to the Fc.gamma.RI-expressing cells, but not the
Fc.gamma.RIII-expressing cells, are selected.
[0059] According to a competitive binding assay, candidate ligating
agent is examined for ability to compete with a known labeled
Fc.gamma.RI-binding agent for binding to Fc.gamma.RI. This assay is
used where the direct binding assay is not conveniently employed,
such as where the candidate ligating agent is not easily labeled.
This is true for small synthetic molecules, for example.
[0060] According to the competitive binding assay, unlabeled
candidate ligating agent is added to Fc.gamma.RI-transfected cells
at 4.degree. C. for 30 minutes. Following this incubation,
FITC-labeled human IgG1, which binds specifically to human
Fc.gamma.RI, is added for an additional 30 minutes at 4.degree. C.
After washing, the cells are fixed in paraformaldehyde and
processed for flow cytometry. Control cells which have not been
exposed to ligating agent will bind FITC-labeled IgGI and
fluoresce. Cells which bind ligating agent via the Fc.gamma.RI will
exhibit decreased fluorescence. Reagents testing positive as
ligating agents for Fc.gamma.RI will yield a decreased
fluorescence.
[0061] Ligation of the Fc.gamma.RI receptor may be accomplished by
contacting the cells with a ligating agent for the receptor.
According to one embodiment of the invention, the ligating agent
comprises an IgG immune complex which is capable of cross-linking
the Fc.gamma.RI receptor. By "immune complex" is meant a
macromolecular complex comprising IgG antibody molecules bound
together by antigen. The IgG antibody in the immune complex may be
polyclonal or monoclonal. Such cross-linking immune complexes
contain at least two IgG antibody molecules, or at least two
fragments of IgG antibody molecules which maintain ability to be
bound by the Fc.gamma.RI receptor. Antibody fragments are typically
generated by treatment of antibodies with an enzyme such as papain
or pepsin. Such antibody fragmentation methods are well-known to
those skilled in the art. The antibody of the immune complex may
comprise one or more antibody fragments which maintain the ability
to form immune complexes, i.e., bind antigen, and which maintain
the ability to be bound and be bound by the Fc.gamma.RI receptor.
Thus, the antigen binding sites on the antibody must be preserved
to the extent necessary to bind antigen, and the Fc segment of the
antibody must be preserved to the extent necessary to comprise a
specific recognition site for the Fc.gamma.RI receptor.
[0062] It may be appreciated that the antibody of the immune
complex may comprise a monovalent antibody or antibody fragment, in
addition to or in lieu of multivalent antibodies and antibody
fragments. By "multivalent" is meant antibody which is at least
divalent, that is, has at least two antigen binding sites. This is
because the spanning of neighboring Fc.gamma.RI receptors on
macrophages by immune complex ligating agents occurs by receptor
binding to the antibody Fc region. So long as the immune complex
contains at least two such antibody molecules, and thus at least
two Fc segments available for binding to Fc.gamma.RI neighboring
Fc.gamma.RI receptors on the cell, cross-linking of those receptors
will occur, thereby inducing stimulation of IL-10 secretion by the
cell.
[0063] Preferably, the immune complex comprises an protein-antibody
complex or a polysaccharide-antibody complex. The antigen should
not be toxic to the host. The antigen should be sufficiently small
in size so as not to induce immune complex disease, such as
glomerulonephritis. Preferably, the antigen consists of a peptide
derived from a nontoxic protein such as albumin, combined with
antibodies which react with specific portions of the peptide.
Alternatively, a synthetic saccharide complexed with human IgG2,
for example, may be utilized as the immune complex.
[0064] According to another embodiment of the invention, the
ligating agent comprises an IgG multimer capable of cross-linking
the Fc.gamma.RI receptor. By "multimer" is meant an association of
two or more IgG antibody molecules, or two or more IgG fragments
containing the Fc region. The multimer thus contains two or more
antibody Fc regions.
[0065] Antibodies can be induced to form multimers according to
well-known techniques. Dimeric IgG or trimeric IgG is preferred, or
a mixture thereof. Dimers and trimers are preferred because of the
adverse side effects associated with formation of higher multimers,
which may result in the formation of large complexes or
"aggregates". IgG aggregates may cause the release of
anaphylatoxins into the bloodstream via complement activation.
Glomerulonephritis is also known to result when large immune
complexes form in the kidneys. Human IgG for this purpose may be
obtained in large quantities from pooled blood or outdated plasma.
IgG preparations for intravenous administration are commercially
available. The use of pooled human IgG is possible because the
antigenic specificity of the IgG is irrelevant for purposes of this
embodiment of the invention.
[0066] The antibodies comprising the IgG multimers may comprise
monoclonal or polyclonal antibodies, and may comprise whole
antibody molecules or fragments thereof which maintain the ability
to be bound by the Fc.gamma.RI receptor.
[0067] IgG dimers and trimers are formed by allowing formation of
multimeric forms of IgG of various sizes, followed by selection of
dimers and trimers by size exclusion chromatography, using standard
chromatographic techniques. IgG multimers are spontaneously
generated by combining IgG monomer from different individuals. IgG
dimers are prevalent in Ig prepared from pooled plasma, whereas Ig
prepared from single-donor plasma is virtually monomeric.
[0068] IgG preparations containing multimers are prepared according
to well-known techniques, beginning with pooled plasma. Therapeutic
immunoglobulins are prepared from large pools of human plasma by
the CohnOncley process, which relies on selective precipitation
with ethanol at sub-zero temperatures (Cohn et al., J. Am. Chem.
Soc. 68,459 (1946); Oncley et al., J. Am. Chem. Soc. 71, 541 (1949)
According to the methodology of Kistler and Nitschmann, Vox Sang.
7, 414 (1962), proteins present in plasma, including
immunoglobulins, may be selectively precipitated through
manipulation of pH, protein concentration, alcohol concentration,
ionic strength and temperature. Fraction II of the Cohn-Onclay
process (fraction GC of the Kistler-Nitschmann scheme) consists of
essentially pure IgG, with only trace amounts of other plasma
proteins such as IgA or IgM.
[0069] Contrary to prior methods for forming intramuscular and
intravenous Ig, which seek to avoid formation of IgG dimers, the
composition of the therapeutic IgG preparation of the present
invention is manipulated to select for IgG dimers and trimers, over
monomers. Factors which facilitate dimer formation are discussed by
Tankersley, Immunological Reviews 139:159-72 (1994), the entire
disclosure of which is incorporated herein by reference. The
exposure of monomeric IgG to oxygen radicals causes aggregate
formation (Kleinveld et al., Scand. J. Rheumatology S75:157-163
(1988) (incorporated herein by reference). Thus, exposure of a
solution of IgG to UV light can be used to favor multimer
formation.
[0070] IgG multimers, preferably IgG dimers, may also be generated
by cold-induced polymerization of IgG monomers, as described by
Vialtel et al., J. Biol. Chem. 257:3811-3818 (1982), the entire
disclosure of which is incorporated herein by reference. IgG is
cooled, and then dimers and trimers are separated from monomers and
aggregates by size exclusion chromatography.
[0071] IgG dimers and trimers may be selected from IgG multimer
mixtures according to standard chromatographic techniques, such as
described by Lee et al., J. Chromatography 444:141-52 (1988), the
entire disclosure of which is incorporated herein by reference. For
example, separation of IgG monomer, dimer and aggregate components
from an IgG mixture may be achieved at ambient temperature by
isocratic elution with a mobile phase consisting of 0.2 M dibasic
potassium phosphate (pH 7.0) containing 0.02% sodium azide. A
typical run time is 40 minutes, using the following size-exclusion
chromatography system: two Beckman Spherogel.TM. TSK 3000 SW
columns connected in series (60 cm.times.7.5 mm combined length), a
Waters Model 6000 A pump (set at 00.5 ml/min.), a Waters Model 440
absorbance detector (set at 280 nm and 0.5 a.u.f.s.), a Waters WISP
Model 710B autoinjector (set at 200-.mu.l injections), and a
Houston Instruments Omniscribe recorder (set at 0.1 inches/min.).
IgG aggregate, dimer, and monomer peaks are eluted at retention
times of 18.2, 21.6, and 25.8 minutes, respectively.
[0072] While IgG preparations have been available for human
therapeutic use, such preparations predominately comprise IgG
monomer. For utilization in the practice of the present invention,
such preparations are first enriched for dimers and/or trimers. At
least about 50%, preferably at least about 80%, more preferably at
least about 90%, most preferably about 95%, of the IgG content of
the preparation, by weight, should comprise IgG dimer, trimer or
combination thereof. IgG dimers are preferred over IgG trimers.
[0073] IgG dimers and trimers may also be formed by chemically
crosslinking IgG monomers with covalent cross-linking agents. One
such agent is the bifunctional cross-linker
dithiobis(succinimidylpropriionate- ) ("DSP"). IgG may be
cross-linked in this fashion according to the method of Wright et
al, Biochem. J. 187:767-774 (1980), and 187:775-780 (1980), the
entire disclosures of which are incorporated herein by reference.
Other commercially available cross-linking agents (for example from
Pierce Chemical Company, Rockford, Ill.) may be substituted for
DSP. Such cross-linking agent include the cross-linking agents
identified below.
[0074] According to another embodiment of the invention, the
ligating agent comprises an Fc fragment multimer comprising two or
more Fc fragments which are coupled together. Preferably, the
fragments arejoined by a linking group forming a covalent bond.
Covalently cross-linked Fc fragment dimers may be prepared by
utilizing homobifunctional cross-linking reagents, e.g.,
disuccinimidyl tartrate, disuccinimidyl suberate, ethylene
glycolbis(succinimidyl succinate), 1,5-difluoro-2,4-dinitrobenzene
("DFNB"), 4,4'-diisothiocyano-2,2'-disulf- onic acid stilbene
("DIDS"), and bismaleimidohexane ("BMH").
[0075] Alternatively, heterobifunctional cross-linking reagents may
be employed. Such agents include, for example,
N-succinimidyl-3-(2-pyridyldi- thio)propionate ("SPDP"),
sulfosuccinimidyl-2-(pazidosalicylamido)ethyl-1--
3'-dithiopropionate ("SASD", Pierce Chemical Company, Rockford,
Ill.), N-maleimidobenzoyl-N-hydroxy-succinimidyl ester ("MBS"),
m-maleimidobenzoylsulfosuccinimide ester ("sulfo-MBS"),
N-succinimidyl(4-iodoacetyl)aminobenzoate ("SIAB"), succinimidyl
4-(N-maleimidomethyl)-cyclohexane-1-carboxylate ("SMCC"),
succinimidyl-4-(pmaleimidophenyl)butyrate ("SMPB"),
sulfosuccinimidyl-(4-iodoacetyl)aminobenzoate ("sulfo-SIAB"),
sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate
("sulfo-SMCC"), sulfosuccinimidyl 4-(p-maleimidophenyl)-butyrate
("sulfo-SMPB"), bromoacetyl-p-aminobenzoyl-Nhydroxy-succinimidyl
ester, iodoacetyl-N-hydroxysuccinimidyl ester, and the like.
[0076] For heterobifunctional cross-linking, a Fc fragment is
derivatized with, e.g., the N-hydroxysuccinimidyl portion of the
bifunctional reagent, and the derivatized Fc fragment is purified
by gel filtration. Next, a second Fc fragment is reacted with the
second functional group of the bifunctional reagent, assuring a
directed sequence of binding between components of the Fc
dimer.
[0077] Typical heterobifunctional cross-linking agents for forming
proteinprotein conjugates have an amino-reactive
N-hydroxysuccinimide ester (NHS-ester) as one functional group and
a sulfhydryl reactive group as the other functional group. First,
epsilon-amino groups of surface lysine residues of the first Fc
fragment are acylated with the NHS-ester group of the cross-linking
agent. The second Fc fragment, possessing free sulfhydryl groups,
is reacted with the sulfhydryl reactive group of the cross-linking
agent to form a covalently crosslinked dimer. Common thiol reactive
groups include maleimides, pyridyl disulfides, and active halogens.
For example, MBS contains a NHS-ester as the amino reactive group,
and a maleimide moiety as the sulfhydryl reactive group.
[0078] Photoactive heterobifunctional cross-linking reagents, e.g.,
photoreactive phenyl azides, may also be employed. One such
reagent, SASD, may be linked to an Fc fragment via its NHS-ester
group.
[0079] As an alternative to intact IgG, or multimers comprising
native IgG Fc fragments, the ligating agent may comprise either
synthetic or recombinant peptides which comprise the Fc region. For
example, a single recombinant peptide may be designed comprising
two Fc regions joined by an appropriate spacer segment.
[0080] According to another embodiment of the invention, the
ligating agent comprises a multivalent antibody against the
Fc.gamma.RI receptor. The antibody may be directed against any
determinant on the Fc.gamma.RI receptor. Preferably, the
determinant is unique to the Fc.gamma.RI receptor, and not shared
by Fc.gamma.RII or Fc.gamma.RIII. The multivalent antibody may be
monoclonal or polyclonal. The antibody may comprise intact
antibody, or fragments capable of binding antigen, providing the
fragments are divalent, e.g., F(ab').sub.2 and Facb fragments. For
use as ligating agents in human subjects, such antibodies would be
of animal origin, but would be preferably "humanized". Humanized
murine antibodies have been prepared in which only the minimum
necessary parts of the mouse antibody, the
complementarity-determining regions (CDRs), are combined with human
V region frameworks and human C regions (Jones et al., Nature 321,
522-525, 1986; Verhoeyen et al., Science 239, 1534-1536, 1988;
Reichmann et al., 322, 323-327, 1988; Hale et al., Lancet 2,
1394-1399, 1988; Queen et al., Proc. Natl. Acad. Sci. USA 86,
10029-10033, 1989). The entire disclosures of the aforementioned
papers are incorporated herein by reference. This technique results
in the reduction of the xenogeneic elements in the humanized
antibody to a minimum. Rodent antigen binding sites are built
directly into human antibodies by transplanting only the antigen
binding site, rather than the entire variable domain, from a rodent
antibody. This technique is available for production of chimeric
rodent/human anti-Fc.gamma.RI antibodies of reduced human
immunogenicity.
[0081] The ligating may be administered to patients in
circumstances where it is desired to obtain a therapeutic
anti-inflammatory effect, or to inhibit or prevent an undesired
IL-12-mediated proinflammatory response.
[0082] As an alternative to administering an exogenous ligating
agent, it is possible to form the ligating agent in situ in the
body of the patient, by administering antibody to a target antigen.
Upon uptake by the body, the antibody combines with its target
antigen to form an immune complex including the target antigen.
Such in situ-formed immune complexes comprising two or more IgG
antibodies function as a ligating agents to ligate Fc.gamma.RI
receptors on macrophages to induce IL-10, and in turn inhibit
IL-12, production.
[0083] The target antigen for achieving formation of immune
complexes in vivo is selected according to the etiology of the
disease. The specificity of the therapeutic antibody is designed
accordingly. For example, an immune response to DNA and chromatin
is frequently observed in autoimmune diseases such as systemic
lupus erythematosus (SLE). IgG antibody specific for DNA or
chromatin may be administered to such individuals. The antibody
will form a complex only when the autoimmune antigen is present,
i.e., only during episodes of autoimmune flare-up. Thus, IgG may be
administered as an anti-inflammatory agent which would be
functional only when needed, that is, when the autoimmune antigen
is expressed. Antibody that does not form complexes with the
autoimmune antigen is cleared from the circulation according to the
half-life of the immunoglobulin. The antibodies are preferably
humanized to reduce their antigenicity and to increase their
half-life in plasma.
[0084] The present invention thus provides a treatment for
autoimmune disorders including moderately acute autoimmune
disorders such as Kawasaki Disease; and chronic autoimmune
disorders such as SLE, rheumatoid arthritis, inflammatory bowel
disease, Sydenham's chorea (post Streptococcal), and autoimmune
hemolytic anemia. For treatment of autoimmune disorders, ligating
agent according to the present invention is administered,
preferably parenterally, most preferably intravenously.
[0085] The ligating agent may be administered as a treatment for
conditions having a proinflammatory component, such as enteric
bacterial infections which produce the pharmacologically active
lipopolysaccharide endotoxin from the bacterial cell wall, known as
endotoxin or LPS. Endotoxin from a wide variety of unrelated
bacterial species behave quite similarly, regardless of the
inherent pathogenicity of the microorganism from which they are
derived or their antigenic structure. In particular, the method of
the present invention is useful for treating the endotoxemia or
septic shock which arises from bacteremia caused by gram-negative
bacilli.
[0086] Bacteremia arises from blood system invasion by enteric
bacteria, most commonly from urinary tract infection, post surgical
disease of the gastrointestinal tract (e.g., following bowel
surgery), infections developing at the site of "cut-downs" and
intravenous catheters, postpartum or postabortal sepsis, and
infection of wounds, ulcer, burns, and internal prosthetic devices.
Shock attributed to endotoxemia is due to the release of the
proinflammatory cytokines IL-1, IL-6, IL-12 and TNF.alpha., most
particularly IL-12. Stimulation of IL-10 secretion by exogenously
administered or in vivo-formed ligating agent can lead to the rapid
downregulation of proinflammatory cytokine release, and the
prevention or abatement of shock symptoms.
[0087] According to another embodiment for treating endotoxemia,
the treatment is carried out by administering an appropriate
antibody which leads to the in vivo formation of ligating agent.
For this purpose, the antibody is preferably directed against
bacterial endotoxin, leading to the in vivo formation of
IgG-endotoxin complexes. These immune complexes are effective in
ligating Fc.gamma.RI receptors on macrophages participating in the
proinflammatory response to the endotoxin. Of course, efforts
should be undertaken to also treat the underlying enteric bacterial
infection, such as described by Kunin in "Enteric Bacterial
Infections", in Textbook of Medicine, P. Beeeson et al. eds., 15th
edition, W. B. Saunders Co., Philadelphia, Pa., 1979,
p.453-457.
[0088] The ligating agent (or antibody to induce in vivo formation
of ligating agent) may be administered by any method which achieves
an adequate distribution of the ligating agent in the target
tissue. The ligating agent or antibody is preferably administered
parenterally, most preferably intravenously or intraarterially. The
administration may take the form of one or more injections or
continuous or semi-continuous infusions. For the treatment of
septic shock, a single bolus injection is contemplated. For the
treatment of immune disorders, one or more injections are utilized,
as needed. For a ligating agent comprising IgG dimers, for example,
a representative dosage may range from about 0.05 to about 1.0
g/kg, more preferably from about 0.1 to about 0.5 g/kg. For
administration of IgG to achieve in situ formation of therapeutic
immune complexes against target antigens such as LPS, the dosage
may range from about 0.1 to about 20 mg/kg, preferably from about 1
to about 10 mg/kg.
[0089] The ligating agent (or antibody) is advantageously
formulated by combination with a pharmaceutically acceptable
carrier suitable for administration of antibodies. For intravenous
administration, the ligating agent or antibody is contained in a
preservative-free sterile saline solution, for example. One or more
additives, such as stabilizers and additives for inhibiting the
formation of antibody aggregates, may are also be included in the
preparation. Representative antiaggregating agents including
polysorbate 80 and tri-n-butyl phosphate. One representative
formulation for intravenous administration of antibody comprises
Dsorbitol (50 mg/ml), human albumin (1 mg/ml), polyethylene glycol
(100 mcg), polysorbate 80 (100 mcg) and tri-n-butyl phosphate.
[0090] The practice of the invention is illustrated by the
following non-limiting examples.
EXAMPLE 1
Effect of Fc.gamma.R Ligation on Mouse Macrophage IL-10
Production
[0091] The production of IL-10 by bone marrow-derived macrophages
(BMM.PHI.) was examined following specific Fc.gamma.R ligation as
follows. The ligating agent comprised IgG-opsonized sheep
erythrocytes.
[0092] A. Macrophages
[0093] Six- to eight-week-old BALB/c and C57BL/6 mice were obtained
from Taconic (Germantown, N.Y.). Bone marrow-derived macrophages
(BMM.PHI.) were established as previously described (Sutterwala et
al., J. Exp. Med. 185:1977-1985, 1997). Briefly, bone marrow cells
were differentiated in DMEM containing 20% L929 cell-conditioned
medium, 10% heat-inactivated (HI)-FCS, 2 mM L-glutamine, 100 U/ml
penicillin G, and 100 .mu.g/ml streptomycin for 5 to 7 days until
uniform monolayers of macrophages were established. Twelve hours
before use, cells were removed from the original plastic petri
dishes by EDTA and were plated in tissue culture-treated six- or
twenty four-well plates (Nunc, Naperville, Ill.) in DMEM containing
10% HI-FCS, 2 mM L-glutamine, 100 U/ml penicillin G, and 100
.mu.g/ml streptomycin (complete medium).
[0094] B. Opsonized Ervthrocvtes
[0095] IgG-opsonized sheep erythrocytes (E-IgG) were generated by
incubating SRBC (Lampire, Pipersville, Pa.) with rabbit anti-SRBC
IgG (Cappel, Durham, N.C.) at nonagglutinating titers for 40
minutes at room temperature. E-IgG were washed and resuspended in
HBSS (GIBCO BRL, Grand Island, N.Y.) prior to their addition to
macrophages. Complement-opsonized erythrocytes (E-C3bi) were
generated by incubating SRBC with culture supernatants of hybridoma
S-S.3 (anti-SRBC IgM/.kappa. [ATCC, Rockville, Md.]) at
nonagglutinating titers for 40 minutes at room temperature.
IgM-opsonized erythrocytes were washed twice with HB S S and
resuspended at 1.times.10.sup.8 cells/ml in HBSS with 10% murine
C5-deficient serum. Following a 15-minute incubation at 37.degree.
C., E-C3bi were washed and resuspended in HBSS prior to their
addition to macrophages. Erythrocytes were added to macrophage
monolayers at a ratio of 20:1.
[0096] C. Macrophage Stimulation
[0097] Monolayers of the BMM.PHI. were washed once with complete
medium, and then stimulated with LPS (Escherichia coli 0127:B8
[Sigma, St. Louis, Mo.]) at a final concentration of 100 ng/ml, in
the presence or absence of opsonized erythrocytes (E-IgG or
E-C3bi).
[0098] D. Competitive Quantitative RT-PCR
[0099] Six hours following the addition of stimuli, total RNA was
extracted from 10.sup.6 BMM.PHI. using RNAzol B according to the
manufacturer's instructions. RNA was reverse transcribed using
Superscript II RT (GIBCO BRL) and random hexamer primers (Promega,
Madison, Wis.). PCR was performed using a multiple
cytokine-containing competitor PQRS, as previously described
(Reiner et al., J. Immunol. Methods 165:37-46, 1993) and a fixed
concentration of competitor in each reaction.
[0100] In brief, concentrations of input cDNAs were first adjusted
using the housekeeping gene hypoxanthine-guanine
phosphoribosyltransferase (HPRT). Input cDNAs were adjusted to
yield comparable ratios of competitor (FIG. 1A, upper band in each
reaction) to wild-type (FIG. 1A, lower band in each reaction)
intensities for the amplification reaction for HPRT. Amplification
products were resolved on 2.0% ethidium-stained agarose gels. The
results are set forth in FIG. 1A. The larger molecular weight
competitor bands provide an internal standard for the relative
amounts of the lower molecular weight experimental cDNAs.
[0101] Constant volumes of normalized cDNAs were then amplified in
the presence of decreasing concentrations of competitor (PQRS),
using primers for IL-10 (Sutterwala et al., J. Exp. Med.
185:1977-1985, 1997). The results are shown in FIG. 1B. The
concentration of the experimental cDNA is represented by the
equivalent intensities of competitor and wild-type bands. The fold
increase in IL-10 levels between BMM.PHI. exposed to LPS or LPS in
combination with E-IgG can be determined by taking the ratio of
their equivalence points. IL-10 mRNA was increased by 4 to
8-fold.
[0102] E. Cytokine ELISA
[0103] Cytokines levels in BMM.PHI. supernatants were measured by
enzyme-linked immunosorbent assay (ELISA) 24 hours following the
addition of stimuli using appropriately diluted cell supernatants.
IL-10 concentrations were determined with a mouse IL-10 ELISA kit
(Genzyme Corp., Cambridge, Mass. or Biosource International,
Camarillo, Calif.) according to the manufacturer's instructions.
The results are shown in FIG. 2. The inset in FIG. 2 results from
BMM.PHI. exposed to either media, LPS or erythrocytes opsonized
with IgG (E-IgG) or C3bi (E-C3bi). The main body of FIG. 2 results
from exposure of BMM4) to LPS alone or LPS in combination with
either E-IgG or E-C3bi. Values in FIG. 2 represent the mean of
three independent experiments, each performed in triplicate,
.+-.SE.
[0104] F. Discussion of Results
[0105] The addition of LPS to monolayers of BMM.PHI. induced a
modest but significant production of IL-10 by macrophages (FIG. 1),
as previously reported (de Waal et al., J. Exp. Med. 174:1209-2120,
1991). The ligation of Fc.gamma.R simultaneously with the addition
of LPS, however, markedly enhanced the production of IL-10. This
enhancement was observed at both the mRNA (FIG. 1A) and protein
(FIG. 2) levels. IL-10 mRNA was increased by 4 to 8-fold (FIG. 1B),
and protein secretion was increased by greater than 6-fold
following Fc.gamma.R ligation (FIG. 2). The induction of IL-10 was
specific to the Fc.gamma.R because ligation of macrophage
complement receptors did not significantly alter IL-10 mRNA (FIG.
1A) or protein (FIG. 2) production.
[0106] Macrophage complement and Fc.gamma.R were also ligated in
the absence of LPS to determine whether receptor ligation alone was
sufficient to trigger IL-10 production (FIG. 2 inset). The ligation
of neither of these receptors was sufficient to induce the
production of significant levels of IL-10. Thus, Fc.gamma.R
ligation on unstimulated macrophages is not sufficient to trigger
the production of IL-10.
Example 2
Effect of Fc.gamma.R Ligation on Macrophage IL-10 Production by
Gene Knockout Mice
[0107] In order to determine the Fc.gamma.R subtype responsible for
IL-10 upregulation, bone marrow derived macrophages (BMM.PHI.) from
gene knockout mice were studied. The FcR y chain is an essential
component of both the Fc.gamma.RI and the Fc.gamma.RIII, and is
required for both receptor assemble and signaling (Takai et al.,
Cell 76:519-529, 1994).
[0108] A. Macrophages
[0109] BMM.PHI. were derived according to the preparative procedure
of Example 1 from each of the following mice: (i) FcR .gamma.
chain-deficient (FcR.gamma..sup.-/-) (Takai et al., Cell
76:519-529, 1994); (ii) Fc.gamma.RII-deficient
(FcRII.gamma..sup.-/-) (Takai et al., Nature 379:346-349, 1996);
(iii) Fc.gamma.RIII-deficient (FcRIII.gamma..sup.-/-) (Hazenbos et
al., Immunity 5:181-188, 1996); (iv) normal C57BL/6 mice.
[0110] B. Macrophage Stimulation and IL-10 ELISA
[0111] BMM.PHI. were exposed to LPS alone or LPS in combination
with either E-IgG or unopsonized erythrocytes (E), as in Example 1.
After 24 hours the supernatant was harvested and IL-10 levels were
determined by ELISA as above. Determinations were performed in
triplicate, and values are expressed as the means .+-.SD. The
results, set forth in FIG. 2, are representative of three separate
experiments.
[0112] C. Discussion of Results
[0113] The FcR .gamma. chain is an essential component of both the
Fc.gamma.RI and the Fc.gamma.RIII, and is required for both
receptor assembly and signaling (Takai et al., Cell
76:519-529,1994). Macrophages from mice lacking the common .gamma.
chain (FcR.gamma..sup.-/-) failed to upregulate IL-10 production
(FIG. 3B), implicating one of these two receptors in this
phenomenon. Macrophages derived from normal mice (FIG. 3A), mice
lacking Fc.gamma.RII (FIG. 3B), and mice lacking Fc.gamma.RIII
(FIG. 3C) were fully capable of upregulating IL-10 production in
response to E-IgG. These results are consistent with the high
affinity Fc.gamma.RI being the mediator of IL-10 induction.
Example 3
Effect of Specific Fc.gamma.RI Ligation on Mouse Macrophage IL-10
Production
[0114] To directly demonstrate the role of Fc.gamma.RI in the
upregulation of IL-10 biosynthesis, erythrocytes were opsonized
with IgG3 (E-IgG3), a subclass of antibody that binds exclusively
to Fc.gamma.RI (Gavin et al., J. Immunol. 160:20-23, 1998).
[0115] A. IG3-Opsonized Erythrocytes
[0116] To specifically opsonize sheep erythrocytes with IgG3, the
erythrocytes were incubated with a 1:10 dilution of ascitic fluid
containing the mAb N-S.7 (anti-SRBC IgG3). The N-S.7 hybridoma was
obtained from the ATCC (Rockville, Md.).
[0117] B. Macrophage Stimulation and IL-10 ELISA
[0118] BMM.PHI. were exposed to either media, E-IgG3, unopsonized
erythrocytes (E) or LPS alone or LPS in combination with either
E-IgG3 or E, according to the procedure of Example 1. After 24
hours the supernatant was harvested and IL-10 levels were
determined by ELISA as above. Determinations were performed in
triplicate, and values are expressed as the means .+-.SD. The
results, set forth in FIG. 4, are representative of two separate
experiments.
[0119] C. Results
[0120] Stimulation of macrophages with LPS and E-IgG3 induced a
5-fold increase in IL-10 production relative to stimulation with
LPS alone (FIG. 4).
Example 4
Suppression of Macrophage IL-12 Production by LPS/Fc.gamma.-induced
IL-10
[0121] The following demonstrates that the levels of IL-10 that
were produced by macrophages in response to Fc.gamma.RI coligation
is adequate to suppress IL-12 production.
[0122] A. Macrophage Stimulation and IL-12 ELISA
[0123] Supernatants from BMM.PHI. exposed to either media or LPS in
combination with E-IgG for 24 hours were harvested and filtered
through a 0.2 .mu.m filter. Supernatants were diluted 1:3 with
media and incubated for 15 minutes at 4.degree. C. either in the
presence or absence of a neutralizing mAb to IL-10 (JESS-2A5; 20
.mu.g/ml). Diluted supernatants were then added to BMM.PHI., that
had been primed with IFN.gamma. (100 U/ml) for 8 hours, and
immediately treated with LPS. After 24 hours the supernatant was
harvested and IL-12(p70) levels were determined by ELISA using mAbs
C 18.2 (anti-murine IL-12 p35) and C 17.15 (biotinylated antimurine
IL-12 p40) as ELISA capture and detection antibodies, respectively,
according to protocols provided by PharMingen (San Diego, Calif.).
Recombinant murine IL-12 (Genzyme Corp.) was used as a standard.
mAbs C18.2 and C17.15 were purified from ascitic fluid (The Wistar
Institute, Philadelphia, Pa.).
[0124] B. Results
[0125] The ELISA results are set forth in FIG. 5. Values represent
the mean of three independent experiments, each performed in
triplicate, .+-.SE. The supernatants from
LPS/Fc.gamma.RI-stimulated BMM.PHI. reduced IL-12 (p70) secretion
to near-background levels. Treating these inhibitory supernatants
with a neutralizing mAb to IL-10 partially restored IL-12(p70)
production. These results indicate that the IL-10 that is produced
by macrophages following LPS/Fc.gamma.R-stimulation is adequate to
inhibit the production of IL-12 by IFN.gamma.-primed
macrophages.
Example 5
Modulation of Macrophage Proinflammatory Responses by IgG-Opsonized
LPS Ligation of Fc.gamma. Receptors
[0126] Cytokine production by macrophages in response to potential
proinflammatory stimuli was examined following Fc.gamma.R
ligation.
[0127] A. Macrophage Stimulation and Cytokine ELISA
[0128] BMM.PHI. were exposed to either media, LPS, or IgG-LPS.
After 24 hours, the supernatant was harvested, and IL-10 and
IL-12(p40) levels were determined by ELISA. Murine IL-10 levels
were determined, as above, with a mouse IL-10 ELISA kit (Genzyme or
Biosource International). Murine IL-12(p40) levels were measured
with a mouse IL-12 ELISA kit (Biosource International) according to
the manufacturer's instructions.
[0129] B. Results
[0130] The ELISA results are set forth in FIGS. 6A and 6B.
Determinations were performed in triplicate, and values are
expressed as the means.+-.SD. Results are representative of four
separate experiments. As expected, LPS induced a potent
proinflammatory response by macrophages characterized by moderate
levels of IL-10 (FIG. 6A) and high levels of IL-12(p40) (FIG. 6B).
In contrast to this, IgG-opsonized LPS induced a different cytokine
response, characterized by higher levels of IL-10 (FIG. 6A) and
only modest levels of IL-12(p40) (FIG. 6B).
Example 6
Modulation of Macrophage Proinflammatory Responses by IgG-Opsonized
LPS Ligation of Fc.gamma. Receptors
[0131] Similar studies were performed using the Gram-negative
bacterium H. influenzae. Cytokine production by macrophages in
response to stimulation with unopsonized or IgG-opsonized H
influenza was examined following Fc.gamma.R ligation.
[0132] A. Onsonization of Heat-Killed Bacteria
[0133] The Eagan clinical isolate of type-b Haemophilus influenzae
has been previously described and characterized (Noel et al., J.
Inf. Dis. 166:178-182, 1992). Organisms were grown for 3 hours at
37.degree. C. in brain-heart infusion broth (Difco, Detroit, Mich.)
supplemented with NAD and hemin and then washed twice in HBSS.
Bacteria were heat killed by incubating at 60.degree. C. for 15
minutes. Bacteria were opsonized by incubation with anti-H.
influenzae poly-serotype antiserum (Difco) at a 1:25 dilution for
15 minutes at room temperature.
[0134] B. Macrophage Stimulation and Cytokine ELISA
[0135] BMM.PHI. were incubated with media alone or with equal
numbers (130 bacteria per macrophage) of either unopsonized or
opsonized heat-killed H. influenzae. After 24 hours, the
supernatant was harvested, and IL-10 and IL-12(p40) levels were
determined by ELISA.
[0136] C. Results
[0137] The ELISA results are set forth in FIGS. 7A (IL-10) and 7B
(IL-12(p40)). Determinations were performed in triplicate, and
values are expressed as the means .+-.SD. Results are
representative of three separate experiments. Unopsonized H.
influenzae induced the production of relatively high levels of both
IL-10 (FIG. 7A) and L-12(p40) (FIG. 7B). IgG-opsonized bacteria,
however, induced a significant decrease in the production of
IL-12(p40) protein, and an increase in the production of IL-10.
Thus, in both the sheep erythrocyte and bacteria in vitro models,
the ligation of Fc.gamma.R by opsonization with IgG resulted in a
reduction in macrophage proinflammatory responses.
Example 7
Modulation of In Vivo Responses to Bacterial Endotoxin
[0138] Studies similar to the in vitro studies performed above were
repeated in experimental animals. These studies were performed in
RAG-1.sup.-/- mice, since recent studies have demonstrated that
normal mice have naturally occurring antibodies to LPS (Reid et
al., J. Immunol. 159:970-975, 1997). IgG opsonization of LPS
reversed the inflammatory cytokine response to LPS in vivo.
[0139] A. In Vivo Challenge with IgG-Opsonized LPS and Cvtokine
ELISA
[0140] RAG-1.sup.-/- mice (The Jackson Laboratory, Bar Harbor, Me.)
received either IgG-LPS or LPS intravenously (tail vein) at a final
LPS dose of 4 .mu.g per mouse. Control LPS was incubated with an
equal volume of HBSS. Mice were bled by retroorbital puncture at
the time intervals up to 24 hours indicated in FIGS. 8A and 8B, and
serum cytokine levels were determined by ELISA. In another
experiment, serum was assayed for cytokines by ELISA at 2, 4 and 8
hours.
[0141] B. Results
[0142] The ELISA results of the 24-hour study are set forth in
FIGS. 8A and 8B. Data show the mean.+-.SD of groups of four
separately handled mice. *P<0.01, and ***P<0.08 (significant
by Rank-Sum Analysis) versus the LPS-treated group as determined by
the Student's t test. The ELISA results of the 8-hour study are set
forth in FIGS. 9A and 9B. Data show the mean.+-.SD of groups of
five separately handled mice. The injection of low level (4 .mu.g)
of LPS into the RAG-1.sup.-/- mice induced the transient production
of relatively high levels of serum IL-12(p40) (FIGS. 8A, 9A), and
only modest levels of IL-10 (FIGS. 8B, 9B). The observation that
RAG-1.sup.-/- mice make high amounts of IL-12 in response to low
levels of LPS is consistent with previous observations that
antibody-deficient mice are hypersusceptible to LPS (Reid et al.,
J. Immunol. 159:970-975, 1997). The injection of IgG-opsonized LPS
into these mice induced an alteration in cytokine profile.
RAG-1.sup.-/- mice injected with IgG-LPS made only modest levels of
IL-12, but they more than doubled their production of IL-10. This
reciprocal alteration in the pattern of cytokine production
suggests that IgG opsonization of LPS not only increases the rate
of LPS clearance through Fc.gamma.R, but in doing so also mediates
a desirable effect by dampening the proinflammatory response of
IL-12 production.
Example 8
Passive Immunization with Anti-LPS Antibody
[0143] Mice were injected intraperitoneally with 100 .mu.l of
anti-LPS antibody (Calbiochem) 2 or 18 hours before being injected
intravenously with LPS (4 .mu.g, E. coli 0128:B12). Control
uninjected mice received LPS alone. Serum was collected at 2, 4,
and 8 hours post LPS injection and assayed for IL-12(p40)
production as in FIG. 10A (solid circles, IL-12 for mice injected
with anti-LPS two hours before LPS injection; solid triangles,
IL-12 for mice injected with anti-LPS eighteen hours before LPS
injection; open circles, IL-12 for control mice receiving LPS
alone). IL-10 production in serum collected 2 hours post LPS
injection is shown in FIG. 10B. Symbols in the figures represent
mean serum cytokine levels from 5 mice .+-.SD.
[0144] The data indicate that the prophylactic administration of
antibody before endotoxemia can prevent the production of
inflammatory cytokines.
[0145] All references cited herein are incorporated by
reference.
[0146] The present invention may be embodied in other specific
forms without departing from the spirit or essential attributes
thereof and, accordingly, reference should be made to the appended
claims, rather than to the foregoing specification, as indication
the scope of the invention.
* * * * *