Roy A. Fava

Revascularization of ischemic tissues by PlGF treatment, and inhibition of tumor angiogenesis, arthritis and atherosclerosis by anti-Flt1

The therapeutic potential of placental growth factor (PlGF) and its receptor Flt1 in angiogenesis is poorly understood. Here, we report that PlGF stimulated angiogenesis and collateral growth in ischemic heart and limb with at least a comparable efficiency to vascular endothelial growth factor (VEGF). An antibody against Flt1 suppressed neovascularization in tumors and ischemic retina, and angiogenesis and inflammatory joint destruction in autoimmune arthritis. Anti-Flt1 also reduced atherosclerotic plaque growth and vulnerability, but the atheroprotective effect was not attributable to reduced plaque neovascularization. Inhibition of VEGF receptor Flk1 did not affect arthritis or atherosclerosis, indicating that inhibition of Flk1-driven angiogenesis alone was not sufficient to halt disease progression. The anti-inflammatory effects of anti-Flt1 were attributable to reduced mobilization of bone marrow–derived myeloid progenitors into the peripheral blood; impaired infiltration of Flt1-expressing leukocytes in inflamed tissues; and defective activation of myeloid cells. Thus, PlGF and Flt1 constitute potential candidates for therapeutic modulation of angiogenesis and inflammation.

VISTA, a novel mouse Ig superfamily ligand that negatively regulates T cell responses

VISTA suppresses T cell proliferation and cytokine production and can influence autoimmunity and antitumor responses in mice.

A Role for the Lymphotoxin/LIGHT Axis in the Pathogenesis of Murine Collagen-Induced Arthritis

A lymphotoxin-β (LTβ) receptor-Ig fusion protein (LTβR-Ig) was used to evaluate the importance of the lymphotoxin/LIGHT axis in the development and perpetuation of arthritis. Prophylactic treatment with the inhibitor protein LTβR-Ig blocked the induction of collagen-induced arthritis in mice and adjuvant arthritis in Lewis rats. Treatment of mice with established collagen-induced arthritis reduced the severity of arthritic symptoms and joint tissue damage. However, in a passive model of anti-collagen Ab-triggered arthritis, joint inflammation was not affected by LTβR-Ig treatment precluding LT/LIGHT involvement in the very terminal immune complex/complement/FcR-mediated effector phase. Collagen-II and Mycobacterium-specific T cell responses were not impaired, yet there was evidence that the overall response to the mycobacterium was blunted. Serum titers of anti-collagen-II Abs were reduced especially during the late phase of disease. Treatment with LTβR-Ig ablated follicular dendritic cell networks in the draining lymph nodes, suggesting that impaired class switching and affinity maturation may have led to a decreased level of pathological autoantibodies. These data are consistent with a model in which the LT/LIGHT axis controls microenvironments in the draining lymph nodes. These environments are critical in shaping the adjuvant-driven initiating events that impact the subsequent quality of the anti-collagen response in the later phases. Consequently, blockade of the LT/LIGHT axis may represent a novel approach to the treatment of autoimmune diseases such as rheumatoid arthritis that involve both T cell and Ab components.

Blockade of lymphotoxin-beta receptor signaling reduces aspects of Sjögren's syndrome in salivary glands of non-obese diabetic mice

IntroductionThe lymphotoxin-beta receptor (LTβR) pathway is important in the development and maintenance of lymphoid structures. Blocking this pathway has proven beneficial in murine models of autoimmune diseases such as diabetes and rheumatoid arthritis. The aim of this study was to determine the effects of LTβR pathway blockade on Sjögren syndrome (SS)-like salivary gland disease in non-obese diabetic (NOD) mice.MethodsThe course of SS-like disease was followed in NOD mice that were given lymphotoxin-beta receptor-immunoglobulin fusion protein (LTβR-Ig) starting at 9 weeks of age. Treatment was given as a single weekly dose for 3, 7, or 10 weeks. Age-matched NOD mice treated with mouse monoclonal IgG1, or not treated at all, were used as controls. The severity of inflammation, cellular composition, and lymphoid neogenesis in the submandibular glands were determined by immunohistochemistry. Mandibular lymph nodes were also studied. Saliva flow rates were measured, and saliva was analyzed by a multiplex cytokine assay. The salivary glands were analyzed for CXCL13, CCL19, and CCL21 gene expression by quantitative polymerase chain reaction.ResultsTreatment with LTβR-Ig prevented the increase in size and number of focal infiltrates normally observed in this SS-like disease. Compared with the controls, the submandibular glands of LTβR-Ig-treated mice had fewer and smaller T- and B-cell zones and fewer high endothelial venules per given salivary gland area. Follicular dendritic cell networks were lost in LTβR-Ig-treated mice. CCL19 expression was also dramatically inhibited in the salivary gland infiltrates. Draining lymph nodes showed more gradual changes after LTβR-Ig treatment. Saliva flow was partially restored in mice treated with 10 LTβR-Ig weekly injections, and the saliva cytokine profile of these mice resembled that of mice in the pre-disease state.ConclusionsOur findings show that blocking the LTβR pathway results in ablation of the lymphoid organization in the NOD salivary glands and thus an improvement in salivary gland function.

Lymphotoxin-beta receptor blockade reduces CXCL13 in lacrimal glands and improves corneal integrity in the NOD model of Sjögren's syndrome

IntroductionIn Sjögrens syndrome, keratoconjunctivitis sicca (dry eye) is associated with infiltration of lacrimal glands by leukocytes and consequent losses of tear-fluid production and the integrity of the ocular surface. We investigated the effect of blockade of the lymphotoxin-beta receptor (LTBR) pathway on lacrimal-gland pathology in the NOD mouse model of Sjögrens syndrome.MethodsMale NOD mice were treated for up to ten weeks with an antagonist, LTBR-Ig, or control mouse antibody MOPC-21. Extra-orbital lacrimal glands were analyzed by immunohistochemistry for high endothelial venules (HEV), by Affymetrix gene-array analysis and real-time PCR for differential gene expression, and by ELISA for CXCL13 protein. Leukocytes from lacrimal glands were analyzed by flow-cytometry. Tear-fluid secretion-rates were measured and the integrity of the ocular surface was scored using slit-lamp microscopy and fluorescein isothiocyanate (FITC) staining. The chemokine CXCL13 was measured by ELISA in sera from Sjögrens syndrome patients (n = 27) and healthy controls (n = 30). Statistical analysis was by the two-tailed, unpaired T-test, or the Mann-Whitney-test for ocular integrity scores.ResultsLTBR blockade for eight weeks reduced B-cell accumulation (approximately 5-fold), eliminated HEV in lacrimal glands, and reduced the entry rate of lymphocytes into lacrimal glands. Affymetrix-chip analysis revealed numerous changes in mRNA expression due to LTBR blockade, including reduction of homeostatic chemokine expression. The reduction of CXCL13, CCL21, CCL19 mRNA and the HEV-associated gene GLYCAM-1 was confirmed by PCR analysis. CXCL13 protein increased with disease progression in lacrimal-gland homogenates, but after LTBR blockade for 8 weeks, CXCL13 was reduced approximately 6-fold to 8.4 pg/mg (+/- 2.7) from 51 pg/mg (+/-5.3) in lacrimal glands of 16 week old control mice. Mice given LTBR blockade exhibited an approximately two-fold greater tear-fluid secretion than control mice (P = 0.001), and had a significantly improved ocular surface integrity score (P = 0.005). The mean CXCL13 concentration in sera from Sjögrens patients (n = 27) was 170 pg/ml, compared to 92.0 pg/ml for sera from (n = 30) healthy controls (P = 0.01).ConclusionsBlockade of LTBR pathways may have therapeutic potential for treatment of Sjögrens syndrome.

Inhibition of arterial lesion progression in CD16-deficient mice: evidence for altered immunity and the role of IL-10

AIMS Given the importance of IgG Fc receptors in immune regulation, we hypothesized that Fcg receptor type III (FcgRIII, CD16) plays an important role in atherogenesis. We therefore analysed the formation of arterial lesions in LDL receptor-deficient (LDLR(-/-)) and FcgRIII(-/-)xLDLR(-/-) double knockout mice at three different points up to 24 weeks of exposure to a high-fat diet. METHODS AND RESULTS Analysis of Oil Red-O-stained sections revealed no difference in lesion formation between strains after 6 weeks of a high-fat diet, and a modest decrease after 14 weeks in double knockouts relative to LDLR(-/-) controls. After 24 weeks, lesion formation was decreased in the aortic root (30%) and innominate artery (50%) in FcgRIII double knockouts relative to LDLR(-/-) controls. Analysis of peripheral CD4+ T-cells by intracellular flow cytometry from double knockouts after 24 weeks of a high-fat diet revealed statistically significant increases in the percentages of cells producing interferon-gamma, interleukin (IL)-10, and IL-4 relative to controls, differences that were also observed by analyses of whole aortas for cytokine mRNA levels. As determined by flow cytometry, FcgRIII deficiency resulted in an expansion of CD4+ cells and an increase in the CD4 to CD8 ratio. Analysis of plasma anti-oxidized LDL (OxLDL) antibodies by chemiluminescent assay revealed that IgG1 and IgG2c titers to OxLDL were increased in FcgRIII (-/-)xLDLR(-/-) double knockouts relative to LDLR(-/-) controls, while total IgG levels were similar. CONCLUSION These results reveal altered immunity in FcgRIII(-/-)xLDLR(-/-) mice and a reduction in lesion formation associated with increased production of IL-10 by an expansion of CD4+ T-cells. The reduction in lesion formation was manifest well after evidence of an immune response to OxLDL, suggesting that FcgRIII contributes to lesion progression in murine atherosclerosis.

AUF1/hnRNP D represses expression of VEGF in macrophages.

Vascular endothelial growth factor (VEGF) expression is regulated by sequence elements in the 3′ UTR of VEGF mRNA. AUF1/hnRNP D suppresses VEGF 3′ UTR–dependent expression. Peptides with arginine–glycine–glycine motifs derived from AUF1 also suppress VEGF expression.

LTBR-Pathway in Sjogren’s Syndrome: CXCL13 Levels and B-cell-Enriched Ectopic Lymphoid Aggregates in NOD Mouse Lacrimal Glands Are Dependent on LTBR

The fact that TNF receptor family members are involved in the control of diverse gene products that effect both pro-inflammatory and homeostatic functions related to immune protection offers multiple targets for clinical intervention in a range of disease contexts. The stunning success of anti-TNF-alpha therapy in the treatment of the inflammatory disease rheumatoid arthritis perhaps best illustrates the vast potential of antagonism of TNF family members in clinical medicine [1]. The involvement of other family members, such as CD40, in many other immune regulated diseases will also no doubt lead to similar success stories. In contrast to the pro-inflammatory pathways controlled by TNF-alpha, our lab has begun to determine whether antagonism of the “homeostatic” pathways in secondary and “ectopic” or tertiary lymphoid tissues that are under the control of the TNF receptor family member lymphotoxin-beta receptor (LTBR) might represent a useful target in the treatment of certain diseases such as Sjogren’s syndrome where frank inflammation is not the primary pathogenic impetus.