B-cell inhibition by cross-linking CD79b is superior to B-cell depletion with anti-CD20 antibodies in treating murine collagen-induced arthritis.

Depletion of B cells with the anti-CD20 antibody rituximab is an established therapy for rheumatoid arthritis. However, rituximab has only moderate efficacy, most likely due to insufficient depletion of B cells in lymphoid organs and expansion of pathogenic B cells. We found that an antibody against mouse CD79b profoundly blocks B-cell proliferation induced via the B-cell receptor, CD40, CD180, and chondroitin sulfate, but not via TLR4 or TLR9. Treatment with anti-CD79b also induces death in resting and activated B cells. B-cell inhibition is mediated by cross-linkage of CD79b, but independent of Fc-receptor engagement. In the model of collagen-induced arthritis, an antibody against mouse CD20 depletes B cells very efficiently but fails to suppress the humoral immune response against collagen and the development of arthritis. In contrast, the antibody against CD79b, and a deglycosylated variant of this antibody, almost completely inhibits the increase in anti-collagen antibodies and the development of arthritis. In mice with established arthritis only the fully glycosylated antibody against CD79b is effective. Our data show that targeting B cells via CD79b is much more effective than B-cell depletion with anti-CD20 antibodies for therapy of arthritis. These findings may have important implications for treatment of B-cell-mediated autoimmune diseases.

Expression changes and novel interaction partners of talin 1 in effector cells of autoimmune uveitis.

Autoimmune uveitis is characterized by crossing of blood-retinal barrier (BRB) by autoaggressive immune cells. Equine recurrent uveitis (ERU) is a valuable spontaneous model for autoimmune uveitis and analyses of differentially expressed proteins in ERU unraveled changed protein clusters in target tissues and immune system. Healthy eyes are devoid of leukocytes. In ERU, however, leukocytes enter the inner eye and subsequently destroy it. Molecular mechanisms enabling cell migration through BRB still remain elusive. Previously, we detected decreased talin 1 expression in blood-derived granulocytes of ERU cases, linking the innate immune system to ERU. Because changes in leukocyte protein expression pattern may play a role in pathological abnormalities leading to migration ability, we aimed at identifying interactors of talin 1 in leukocytes with immunoprecipitation, followed by LC-MS/MS for candidate identification. This enabled us to identify CD90 (Thy1) as novel interactor of talin 1 besides several other interactors. In blood-derived granulocytes from healthy individuals, CD90 was highly abundant and significantly reduced in ERU, especially in effector cells. Connection between talin 1 and CD90 and their expression differences in inflammation is an interesting novel finding allowing deeper insight into immune response of innate immune system and granulocyte migration ability in this organ-specific autoimmune disease.

Miscellaneous vitreous-derived IgM antibodies target numerous retinal proteins in equine recurrent uveitis.

OBJECTIVE: In equine recurrent uveitis (ERU), immune reactions are directed toward known antigens like S-antigen, interphotoreceptor retinoid-binding protein, and cellular retinalaldehyde-binding protein, and anti-retinal antibodies were detected in vitreous samples. The aim of this study was the investigation of intraocular immunoglobulin M (IgM) reactivities to retinal proteome. PROCEDURES: Retina was separated by one- and two-dimensional gel electrophoresis and blotted semidry on PVDF membranes. To identify intraocular IgM antibody responses to retinal tissue, blots were incubated with vitreous samples of ERU-diseased horses (n = 50) and healthy controls (n = 30), followed by an HRP-labeled secondary antibody specific for equine IgM. Noticeable 2D western blot signals were aligned on a 2D gel of retinal proteome, excised, and subsequently identified by tandem mass spectrometry. RESULTS: Interestingly, frequent and very miscellaneous IgM response patterns to the retinal proteome in 68% of ERU vitreous samples were detected. Binding of IgM antibodies was localized at 17 different molecular weights. The most frequently detected signal, in 21 of the 50 samples, was located at 49 kDa. Comparing the samples interindividually between one and up to nine different signals in one sample could be observed. All healthy vitreous samples were devoid of IgM antibodies. Analysis of targeted spots with mass spectrometry led to the clear identification of 11 different proteins (corresponding to 16 different spots). One candidate could not be discovered so far. CONCLUSION: The considerable IgM response to retinal proteins demonstrates an ongoing immune response, which might contribute to the remitting relapsing character of ERU. Novel identified target proteins point to a diverse response pattern of individual ERU cases.

Comprehensive cardiac phenotyping in large animals: comparison of pressure-volume analysis and cardiac magnetic resonance imaging in pig post-myocardial infarction systolic heart failure.

Large animal ischemic cardiomyopathy models are widely used for preclinical testing of promising novel therapeutic approaches. Pressure volume (PV) loop analysis and cardiac magnetic resonance imaging (CMRI) allow functional and morphological phenotyping. In this study we performed a comparative analysis of both methods highlighting the strength of each and their synergistic potential. Myocardial infarction (MI) was created in German farm pigs (German Landrace) by 2 h LCX occlusion (n = 11) and subsequent reperfusion. Cardiac function was assessed by PV-loops and CMRI 56 and 112 days post-MI. Two hours occlusion of the LCX led to mid-size left ventricular (LV) MI represented by high-sensitive troponin T (hsTnT) 3 days post-MI, correlating well with cardiac CMRI late enhancement. CMRI determined end-diastolic and end-systolic volumes significantly increased post-MI, while ejection fraction was reduced in infarcted animals compared to the sham group (n = 6). PV-loop derived preload-insensitive parameters of systolic and diastolic function were diminished post-MI compared to sham animals while preload-dependent parameters only deteriorated in advanced HF. PV-loop analysis significantly correlates with CMRI analysis of cardiac function in pig post-MI ischemic cardiomyopathy. PV-Loop analysis accurately quantifies LV volumetry and function in post-MI HF, and thus eccentric LV morphology. PV-loop analysis correlates well to cardiac MRI. Preload-insensitive parameters show high sensitivity to quantify HF while preload-sensitive parameters are not able to quantify early-stages of LV HF.

Equine recurrent uveitis--a spontaneous horse model of uveitis.

Equine recurrent uveitis (ERU) is an autoimmune disease that occurs with a high prevalence (10%) in horses. ERU represents the only reliable spontaneous model for human autoimmune uveitis. We already identified and characterized novel autoantigens (malate dehydrogenase, recoverin, CRALBP) by analyzing the autoantibody-binding pattern of horses affected by spontaneous recurrent uveitis (ERU) to the retinal proteome. CRALBP also seems to be relevant to human autoimmune uveitis. Proteomic screening of vitreous and retinal samples from ERU diseased cases in comparison to healthy controls has led to the identification of a series of differentially regulated proteins, which are functionally linked to the immune system and the maintenance of the blood-retinal barrier.

Major retinal autoantigens remain stably expressed during all stages of spontaneous uveitis.

Equine recurrent uveitis (ERU) is a valuable model for autoimmune diseases, since it develops frequently and occurs spontaneously. We investigated the overall expression level of three major retinal autoantigens in normal retinas and various ERU stages. Analysis of retinal proteomes of both, healthy and diseased retinas revealed an almost unaffected expression of IRBP, S-antigen and cRALBP in ERU cases. Validation of these findings with western blots and immunohistochemistry confirmed constant to increased expression of these autoantigens, although loss of their physiological expression sites within retina is evident. In contrast to stable expression of autoantigens, rhodopsin, the major component of phototransduction in photoreceptors, disappeared from destructed retinas. These results explain persistent uveitic attacks even in severely damaged eyes and draw the attention to further investigations of biological pathways and regulations in autoimmune target tissues.

CRALBP is a highly prevalent autoantigen for human autoimmune uveitis.

Cellular retinaldehyde binding protein (CRALBP) is an autoantigen in spontaneous equine recurrent uveitis. In order to test whether CRALBP contributes to human autoimmune uveitis, the specificity of antibodies from human uveitis patient's sera was first evaluated in two-dimensional (2D) Western blot analysis. Subsequent identification of the immunoreactive proteins by mass spectrometry resulted in the identification of CRALBP as a putative autoantigen. Additionally, sera from human uveitis and control patients were by Western blot using purified human recombinant CRALBP. Anti-CRALBP autoantibodies occur more frequently (P<.01) in human uveitis patients than in normal controls. Thirty out of 56 tested uveitis patient's sera contained autoantibodies reactive against CRALBP, compared to only four out of 23 normal control subjects. The presence of CRALBP autoantibodies in 54% of tested uveitis patients supports CRALBP as a possible autoantigen in human autoimmune uveitis.

Multiple active states and oligomerization of CCR5 revealed by functional properties of monoclonal antibodies.

CC-chemokine receptor 5 (CCR5) is the principal coreceptor for macrophage-tropic strains of human immunodeficiency virus type 1 (HIV-1). We have generated a set of anti-CCR5 monoclonal antibodies and characterized them in terms of epitope recognition, competition with chemokine binding, receptor activation and trafficking, and coreceptor activity. MC-4, MC-5, and MC-7 mapped to the amino-terminal domain, MC-1 to the second extracellular loop, and MC-6 to a conformational epitope covering multiple extracellular domains. MC-1 and MC-6 inhibited regulated on activation normal T cell expressed and secreted (RANTES), macrophage inflammatory polypeptide-1beta, and Env binding, whereas MC-5 inhibited macrophage inflammatory polypeptide-1beta and Env but not RANTES binding. MC-6 induced signaling in different functional assays, suggesting that this monoclonal antibody stabilizes an active conformation of CCR5. Flow cytometry and real-time confocal microscopy showed that MC-1 promoted strong CCR5 endocytosis. MC-1 but not its monovalent isoforms induced an increase in the transfer of energy between CCR5 molecules. Also, its monovalent isoforms bound efficiently, but did not internalize the receptor. In contrast, MC-4 did not prevent RANTES binding or subsequent signaling, but inhibited its ability to promote CCR5 internalization. These results suggest the existence of multiple active conformations of CCR5 and indicate that CCR5 oligomers are involved in an internalization process that is distinct from that induced by the receptor's agonists.

Evaluation of decellularized human umbilical vein (HUV) for vascular tissue engineering - comparison with endothelium-denuded HUV.

Human umbilical vessels have been recognized as a valuable and widely available resource for vascular tissue engineering. Whereas endothelium-denuded human umbilical veins (HUVs) have been successfully seeded with a patient-derived neoendothelium, decellularized vessels may have additional advantages, due to their lower antigenicity. The present study investigated the effects of three different decellularization procedures on the histological, mechanical and seeding properties of HUVs. Vessels were decellularized by detergent treatment (Triton X-100, sodium deoxycholate, IGEPAL-CA630), osmotic lysis (3 m NaCl, distilled water) and peroxyacetic acid treatment. In all cases, nuclease treatments were required to remove residual nucleic acids. Decellularization resulted in a partial loss of fibronectin and laminin staining in the subendothelial layer and affected the appearance of elastic fibres. In addition to removing residual nucleic acids, nuclease treatment weakened all stainings and substantially altered surface properties, as seen in scanning electron micrographs, indicating additional non-specific effects. Detergent treatment and osmotic lysis caused failure stresses to decrease significantly. Although conditioned medium prepared from decellularized HUV did not severely affect endothelial cell growth, cells seeded on decellularized HUV did not remain viable. This may be attributed to the partial removal of essential extracellular matrix components as well as to changes of surface properties. Therefore, decellularized HUVs appear to require additional modifications in order to support successful cell seeding. Replacing the vessels' endothelium may thus be a superior alternative to decellularization when creating tissue-engineered blood vessels with non-immunogenic luminal interfaces.

Nonthermal effects of lifelong high-frequency electromagnetic field exposure on social memory performance in rats.

We are today surrounded almost constantly by high-frequency electromagnetic fields (EMFs) from mobile communications base stations. To date, however, there has been little concern regarding nonthermal effects of EMFs on cognition. In the present study, male and female rats were subjected to continuous far-field exposure to a frequency of 900-MHz (Global System for Mobile Communications [GSM]) or 1.966-GHz (Universal Mobile Telecommunications System [UMTS]) at 0.4 W/kg. Memory performance of adult EMF-exposed and sham-exposed female rats (at 6 months of age) and male rats (at 3 and 6 months of age) was tested using a social discrimination procedure. For this procedure, a target juvenile male was introduced to the subject's home cage for 4 min (Trial 1). After 30 min, the same target animal and a novel juvenile male were simultaneously presented to the subject for 4 min (Trial 2). Differences in sniffing duration to the familiar and novel target rats during Trial 2 were used to assess memory performance. EMF-exposed females exhibited no differences in sniffing duration compared with controls. In contrast, the sniffing durations of EMF-exposed males at 3 months of age were significantly affected. At 6 months of age, GSM-, but not UMTS-, exposed male adults showed a memory performance deficit. These findings provide new insight into the nonthermal effects of long-term high-frequency EMF exposure on memory.

Chondroitin sulfate activates B cells in vitro, expands CD138+ cells in vivo, and interferes with established humoral immune responses.

Glycosaminoglycans have anti-inflammatory properties and interact with a variety of soluble and membrane-bound molecules. Little is known about their effects on B cells and humoral immune responses. We show that CS but not dextran or other glycosaminoglycans induces a pronounced proliferation of B cells in vitro compared with TLR4 or TLR9 ligands. With the use of inhibitors and KO mice, we demonstrate that this proliferation is mediated by the tyrosine kinases BTK and Syk but independent of CD44. Antibodies against Ig-α or Ig-ß completely block CS-induced B cell proliferation. Injection of CS in mice for 4-5 days expands B cells in the spleen and results in a marked increase of CD138(+) cells in the spleen that is dependent on BTK but independent of CD4(+) T cells. Long-term treatment with CS for 14 days also increases CD138(+) cells in the bone marrow. When mice were immunized with APC or collagen and treated with CS for up to 14 days during primary or after secondary immune responses, antigen-specific humoral immune responses and antigen-specific CD138(+) plasma cells in the bone marrow were reduced significantly. These data show that CD138(+) cells, induced by treatment with CS, migrate into the bone marrow and may displace other antigen-specific plasma cells. Overall, CS is able to interfere markedly with primary and fully established humoral immune responses in mice.

Vasodilator phosphostimulated protein (VASP) protects endothelial barrier function during hypoxia.

The endothelial barrier controls the passage of solutes from the vascular space. This is achieved through active reorganization of the actin cytoskeleton. A central cytoskeletal protein involved into this is vasodilator-stimulated phosphoprotein (VASP). However, the functional role of endothelial VASP during hypoxia has not been thoroughly elucidated. We determined endothelial VASP expression through real-time PCR (Rt-PCR), immunhistochemistry, and Western blot analysis during hypoxia. VASP promoter studies were performed using a PGL3 firefly luciferase containing plasmid. Following approval by the local authorities, VASP ( -/- ) mice and littermate controls were subjected to normobaric hypoxia (8% O(2), 92% N(2)) after intravenous injection of Evans blue dye. In in vitro studies, we found significant VASP repression in human microvascular and human umbilical vein endothelial cells through Rt-PCR, immunhistochemistry, and Western blot analysis. The VASP promoter construct demonstrated significant repression in response to hypoxia, which was abolished when the binding of hypoxia-inducible factor 1 alpha was excluded. Exposure of wild-type (WT) and VASP ( -/- ) animals to normobaric hypoxia for 4 h resulted in an increase in Evans blue tissue extravasation that was significantly increased in VASP ( -/- ) animals compared to WT controls. In summary, we demonstrate here that endothelial VASP holds significant importance for endothelial barrier properties during hypoxia.

Complement factor B expression profile in a spontaneous uveitis model.

Equine recurrent uveitis serves as a spontaneous model for human autoimmune uveitis. Unpredictable relapses and ongoing inflammation in the eyes of diseased horses as well as in humans lead to destruction of the retina and finally result in blindness. However, the molecular mechanisms leading to inflammation and retinal degeneration are not well understood. An initial screening for differentially regulated proteins in sera of uveitic cases compared to healthy controls revealed an increase of the alternative pathway complement component factor B in ERU cases. To determine the activation status of the complement system, sera were subsequently examined for complement split products. We could demonstrate a significant higher concentration of the activation products B/Ba, B/Bb, Bb neoantigen, iC3b and C3d in uveitic condition compared to healthy controls, whereas for C5b-9 no differences were detected. Additionally, we investigated complement activation directly in the retina by immunohistochemistry, since it is the main target organ of this autoimmune disease. Interestingly, infiltrating cells co-expressed activated factor Bb neoantigen, complement split product C3d as well as CD68, a macrophage marker. In this study, we could demonstrate activation of the complement system both systemically as well as in the eye, the target organ of spontaneous recurrent uveitis. Based on these novel findings, we postulate a novel role for macrophages in connection with complement synthesis at the site of inflammation.

Kininogen in autoimmune uveitis: decrease in peripheral blood stream versus increase in target tissue.

PURPOSE: Equine recurrent uveitis (ERU) is an incurable disease affecting the inner eye that leads to blindness, through activated T cells that pass the blood-retinal barrier and destroy the retina. Serum markers are a desirable choice for monitoring development of disease, as serum is easy accessible and the markers could serve to predict the beginning of disease or an imminent relapse. METHODS: In this study, serum proteomes (depleted of high-abundance serum proteins) of horses with ERU and healthy controls were compared with the 2-D DIGE (two-dimensional gel electrophoresis) technique to identify differentially expressed proteins. The expression pattern of a candidate protein in retina and vitreous was validated by Western blots and immunohistochemistry. RESULTS: Ten differentially expressed proteins could be identified by mass spectrometry (MALDI-TOF/TOF). Five proteins--IgM, IgG4 hc, serotransferrin, alpha-2HS-glycoprotein, and complement factor B--were upregulated in the uveitic state, whereas the five proteins albumin, apolipoprotein A-IV and H, IgG5 hc, and high-molecular-weight kininogen (HK) showed a significantly lower expression in sera of uveitis cases. Of interest, kininogen was significantly upregulated in the target tissues vitreous and retina. HK is a plasma protein with multiple physiological functions, with an important role in inflammation and promoting neovascularization. Most interesting is the as of yet unaddressed association of HK with uveitis. Immunohistochemistry showed coexpression of kininogen and VEGF in inflamed eyes. CONCLUSIONS: Since neovascularization plays a major role in the pathogenesis of uveitis, the identification of a proangiogenic factor in the retina presents an important finding and may contribute to elucidating the pathogenesis of uveitis.

Serum PEDF levels are decreased in a spontaneous animal model for human autoimmune uveitis.

Identification of biomarkers is of critical relevance toward improving diagnosis and therapy of autoimmune disorders. Serum markers are a desirable choice as sera are easily accessible and the development of assays for routine clinical detection prompts feasible. Autoimmune uveitis, a recurrent disease affecting the eye, is characterized by returning inflammatory attacks of the inner eye followed by variable periods of quiescent stages. Spontaneous equine recurrent uveitis (ERU) is the equine equivalent and serves as a model for the human disease. To identify potential biomarker candidates, we first systematically compared the proteomes of individual ERU cases with healthy controls by proteomic profiling using 2-D difference-gel-electrophoresis (2-D DIGE) followed by tandem mass spectrometry. A total of seven differentially expressed proteins were identified. Besides the upregulation of IgG and the significant lower expression of albumin, Antithrombin III, and Vitamin D binding protein, we found complement components C1q and C4, to be downregulated in uveitic state. Interestingly, Pigment epithelium-derived factor (PEDF), a marker already detected by 2DE differential proteome analysis in ERU target tissues, vitreous and retina, was found to be also significantly downregulated in sera. The lower expression of PEDF in sera of horses with uveitis could be verified in a cohort of 116 ERU cases and 115 healthy controls. Our findings of a significant lower PEDF expression in ERU cases also in the periphery of the eye proves PEDF as a promising uveitis biomarker.

Important role of interleukin-3 in the early phase of collagen-induced arthritis.

OBJECTIVE: Activation of basophils contributes to memory immune responses and results in exacerbation of collagen-induced arthritis (CIA). We undertook the present study to analyze the production and biologic effects of interleukin-3 (IL-3), a strong activator of basophils, in CIA. METHODS: Arthritis was induced by immunization with type II collagen. Mice were treated with blocking monoclonal antibodies against IL-3 or with recombinant IL-3. Clinical scoring, histologic analysis, fluorescence-activated cell sorter analysis, enzyme-linked immunosorbent assay, and cell culturing were performed to assess disease activity and IL-3 production. RESULTS: IL-3 was produced in large quantities by collagen-specific CD4+ T cells in the spleen and was present in the synovial tissue during onset of arthritis, but was down-regulated in paws with severe inflammation. Blockade of IL-3 during the time of arthritis onset resulted in profound improvement of the disease, with reductions in synovial leukocyte and cytokine levels, peripheral blood basophil levels, and anticollagen antibody titers. Blockade of IL-3 during the late phase of arthritis had no beneficial effect. Administration of recombinant IL-3 during onset of arthritis induced a marked exacerbation of the disease, with increased peripheral blood basophil and plasma IL-6 levels and increased titers of anticollagen antibody. In studies of the regulation of IL-3 expression in CD4+ T cells, IL-6 and IL-4 suppressed the release of IL-3 by activated CD4+ T cells, whereas lipopolysaccharide and CpG DNA up-regulated IL-3 secretion in activated CD4+ T cells by acting on costimulatory cells. CONCLUSION: Taken together, the present results demonstrate for the first time that IL-3 has an important role in the early phase of CIA.

Discovering novel targets for autoantibodies in dilated cardiomyopathy.

There is increasing evidence that a large proportion of dilated cardiomyopathy (DCM) cases are mediated by autoimmune processes. Since DCM is a fatal disorder with rapid aggravation and is the leading cause of heart transplantation, further insights into disease pathogenesis are needed. Recent studies have separated the pathogenic capacity of autoantibodies and initial clinical trials removing such autoantibodies via immunoadsorption have been promising. In order to elucidate the full autoantibody repertoire involved in DCM, we applied an autoantibody screening test using ventricular and atrial proteomes as autoantigenic sources and subsequently tested the autoantibody-binding patterns of sera from dogs with spontaneous DCM. With this method, we detected five potentially DCM-related autoantigens which were identified by MS as being: myosin heavy chain cardiac muscle alpha isoform, alpha cardiac actin, mitochondrial aconitate hydratase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and brain glycogen phosphorylase (GPBB). The recovery of two known DCM autoantigens (myosin heavy chain and alpha cardiac actin) and the discovery of three novel autoantigens (mitochondrial aconitate hydratase, GADPH, and GPBB) underscore the efficacy of this experimental method and the significance of the spontaneous canine DCM model.

Retinal Mueller glial cells trigger the hallmark inflammatory process in autoimmune uveitis.

Spontaneous equine recurrent uveitis (ERU) is an incurable autoimmune disease affecting the eye. Although retinal-autoantigen specific T-helper 1 cells have been demonstrated to trigger disease progression and relapses, the molecular processes leading to retinal degeneration and consequent blindness remain unknown. To elucidate such processes, we studied changes in the total retinal proteome of ERU-diseased horses compared to healthy controls. Severe changes in the retinal proteome were found for several markers for blood-retinal barrier breakdown and whose emergence depended upon disease severity. Additionally, uveitic changes in the retina were accompanied by upregulation of aldose 1-epimerase, selenium-binding protein 1, alpha crystallin A chain, phosphatase 2A inhibitor (SET), and glial fibrillary acidic protein (GFAP), the latter indicating an involvement of retinal Mueller glial cells (RMG) in disease process. To confirm this, we screened for additional RMG-specific markers and could demonstrate that, in uveitic retinas, RMG concomitantly upregulate vimentin and GFAP and downregulate glutamine synthetase. These expression patterns suggest for an activated state of RMG, which further downregulate the expression of pigment epithelium-derived factor (PEDF) and begin expressing interferon-gamma, a pro-inflammatory cytokine typical for T-helper 1 cells. We thus propose that RMG may play a fatal role in uveitic disease progression by directly triggering inflammatory processes through the expression and secretion of interferon-gamma.

Down-regulation of pigment epithelium-derived factor in uveitic lesion associates with focal vascular endothelial growth factor expression and breakdown of the blood-retinal barrier.

Spontaneous equine recurrent uveitis (ERU) is an incurable autoimmune disease affecting the eye. Identifying biological markers or pathways associated with this disease may allow the understanding of its pathogenesis at a molecular level. The vitreous is the body fluid closest to the disease-affected tissue and possibly also an effector of pathological processes relevant for ERU. Surgical removal of vitreous leads to cessation of relapses in spontaneous uveitis of both man and horse, therefore vitreous composites are likely to contribute to disease progression. Uveitic vitreous is likely to contain potential biomarkers in relatively undiluted quantities. With the goal to identify these markers, we systematically compared vitreous from healthy and disease-affected eyes by proteomic profiling. Nine differentially expressed proteins were identified, that are functionally related to immune response, inflammation, and maintenance of the blood-retinal barrier. One of these, pigment epithelium-derived factor, a protein involved in maintaining a proper blood-retina barrier as well as protecting from neoangiogenesis was additionally found to be down-regulated within uveitic retinal lesions whereas, conversely, vascular endothelial growth factor was found to be up-regulated at these sites. Together, these changes point to as of yet undiscovered biological pathways involved in the pathogenesis of this autoimmune disease.

Targeting of Gr-1+,CCR2+ monocytes in collagen-induced arthritis.

OBJECTIVE: The chemokine receptor CCR2 is highly expressed on monocytes and considered a promising target for treatment of rheumatoid arthritis. However, blockade of CCR2 with a monoclonal antibody (mAb) during progression of collagen-induced arthritis results in a massive aggravation of the disease. In this study we investigated why CCR2 antibodies have proinflammatory effects, how these effects can be avoided, and whether CCR2+ monocytes are useful targets in the treatment of arthritis. METHODS: Arthritis was induced in DBA/1 mice by immunization with type II collagen. Mice were treated with mAb against CCR2 (MC-21), IgE, or isotype control antibodies at various time points. Activation of basophils and depletion of monocyte subsets were determined by fluorescence-activated cell sorter analysis and enzyme-linked immunosorbent assay. RESULTS: Crosslinkage of CCR2 activated basophils to release interleukin-6 (IL-6) and IL-4. In vivo, IL-6 release occurred only after exposure to high doses of MC-21, whereas application of low doses of the mAb circumvented the release of IL-6. Regardless of the dose level used, the antibody MC-21 efficiently depleted Gr-1+,CCR2+ monocytes from the synovial tissue, peripheral blood, and spleen of DBA/1 mice. Activation of basophils with high doses of MC-21 or with antibodies against IgE resulted in a marked aggravation of collagen-induced arthritis and an increased release of IL-6. In contrast, low-dose treatment with MC-21 in this therapeutic setting had no effect on IL-6 and led to marked improvement of arthritis. CONCLUSION: These results show that depletion of CCR2+ monocytes may prove to be a therapeutic option in inflammatory arthritis, as long as the dose-dependent proinflammatory effects of CCR2 mAb are taken into account.