WO2007094776A1

WO2007094776A1 - Compositions and methods for suppressing fibrocyte differentiation

Info

Publication number: WO2007094776A1
Application number: PCT/US2006/005229
Authority: WO
Inventors: Richard Gomer; Darrell Pilling
Original assignee: William Marsh Rice University
Current assignee: William Marsh Rice University
Priority date: 2006-02-15
Filing date: 2006-02-15
Publication date: 2007-08-23
Anticipated expiration: 2008-08-15

Abstract

The present invention relates to the ability of anti-FcγR antibodies and aggregated or cross-linked IgG to suppress fibrocytes differentiation. Methods and compositions for suppressing fibrocyte differentiation using these antibodies are provided. These methods are useful in a variety of applications including treatment and prevention of conditions resulting from fibrosis in the kidney, liver, lung, heart, eye, uterus, tumors, and wounds.

Description

COMPOSITIONS AND METHODS FOR SUPPRESSING FIBROCYTE

DIFFERENTIATION

FIELD OF THE 13SfVENTION

The present invention relates to the ability of anti-FcγR antibodies and cross-linked IgG to suppress differentiation of fibrocytes. Accordingly, it may include compositions and methods for suppressing such differentiation. These compositions and methods may be useful in a variety of applications in which decreased fibrocyte formation is beneficial, such as treatment of fibrosing diseases and asthma. BACKGROUND Fibrocytes

Inflammation is the coordinated response to tissue injury or infection. The initiating events are mediated by local release of chemotactic factors, platelet activation, and initiations of the coagulation and complement pathways. These events stimulate the local endothelium, promoting the extravasation of neutrophils and monocytes. The second phase of inflammation is characterized by the influx into the tissue of cells of the adaptive immune system, including lymphocytes. The subsequent resolution phase, when apoptosis of the excess leukocytes and engulfment by tissue macrophages takes place, is also characterized by repair of tissue damage by stromal cells, such as fibroblasts.

In chronic inflammation, the resolution of inflammatory lesions is disordered, with the maintenance of inflammatory cells, fibroblast hyperplasia, and eventual tissue destruction. The mechanisms that lead to these events are complex, but include enhanced recruitment, survival and retention of calls and impaired emigration.

The source of fibroblasts responsible for repair of wound lesions or in other fibrotic responses is controversial. The conventional hypothesis suggests that local quiescent fibroblasts migrate into the affected area, produce extracellular matrix proteins, and promote wound contraction or fibrosis. An alternative hypothesis is that circulating fibroblast precursors (called fibrocytes) present within the blood migrate to the sites of injury or fibrosis, where they differentiate and mediate tissue repair and other fibrotic responses .

Fibrocytes are fibroblast-like cells that appear to participate in wound healing and are present in pathological lesions associated with asthma, pulmonary fibrosis and scleroderma. Fibrocytes are known to differentiate from a CD14+ peripheral blood monocyte precursor population. Fibrocytes may also differentiate from other sources. Fibrocytes express markers of both hematopoietic cells (CD45, MHC class II, CD34) and stromal cells (collagen types I and III and fibronectin) . Fibrocytes at sites of tissue injury secrete inflammatory cytokines, extracellular matrix proteins and promote angiogenesis and wound contraction. Fibrocytes are also associated with the formation of fibrotic lesions after infection or inflammation, and are implicated in fibrosis associated with autoimmune diseases. Fibrocytes are associated with a variety of processes and diseases including scleroderma, keloid scarring, rheumatoid arthritis, lupus, nephrogenic fibrosing dermopathy, and idiopathic pulmonary fibrosis. They play a role in the formation of fibrotic lesions after Schistosoma japonicum infection in mice and are also implicated in fibrosis associated . with autoimmune diseases. Fibrocytes have also been implicated in pathogenic fibrosis associated with radiation damage, Lyme disease and pulmonary fibrosis. CD34+ fibrocytes have also been associated with stromal remodeling in pancreatitis and stromal fibrosis, whereas lack of such fibrocytes is associated with pancreatic tumors and adenocarcinomas. Fibrosis additionally occurs in asthma patients and possibly other pulmonary diseases such as chronic obstructive pulmonary disease when fibrocytes undergo further differentiation into myofibroblasts. Fibrocytes may also play a role in a variety of conditions, likely even some in which fibrocyte formation is not currently known. Some additional conditions may include congestive heart failure and other post-ischemic conditions, such as cardiac fibrosis, post-surgical scarring including abdominal adhesions, corneal refraction surgery, and wide angle glaucoma trabeculotomy.

Fibrocytes are important in the formation of tumors, particularly stromal tissue in tumors. Recent evidence also suggests that fibrocytes may further differentiate into adipocytes and thus play a role in obesity.

It has been previously identified that fibrocytes may differentiate from CD14+ peripheral blood monocytes, and the presence of human serum dramatically delays this process. The factor in human serum that inhibits fibrocyte differentiation is serum amyloid P (SAP) . SAP, a member of the pentraxin family of , proteins that includes C-reactive protein (CRP) , is produced by the liver, secreted into the blood, and circulates in the blood as stable pentamers . SAP binds to receptors for the Fc portion of IgG antibodies (FCYR) on a variety of cells and may effectively crosslink FcγR without additional proteins because SAP is a pentameric protein with five potential FcγR binding sites per molecule. As SAP binds to FcγR, intracellular signaling events consistent with FcγR activation are initiated.

Anti-FcγR Antibodies

Anti-FcγR antibodies are IgG antibodies that bind to receptors for the Fc portion of IgG antibodies (FcγR) . The anti-FcγR antibodies bind through their variable region, and not through their constant (Fc) region. However, IgG from the appropriate source (e.g. human IgG for human receptors) may normally bind to FcγR through its Fc region. FcγR are found on the surface of a variety of hematopoietic cells. There are four distinct classes of FcγR. FcγRI (CD64) is expressed by peripheral blood monocytes and binds monomeric IgG with a high affinity. FcγRII (CD32) and FcγRIII (CD16) are low affinity receptors for IgG and only efficiently bind aggregated IgG. FcγRII is expressed by peripheral blood B cells and monocytes, whereas FcγRIII is expressed by NK cells and a subpopulation of monocytes. FcγRIV was recently identified in mice and is present on murine peripheral blood monocytes and neutrophils, macrophages and dendritic cells and efficiently binds murine IgG2a and IgG2b antibodies. There is a putative human FcγRIV gene, but the biological function of the protein, such as ligand specificity and cellular expression is, as yet unknown.

Peripheral blood monocytes express both FcγRI and FcγRII (a subpopulation of monocytes express FcγRIII) , whereas tissue macrophages express all three classical FcγR. Clustering of FcγR on monocytes by IgG, either bound to pathogens or as part of an immune complex, initiates a wide variety of biochemical events.

FcγR activation and induction of intracellular signaling pathways may occur when multiple FcγR are cross-linked or aggregated. This FcγR activation leads to a cascade of signaling events initiated by two main kinases. The initial events following FcγR activation involve the phosphorylation of intracellular immunoreceptor tyrosine activation motifs (ITAMs) present on the cytoplasmic tail of FcγRII or the FcR-γ chain associated with FcγRI and FcγRIII, by Src-related tyrosine kinases (SRTK) . In monocytes, the main Src- kinases associated with FcγRI and FcγRII are hck and lyn. The phosphorylated ITAM then recruit cytoplasmic SH2 -containing kinases, especially Syk, to the ITAMs and Syk then activates a series of downstream signaling molecules .

Anti-FcγR antibodies for FcγRI (anti-FcγRI) and for FcγRII (anti-FcγRII) are able to bind to either

FcγRI or FcγRII, respectively. These FcγR may then be cross-linked by the binding of additional antibodies or other means. This process initiates intracellular signaling events consistent with FcγR activation.

Scleroderma

Scleroderma is a non-inherited, noninfectious disease that has a range of symptoms. It involves the formation of scar tissue containing fibroblasts in the skin and internal organs . The origin of the fibroblasts is unknown. In mild or early cases of scleroderma, there is a hardening of the skin, fatigue, aches and sensitivity to cold. In more severe and later stages, there is high blood pressure, skin ulcers, difficulty moving joints, and death from lung scarring or kidney failure. Approximately 300,000 people in the U.S. have scleroderma. The disease has similarities to lupus and rheumatoid arthritis. There is no cure or significant treatment for scleroderma and even diagnosis is difficult because there is no clinical test.

Nephrogenic fibrosing dermopathy

Nephrogenic fibrosing dermopathy (NFD) is a newly recognized scleroderma-like fibrosing skin condition. It develops in patients with renal insufficiency. Yellow scleral plaques and circulating antiphospholipid antibodies have been proposed as markers of NFD. Dual immunohistochemical staining for CD34 and pro-collagen in the spindle cells of NFD suggest that the dermal cells of NFD may represent circulating fibrocytes recruited to the dermis. Therefore, inhibition of fibrocyte formation may alleviate symptoms of this disease.

Asthma

Asthma affects more than 100 million people worldwide, and its prevalence is increasing. Asthma appears to be caused by chronic airway inflammation. One of the most destructive aspects of asthma is ^'remodeling of the airways in response to chronic inflammation. This remodeling involves thickening of the lamina reticularis (the subepithelial reticular basement membrane surrounding airways) due to fibrosis. The airway passages then become constricted due to the thickened airway walls.

The thickened lamina reticularis in asthma patients contains abnormally high levels of extracellular matrix proteins such as collagen I, collagen III, collagen V, fibronectin and tenascin. The source of these proteins appears to be a specialized type of fibroblast called myofibroblasts. In asthma patients, CD34+/collagen 1+ fibrocytes accumulate near the basement membrane of the bronchial mucosa within 4 hours of allergen exposure. 24 hours after allergen exposure, labeled monocytes/fibrocytes have been observed to express α-smooth muscle actin, a marker for myofibroblasts. These observations suggest that in asthma patients allergen exposure causes fibrocytes from the blood to enter the bronchial mucosa, differentiate into myofibroblasts, and then cause airway wall thickening and obstruct the airways. Further, there is a correlation between having a mutation in the regulatory regions of the genes encoding monocyte chemoattractant protein 1 or TGFβ-1 and the severity of asthma. This also suggests that recruitment of monocytes and appearance of myofibroblasts lead to complications of asthma.

Thickening of the lamina reticularis distinguishes asthma from chronic bronchitis or chronic obstructive pulmonary disease and is found even when asthma is controlled with conventional medications. An increased extent of airway wall thickening is associated with severe asthma. No medications or treatments have been found to reduce thickening of the lamina reticularis. However, it appears likely that reducing the number of myofibroblasts found in the airway walls may reduce thickening or help prevent further thickening.

Idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a unique type of chronic fibrosing lung disease of unknown etiology. The sequence of the pathogenic mechanisms is unknown, but the disease is characterized by epithelial injury and activation, the formation of distinctive subepithelial fibroblast/myofibroblast foci, and excessive extracellular matrix accumulation. These pathological processes usually lead to progressive and irreversible changes in the lung architecture, resulting in progressive respiratory insufficiency and an almost universally terminal outcome in a relatively short period of time. While research has largely focused on inflammatory mechanisms for initiating the fibrotic response, recent evidence strongly suggests that disruption of the alveolar epithelium is an underlying pathogenic event. Given the role played by fibrocytes in wound healing and their known role in airway wall thickening in asthma, it appears likely that overproduction of fibrocytes may be implicated in IPF. SUMMARY

The present invention may include compositions and methods for suppressing fibrocyte differentiation. It may particularly relate to suppressing fibrocyte differentiation from monocytes.

In selected embodiments, fibrocyte differentiation in a target location may be suppressed by providing anti-FcγRI antibodies and/or anti-FcγRII antibodies that are able to cross-link FoγR. The target location may be located in vitro or in vivo. Specifically, the target location may be located in a mammal, such as a human patient.

Jn vivo, the target location may include an entire organism or a portion thereof and the composition may be administered systemically or it may be confined to a particular area, such as an organ or tissue. A decrease in or suppression of differentiation of fibrocytes may alleviate symptoms of numerous fibrosing diseases or other disorders caused by fibrosis. In a specific embodiment, administration of anti-FcγR antibodies may be used to treat the effects of unwanted differentiation of fibrocytes. For example, it may be used to treat fibrosis in the kidney, liver, lung, heart, eye, uterus, tumors, and wounds.

BRIEF DESCRIPTION OF THE DRAWINGS The following figures form part of the present specification and are included to further demonstrate certain aspects of the present invention.

FIGURE 1 shows the effects of cross-linked and non-cross-linked anti-FcγR antibodies on fibrocyte differentiation from Peripheral Blood Mononuclear Cells (PBMC) . PBMC at 2.5 x 10⁵ cells per ml were cultured in serum-free medium for 5 days in the presence or absence of 1 μg/ml of the indicated F(ab')2 anti-FcγR or control IgGl antibodies, in the presence (black bars) or absence (white bars) of 500 ng/ml goat F(ab')2 anti-mouse IgG, which cross-links the F(ab')2- Cells were then air-dried, fixed, stained, and fibrocytes were enumerated by morphology. FIGURE 2 shows the effects of SRTK and Syk inhibitors on the ability of anti-FcγR antibodies on fibrocyte differentiation from PBMC. PBMC were incubated for 60 minutes at 4°C with 10 nM PP2 , PP3 , or Syk inhibitor. PBMC at 2.5 x 10⁵ cells per ml were then cultured in serum-free medium for 5 days in the presence or absence of 1 μg/ml of the indicated murine F(ab')₂ anti-FcγR antibodies, in the presence or absence of 500 ng/ml goat F(ab')2 anti-mouse IgG. Results are expressed as the mean ± SD of the number of fibrocytes per 2.5 x 10⁵ cells (one of two separate donors) .

FIGURE 3 shows the effects of FcyR aggregation and the effects of SRTK and Syk on fibrocyte differentiation from monocytes. PBMC were at 2.5 x 10⁵ cells per ml were incubated for 60 minutes at 37°C. Non-adherent cells were then removed by pipetting, resulting in a substantially monocyte cell sample. The adherent monocytes were incubated for 60 minutes at 4⁰C in the presence or absence of 10 nM PP2 , PP3 or Syk inhibitor. Monocytes were then washed twice and cultured in the presence or absence of heat-aggregated human IgG for 60 minutes at 4⁰C. This IgG was not an anti-FcγR IgG, but instead was able to bind through its Fc region. The monocytes were then washed twice, and the non-adherent cells were replaced to a final concentration of 2.5 x 10⁵ cells per ml and then cultured for 5 days at 37°C in serum-free medium. Results are expressed as the mean ± SEM of the number of fibrocytes per 2.5 x 10⁵ cells (n=3 separate donors.

FIGURE 4 shows the effects of cross-linking IgG and other antibody isotypes and on fibrocyte differentiation. In FIGURE 4A, PBMC were incubated with the indicated concentrations of monomeric human IgG for 60 seconds. PBMC were then washed and incubated in the presence (white boxes) or absence (black boxes) of 500 ng/ml goat F(ab^')2 anti-human IgG. PBMC were then cultured at 2.5 x 10⁵ cells per ml in serum-free medium for 5 days. PBMC were then air- dried, fixed, stained, and fibrocytes were enumerated by morphology. Results are expressed as the mean ± SEM of number of fibrocytes per 2.5 x 10^Ξ cells (n=4 separate donors) .

In FIGURE 4B, PBMC were cultured as in FIGURE 4A in the presence of the indicated concentrations of heat-aggregated human IgG or heat-aggregated human F(ab^')2- Results are expressed as the + SEM of number of fibrocytes per 2.5 x 10^s cells (n=3 separate donors) .

In FIGURE 4C, PBMC were cultured as in Figure 4A in the presence of 20 μg/ml of native or heat- aggregated human IgA, IgE, IgG or IgM.

DETAILED DESCRIPTION

The regulation of events leading to fibrosis involves the proliferation and differentiation of fibrocytes. Fibrocytes are a distinct population of fibroblast-like cells derived from peripheral blood monocytes that normally enter sites of tissue injury to promote angiogenesis and wound healing. Fibrocytes differentiate from CD14+ peripheral blood monocytes, and may differentiate from other PBMC cells. The presence of anti-FcγRI and anti-FcγRII antibodies may inhibit or at least partially delay this process.

Anti-FcγR antibodies are IgG antibodies that bind to specifically to FcγR through their F(ab) (variable) region. Compositions containing anti-FcγRI antibodies and/or anti-FcγRII antibodies, and/or cross-linked or aggregated IgG, which may bind to FcγR through the Fc region, may be used to suppress the differentiation of fibrocytes in inappropriate locations and in fibrosing disorders and chronic inflammatory conditions, inter alia.

Compositions may be applied locally or systemically. In specific embodiments, compositions containing approximately 1 μg/ml anti-FcγR antibodies may be effective to inhibit fibrocyte differentiation by approximately 50%. In other embodiments, compositions may contain an amount sufficient to deliver 1 μg/ml anti-FcγR antibodies to the target tissue. In other specific embodiments, compositions may contain as little as 0.1 μg ml cross-linked or aggregated IgG.

Anti-FcγR antibodies used in examples of the present disclosure include anti-FcγRI antibodies and anti-FcγRII antibodies. Cross-linked or aggregated IgG may include any IgG able to bind the target FcγR through its Fc region, provided that at least two such IgG antibodies are physically connected to one another. Antibodies of both types may include whole antibodies or a portion thereof, preferably the portion functional in suppression of fibrocyte differentiation. For example, they may include any antibody portion able to cross-link FcγR. This may include aggregated or cross-linked antibodies or fragments thereof, such as aggregated or cross-linked whole antibodies, F(ab')2 fragments, and possible even Fc fragments.

Aggregation or cross-linking of antibodies may be accomplished by any known method, such as heat or chemical aggregation. Any level of aggregation or cross-linking may be sufficient, although increased aggregation may result in increased fibrocyte suppression. Antibodies may be polyclonal or monoclonal, such as antibodies produced from hybridoma cells. Compositions and methods may employ mixtures of antibodies, such as mixtures of multiple monoclonal antibodies, which may be cross-linked or aggregated to like or different antibodies. Anti-FcγR antibodies may include any isotype of antibody.

The compositions of the present invention may be supplied to a target location from an exogenous source, or they may be made in vivo by cells in the target location or cells in the same organism as the target location.

Compositions of the present invention may be in any physiologically appropriate formulation. They may be administered to an organism by injection, topically, by inhalation, orally or by any other effective means. The same compositions and methodologies described above to suppress differentiation of fibrocytes may also be used to treat or prevent conditions resulting from inappropriate fibrocyte differentiation. For example, they may treat or prevent a condition occurring in the kidney, liver, lung, heart, eye, uterus, a tumor, or a wound. Specifically, they may treat or prevent fibrosis resulting from conditions including but not limited to: scleroderma, keloid scarring, rheumatoid arthritis, lupus, nephrogenic fibrosing dermopathy, renal interstitial fibrosis, such as that resulting from immune complex disease, FSGS, HIV nephritis, or Lupus nephritis, fibrotic lesions such as those formed after Schistosoma japonicum infection, autoimmune diseases, pathogenic fibrosis, Lyme disease, stromal remodeling in pancreatitis and stromal fibrosis, asthma, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, pulmonary fibrosis, post trabeculectomy fibrosis, neonatal bronchopulmonary dysplasia, diabetic nephropathy, uterine fibroids, ovarian fibrosis, other fibrocystic formations, corneal fibrosis or other eye fibrosis, such as that resulting from corneal refraction surgery, fibrosis resulting from congestive heart failure and other post-ischemic conditions, such as cardiac fibrosis, post-surgical scarring including abdominal adhesions, wide angle glaucoma trabeculotomy, congestive cardiomyopathy, post-myocardial infarction scarring, restenosis, and fibrosis in tumors, such as formation of stromal tissue. In some such fibrosing diseases fibrocytes may not represent an end-stage of fibrosis. For example, in asthma, fibrocytes further differentiate into myofibroblasts, which persist in thickened airway walls. In patients with obesity, fibrocytes further differentiate into adipocytes and thus cause or exacerbate the condiction.

The invention also includes a method of inhibiting or suppressing fibrocyte differentiation or treating or preventing a condition by cross-linking FcyRI and/or FcγRII through the use of anti-FcγR antibodies and/or through aggregated or cross-linked IgG.

Anti-FcγR antibodies may be administered in a dose of approximately 1.0 μg/mL, in an amount sufficient to deliver 1 μg/ml anti-FcγR antibodies to the target tissue, or in another dose sufficient to inhibit fibrocyte differentiation without causing an undesirable amount of cell death in the patient. Aggregated or cross-linked IgG may be administered in an amount sufficient to deliver at least 0.1 μg/ml IgG to the target tissue, or in another dose sufficient to inhibit fibrocyte differentiation without causing an undesirable amount of cell death in the patient.

In a particular embodiment, pulmonary fibrosis or other pulmonary fibrosing diseases may be treated by administration of anti-FcγR antibodies and/or aggregated or cross-linked IgG. Treatment may reduce cellular growth associated with fibrosis and also collagen deposition. Treatment may prevent further fibrosis or reduce the effects of current fibrosis.

The following examples are included to demonstrate specific embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. EXAMPLES EXAMPLE 1 : FIBROCYTE DIFFERENTIATION ASSAY

Human peripheral blood mononuclear cells (PBMC) were isolated from buffy coats (Gulf Coast Regional Blood Center, Houston, Texas) by Ficoll-Paque Plus (Amersham Biosciences, Piscataway, NJ) . Cells were incubated in serum-free medium (SFM) , which consists of RPMI (Invitrogen, Carlsbad, CA) supplemented with 10 mM HEPES (Invitrogen) , 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 1 x ITS-3 (500 μg/ml bovine serum albumin, 10 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml sodium selenite, 5 μg/ml linoleic acid, and 5 μg/ml oleic acid; Sigma-Aldrich, St. Louis, MO) . Normal human serum (Sigma-Aldrich) was added at 1%. PBMC were cultured in flat-bottomed 96 well tissue culture plates (Type 353072, BD Biosciences Discovery Labware, Bedford, MA) in 200 μl volumes at 2.5 x 10⁵ cells per ml in a humidified incubator containing 5% CO₂ at 37°C for 5 days. Fibrocytes were identified by morphology in viable cultures as adherent cells with an elongated spindle-shaped morphology as distinct from lymphocytes or adherent monocytes. Enumeration of fibrocytes was performed on cells cultured for 5 days. Cells were air dried, fixed in methanol and stained with eosin and methylene blue (Hetna 3 Stain, Fisher Scientific, Hampton, NH) . Fibrocytes from duplicate wells were counted in five different 900 μm diameter fields per well, using the above criteria of an elongated spindle-shape and the presence of an oval nucleus. All cultures were counted by at least two independent observers . The number of fibrocytes observed was 1.2 ± 0.6 x 10⁴ (mean ± SD, n = 12 healthy individuals) fibrocytes per ml of peripheral blood, with a range of 3.7 x 10³ to 2.9 x 10⁴ fibrocytes per ml . These results indicate that fibrocyte precursors account for approximately 1% of the total peripheral blood mononuclear cells.

EXAMPLE 2: ANTIBODIES, PROTEINS, AND INHIBITORS

Human IgA, IgG, IgM, and IgG F(ab')2 fragments were from Jackson ImmunoResearch Laboratories, West Grove, PA. Goat F(ab')₂ anti-human IgG, goat F(ab')2 anti-murine IgG, goat F(ab')2 anti-rabbit IgG, and whole mouse IgGl, whole mouse IgG2a and mouse F(ab')₂ IgGl isotype control antibodies were from Southern Biotechnology Associates Inc., Birmingham, AL. Sheep red blood cells (SRBC) and rabbit anti-SRBC were from ICN, Irvine, CA. F(ab')2 fragments of the blocking monoclonal antibodies to FcγRI (clone 10.1, IgGl isotype) and FcγRII (clone 7.3, IgGl isotype) were from Ancell, Bayport, MN. The following primary monoclonal antibodies were used for immunohistochemistry : anti- CD14 (clone M5E2 , IgG2a, BD-Biosciences, San Diego, CA), anti-CD34 (clone QBendlO, IgGl, GeneTex, San Antonio, TX), CD 43 (clone IGlO, IgGl, BD), pan-CD45 (clone H130, IgGl, BD), anti-prolyl 4-hydrolase (clone 5B5, IgGl, Dako, Carpinteria, CA) , and anti-alpha smooth muscle actin (clone 1A4, IgG2a, Sigma-Aldrich, St. Louis, MO) . Collagen-I was detected using an affinity-purified rabbit polyclonal antibody from Rockland, Gilbertsville, PA. PP2 (AG 1879; 4-Amino-5- (4-chlorophenyl) -7- (t-butyl) pyrazolo [3 , 4-d] pyrimidine) , PP3 4-Amino-7-phenylpyrazol [3, 4-d] pyrimidine) and the Syk inhibitor (3- (1-Methyl-lH-indol-3 -yl-methylene) -2- oxo-2 , 3-dihydro-lH-indole-5-sulfonamide) were from Calbiochem, EMD Biosciences, San Diego, CA.

EXAMPLE 3: INHIBITION OF FIBROCYTE DIFFERENTIATION

To determine if anti-FcγR antibodies activate FoγR to inhibit fibrocyte differentiation, PBMC at 2.5 x 10⁵ per ml were cultured in serum-free medium for 5 days in the presence or absence of 1 μg/ml of free or cross- linked F(ab')₂ antibodies to FcγRI or FcyRII. ±a

To crosslink individual Fc:γR, PBMC were incubated for 30 minutes at 4°C with 1 μg/ml F(ab')₂ anti-FcγRI or F(ab')2 anti-FcγRII, and receptors were then cross- linked by the addition of 500 ng/tnl F(ab')₂ goat anti- mouse IgG for 30 minutes at 4°C . PBMC were then warmed to 37°C and cultured for 5 days.

After the cells were cultured in the presence or absence of free or cross-linked F(ab')2 antibodies to FcγRI or FcγRII, the cells were then air-dried, fixed, stained, and fibrocytes were enumerated by morphology. The results of this example are shown in Figure 1.

Compared to PBMC cultured in the presence of 500 ng/ml goat F(ab')₂ anti-mouse IgG alone, cells cultured in the presence of 500 ng/ml goat F(ab')₂ anti-mouse IgG and anti-FcγRI or anti-FcγRII significantly inhibited fibrocyte differentiation (p<0.01, indicated by **) , as determined by ANOVA (n=3 separate donors) . These results suggest that cross-linking either FcγRI or FcγRII alone significantly inhibited fibrocyte differentiation. However, there was no additional inhibition when both receptors were cross-linked together, suggesting that no synergistic interaction occurs (Figure 1) . These experiments show that fibrocyte differentiation can be inhibited to approximately 50% by the addition of 1 μg/ml anti-FcγR. Greater inhibition could be achieved by incubating PBMC with higher concentrations of anti-FcγR (5 and 10 μg/ml) , however these concentrations of anti-FcγR also led to significant cell death (data not shown) . These results suggest that ligation and cross-linking of FcγRI or FcγRII can inhibit fibrocyte differentiation. Although PBMC may contain various cells that may form fibrocytes, including monocytes, monocyte cultures alone may also differentiate to form fibrocytes. This phenomena is show in Figure 3. Figure 3 also indicates that this fibrocyte differntiation is suppressed by the aggregation of FcγR. Specifically, compared to monocytes cultured in serum free medium (SFM) , aggregated human IgG was able to cross-link FcγR through its Fc region significantly inhibited fibrocyte differentiation (p<0.01), as determined by ANOVA.

EXAMPLE 4: INHIBITION OF FIBROCYTE DIFFERENTIATION IS SYK- AND SRC KINASE DEPENDENT

FcγR activation leads to a cascade of signaling events initiated by two main kinases. The initial events following FcγR aggregation involve the phosphorylation of intracellular immunoreceptor tyrosine activation motifs (ITAM) present on the cytoplasmic tail of FcγRII or the FcRγ chain associated with FcγRI, by src-related tyrosine kinases (SRTK) . In monocytes, the main src-kinases associated with FcγRI and FcγRII are hck and lyn. The phosphorylated ITAM then recruits cytoplasmic SH2-containing kinases, especially Syk, to the ITAMs and Syk then activates a series of downstream signaling molecules.

To determine the roles of SRTK and Syk in the regulation of fibrocyte differentiation, PBMC were pre- incubated with the specific SRTK inhibitor PP2, PP3 as a control for PP2, or the specific Syk inhibitor 3- (1- methyl -lH-indol -3-yl -methylene) -2 -oxo-2 , 3 -dihydro-lH- indole-5-sulfonamide, before the addition of anti-FcγR antibodies. This Syk inhibitor was used instead of the standard Syk inhibitor piceatannol, as piceatannol at concentrations used to inhibit Syk in whole cells (10 μM) also inhibits a variety of other enzymes and transcription factors. These proteins include the catalytic subunit of protein kinase A, protein kinase C, myosin light chain kinase, TNF-induced NF-kB activation, and interferon α-mediated signaling via STAT proteins.

Inhibition of fibrocyte differentiation by activating either FcγRI or FcγRII alone or both receptors together was dependent on STRK and Syk, as the inhibition was lost when PBMC were pre-incubated with either PP2 or the Syk inhibitor (Figure 2) .

Compared to control cultures or cultures incubated with of 500 ng/ml goat F(ab')2 anti-mouse IgG (X-linker only), PBMC cultured with 500 ng/ml goat F(ab')₂ in addition to anti-mouse IgG anti-FcγRI, anti-FcγRII or both antibodies significantly inhibited fibrocyte differentiation (p<0.05), as determined by ANOVA. The presence of PP2 or Syk inhibitor, but not the control compound PP3 , inhibited this inhibition. These data suggest that anti-FcγR antibodies inhibit fibrocyte differentiation through a pathway involving both Syk and SRTK.

Similar results were found when monocyte samples, rather than PBMC were used to perform tests. Specifically, in Figure 3, compared to monocytes incubated with 10 μg/ml aggregated human IgG (able to bind to FcγR through its Fc region) , pre-incubation with PP2 (p<0.01) or Syk inhibitor (p<0.05) significantly inhibited the ability of IgG to inhibit fibrocyte differentiation as determined by ANOVA.

EXAMPLE 5: IgG IMMUNE COMPLEXES INHIBIT FIBROCYTE DIFFERENTIATION In addition to FcγR, monocytes express IgA receptors, low numbers of IgE receptors, and the recently characterized IgM receptor. To determine if other immunoglobins inhibit fibrocyte differentiation, native or heat-aggregated IgA, IgE, IgG or IgM were added to PBMC. The results of this example are shown in Figure 4C. Only heat-aggregated IgG, but not monomeric IgG or monomeric or heat-aggregated IgA, IgE or IgM, could inhibit fibrocyte differentiation. This suggests that ligation and cross-linking of FoγR receptors is an inhibitory signal for fibrocyte differentiation, but that ligation of the other immunoglobin receptors has no effect on fibrocyte differentiation .

EXAMPLE 6: CROSS-LINKED IgG INHIBITS FIBROCYTE DIFFERENTIATION

PBMC were incubated with the indicated various concentrations of monomeric human IgG for 60 seconds. PBMC were then washed and incubated in the presence or absence of 500 ng/ml goat F(ab^')2 anti-human IgG. PBMC were then cultured at 2.5 x 10⁵ cells per ml in serum- free medium for 5 days. PBMC were then air-dried, fixed, stained, and fibrocytes were enumerated by morphology. Results are shown in Figure 4A.

Specifically, compared to monomeric IgG, cross-linked human IgG clearly inhibited fibrocyte differentiation as compared to non-cross-linked IgG at 0.1 μg/ml . At 10 and 100 μg/ml inhibition of differentiation was significant (p = 0.03 and p = 0.003, respectively, as determined by Student's t test. Additional experiments using sheep red blood cells (SRBC) either opsinized or not opsonized with rabbit anti-SRBC IgG indicated that the opsonized SRBC significantly inhibited fibrocyte differentiation (p = 0.018) (data not shown) .

PBMC were also cultured as above in the presence of the indicated concentrations of heat-aggregated human IgG or heat-aggregated human F(ab^')₂. Results of this example are shown in Figure 4B. Compared to heat- aggregated human F(ab^')₂, heat-aggregated whole IgG significantly inhibited fibrocyte differentiation at concentrations of 25 μg/ml and higher, as determined by Student's t test.

Although only exemplary embodiments of the invention are specifically described above, it will be appreciated that modifications and variations of these examples are possible without departing from the spirit and intended scope of the invention.

Claims

1. A fibrocyte suppression composition comprising: an anti-FcγRI antibody able to inhibit fibrocyte differentiation; and a pharmaceutically acceptable carrier able to deliver the antibody to a target location in a mammal in a concentration sufficient to inhibit fibrocyte differentiation in the target location.

2. The composition of claim 1, wherein the anti- FcγRI antibody has a concentration of at least approximately 1.0 μg/ml .

3. The composition of claim 1, wherein the anti- FcγRI antibody comprises a monoclonal antibody.

4. The composition of claim 1, wherein the anti- FcγRI antibody comprises an antibody fragment.

5. The composition of claim 1, wherein the anti- FcγRI antibody comprises an aggregated or cross-linked antibody.

6. The composition of claim 1, wherein the pharmaceutically acceptable carrier is suitable for administration to the mammal by an injection.

7. A fibrocyte suppression composition comprising: an anti-FcγRII antibody able to inhibit fibrocyte differentiation; and a pharmaceutically acceptable carrier able to deliver the antibody to a target location in a mammal in a concentration sufficient to inhibit fibrocyte differentiation in the target location.

8. The composition of claim 7, wherein the anti- FcγRII antibody has a concentration of at least approximately 1.0 μg/ml .

9. The composition of claim 7, wherein the anti- FcyRII antibody comprises a monoclonal antibody.

10. The composition of claim 7, wherein the anti- FcγRII antibody comprises an antibody fragment.

11. The composition of claim 7, wherein the anti- FcγRII antibody comprises an aggregated or cross-linked antibody.

12. The composition of claim 7, wherein the pharmaceutically acceptable carrier is suitable for administration to the mammal by an injection.

13. A method of suppressing fibrocyte formation comprising administering an antibody selected from the group consisting of anti-FcγR antibody, an aggregated IgG antibody, a cross-linked IgG antibody or any combinations thereof to a target location in an amount and for a time sufficient to suppress fibrocyte differentiation in the target location.

14. The method of claim 13, wherein the anti-FcγR antibody comprises an anti-FcγRi antibody.

15. The method of claim 13, wherein the anti-FcγR antibody comprises an anti-FcγRII antibody.

16. The method of claim 13, wherein the amount of anti-FcγR antibody is at a concentration of at least approximately 1.0 μg/ml .

17. The method of claim 13, wherein the amount of IgG antibody is at a concentration of at least approximately 10 μg/ml.

18. The method of claim 13 , wherein the target location is in a mammal.

19. The method of claim 13, further comprising administering the antibody by intravenous injection.

20. The method of claim 13, wherein the mammal has a fibrosing disease.

21. The method of claim 13, wherein the mammal has a condition resulting from fibrosis in the kidney, liver, lung, heart, eye, uterus, tumors, and wounds.