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

Vascular permeability factor (VPF, also known as vascular endothelial growth factor or VEGF), is a potent microvascular permeability enhancing cytokine and a selective mitogen for endothelial cells. It has been implicated in tumor angiogenesis and ascites fluid accumulation. Since development of the destructive synovial pannus in rheumatoid arthritis (RA) is associated with changes in vascular permeability (synovial fluid accumulation), synovial cell hyperplasia, and angiogenesis, we examined synovial fluids (SFs) and joint tissue for the expression and local accumulation of VPF/VEGF. VPF/VEGF was detected in all of 21 synovial fluids examined and when measured by an immunofluorimetric assay, ranged from 6.9 to 180.5 pM. These levels are biologically significant, since < 1 pM VPF/VEGF can elicit responses from its target cells, endothelial cells. Levels of VPF/VEGF were highest in rheumatoid arthritis fluids (n = 10), with a mean value (+/- SEM) of 59.1 +/- 18.0 pM, vs. 21.4 +/- 2.3 pM for 11 SFs from patients with other forms of arthritis (p = 0.042). In situ hybridization studies that were performed on joint tissues from patients with active RA revealed that synovial lining macrophages strongly expressed VPF/VEGF mRNA, and that microvascular endothelial cells of nearby blood vessels strongly expressed mRNA for the VPF/VEGF receptors, flt-1 and KDR. Immunohistochemistry performed on inflamed rheumatoid synovial tissue revealed that the VPF/VEGF peptide was localized to macrophages within inflamed synovium, as well as to microvascular endothelium, its putative target in the tissue. Together, these findings indicate that VPF/VEGF may have an important role in the pathogenesis of RA.

The lymphotoxin axis is important for the maintenance of several specialized lymphoid microenvironments in secondary lymphoid tissue. Lymphoid-tissue architecture is highly plastic and requires continual homeostatic signaling to maintain its basal functional state. The cellularity of lymph nodes in adult mice was reduced by systemic blockade of lymphotoxin-beta receptor (LTbeta R) signaling with a soluble decoy receptor both in resting and reactive settings. This reduction in cellularity resulted from greatly impaired lymphocyte entry into lymph nodes due to decreased levels of peripheral lymph node addressing (PNAd) and MAdCAM on high endothelial venules (HEV). LTbeta R signaling was required to maintain normal levels of RNA expression of MAdCAM, and also of PNAd by regulating the expression of key enzymes and scaffold proteins required for its assembly. Thus, the homeostatic maintenance of functional HEV status in adult mice relies largely on LTbeta R signaling.

The ligand for the CD40 antigen is a 39-kilodalton protein, gp39, expressed on the surface of activated CD4+ T cells and is essential for thymus-dependent humoral immunity. The role of gp39-CD40 interactions in autoimmune disease was investigated in vivo with the use of an antibody that blocks their interactions (anti-gp39). Arthritis induced in mice by immunization with type II collagen was inhibited by anti-gp39. Anti-gp39 blocked the development of joint inflammation, serum antibody titers to collagen, the infiltration of inflammatory cells into the subsynovial tissue, and the erosion of cartilage and bone. Thus, interference with gp39-CD40 interactions may have therapeutic potential in the treatment of autoimmune disease.

Transforming growth factor ß (TGF ß) is a multifunctional growth regulator with diverse biological effects, including promotion and inhibition of fibroblastic and epithelial cell growth, respectively (1, 2), chemotaxis of dermal fibroblasts and monocytes (3, 4), facilitation of extracellular matrix remodeling by fibroblastic cells (5-7), and effects on proliferation and function of T and B lymphocytes (8-10). These cellular responses appear pertinent to development and/or maintenance ofthe synovial panmus in inflammatory arthritis. Furthermore, subcutaneous injection of purified TGFP into newborn mice results in a multicellular response over 48-72 h (11), which is histologically similar to the synovial panmus seen in rheumatoid arthritis (RA). These findings led us to examine synovial effusions for the presence of TGFß.Volume 169 January 1989 291-296 Materials and Methods Brief Definitive ReportSynovial Fluids . Synovial fluids from the knees of 16 patients seen in the Vanderbilt University Rheumatology Clinic were collected by needle aspiration into heparin-free plastic syringes . Diagnoses determined by a rheumatologist included nine patients with RA, four with osteoarthritis, two with gout, and one patient with avascu1ar necrosis .Most fluids were routinely centrifuged at 1,200 g for 10 min at 4°C to remove cells . Some fluids were subjected to a more rigorous collection procedure involving centrifugation in plastic tubes at 5,000 g in a Sorvall high-speed centrifuge for 20 min, in order to minimize the possibility of contamination with platelet products released in vitro. The cell-and platelet-free supernatants were then collected with a glass pipette that was left in the tube at 37°C . Clotted material that formed around the glass pipette was discarded . The fluids were stored at -70°C until assay.Assaysfor 7GF--ß . A radioreceptor assay for TGF ß was performed using a previously described procedure (12) . Briefly, AKR-2B fibroblasts were plated in six-well culture plates at 1-2 x 105 cells per well using McCoy's 5A medium with 5% FCS . The next day, cells were washed three times with PBS and incubated for 2 h in the presence of 0 .25 ng of '2'I TGF p in 1 .0 ml of binding medium, with or without further additions. Nonspecific binding was determined by the addition of 1 gg of 50% pure unlabeled TGFP .After a 2-h incubation at room temperature with rocking, the cells were washed, released from the plate with collagenase, and cell-bound 125 1TGF-ß was measured in a gamma counter. Results were expressed as nanograms per milliliter TGF0 calculated from a standard curve (see Fig . 1) . The concentrations of TGF ß-competing activity in synovial fluids (Table I) were derived from triplicate determinations at a final dilution that would yield a 40-60% inhibition of binding.Two cell lines were also used in soft agar assays for TGF-ß . AKR-2B cells were assayed in the presence of serum as previously described (13) . Base layers of 0 .8% agar in McCoy's

SUMMARYVascular endothelial growth factor (VEGF) is abundant in synovium and synovial fluids, where it probably contributes to vascular permeability and angiogenesis in arthritic joints. To investigate the probable sources of VEGF in synovium, we compared the ability of several cytokines (TGF-b, plateletderived growth factor (PDGF), IL-1, tumour necrosis factor (TNF), basic fibroblast growth factor (bFGF) that are associated with arthritis and angiogenesis, to stimulate secretion of VEGF protein by human synovial fibroblasts. TGF-b was the strongest inducer of VEGF secretion; six times more VEGF was secreted when cells were stimulated by TGF-b than when stimulated by PDGF or IL-1 for 24 h. TNF-a and bFGF did not stimulate any secretion of VEGF. The stimulatory effects of TGF-b and IL-1 on VEGF secretion were additive. Hypoxic culture alone also stimulated VEGF secretion, but more importantly, hypoxic culture conditions doubled the rate of VEGF secretion stimulated by the cytokines TGF-b and IL-1. When dermal and synovial fibroblasts were stimulated identically by hypoxia and cytokines (TGF-b and IL-1), synovial fibroblasts secreted four times more VEGF than did dermal fibroblasts. Thus in rheumatoid arthritis, the capacity of synovial fibroblasts in the hypoxic environment to secrete large amounts of VEGF in response to cytokines such as TGF-b probably contributes significantly to angiogenesis in the synovium.

We have studied the consequences of introducing human recombinant transforming growth factor beta 1 (hrTGF-beta 1) into synovial tissue of the rat, to begin to better understand the significance of the fact that biologically active TGF-beta is found in human arthritic synovial effusions. Within 4-6 h after the intra-articular injection of 1 microgram of hrTGF-beta 1 into rat knee joints, extensive recruitment of polymorphonuclear leukocytes (PMNs) was observed. Cytochemistry and high resolution histological techniques were used to quantitate the influx of PMNs, which peaked 6 h post-injection. In a Boyden chamber assay, hrTGF-beta 1 at 1-10 fg/ml elicited a chemotactic response from PMNs greater in magnitude than that evoked by FMLP, establishing that TGF-beta 1 is an effective chemotactic agent for PMNs in vitro as well as in vivo. That PMNs may represent an important source of TGF-beta in inflammatory infiltrates was strongly suggested by a demonstration that stored TGF-beta 1 was secreted during phorbol myristate acetate-stimulated degranulation in vitro. Acid/ethanol extracts of human PMNs assayed by ELISA contained an average of 355 ng of TGF/beta 1 per 10(9) cells potentially available for secretion during degranulation of PMNs. [3H]Thymidine incorporation in vivo and autoradiography of tissue sections revealed that widespread cell proliferation was triggered by TGF-beta 1 injection. Synovial lining cells and cells located deep within the subsynovial connective tissue were identified as sources of at least some of the new cells that contribute to TGF-beta 1-induced hyperplasia. Our results demonstrate that TGF-beta is capable of exerting pathogenic effects on synovial tissue and that PMNs may represent a significant source of the TGF-beta present in synovial effusions.