Reversal of acute and chronic synovial inflammation by anti- transforming growth factor beta

Transforming growth factor beta (TGF-beta) induces leukocyte recruitment and activation, events central to an inflammatory response. In this study, we demonstrate that antagonism of TGF-beta with a neutralizing antibody not only blocks inflammatory cell accumulation, but also tissue pathology in an experimental model of chronic erosive polyarthritis. Intraarticular injection of monoclonal antibody 1D11.16, which inhibits both TGF-beta 1 and TGF-beta 2 bioactivity, into animals receiving an arthropathic dose of bacterial cell walls significantly inhibits arthritis. Inhibition was observed with a single injection of 50 micrograms antibody, and a 1-mg injection blocked acute inflammation > 75% compared with the contralateral joints injected with an irrelevant isotype control antibody (MOPC21) as quantitated by an articular index (AI = 0.93 +/- 0.23 for 1D11.16, and AI = 4.0 +/- 0 on day 4; p < 0.001). Moreover, suppression of the acute arthritis achieved with a single injection of antibody was sustained into the chronic, destructive phase of the disease (on day 18, AI = 0.93 +/- 0.07 vs. AI = 2.6 +/- 0.5; p < 0.01). The decreased inflammatory index associated with anti-TGF-beta treatment was consistent with histopathologic and radiologic evidence of a therapeutic response. These data implicate TGF-beta as a profound agonist not only in the early events responsible for synovial inflammation, but also in the chronicity of streptococcal cell wall fragment-induced inflammation culminating in destructive pathology. Interrupting the cycle of leukocyte recruitment and activation with TGF-beta antagonists may provide a mechanism for resolution of chronic destructive lesions.     

Transforming growth factor beta 1 (TGF-beta 1) induced neutrophil recruitment to synovial tissues: implications for TGF-beta-driven synovial inflammation and hyperplasia

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.     

Constitutive production of inflammatory and mitogenic cytokines by rheumatoid synovial fibroblasts

Conditioned media obtained from fibroblasts cultured from rheumatoid and certain other inflammatory synovia were observed to stimulate [3H]thymidine incorporation in an indicator murine fibroblast line. Synovial fibroblasts derived from the joints of patients with osteoarthritis did not display this property. This effect persisted in culture for many weeks and occurred in the absence of co-stimulatory immune cells. Antibody neutralization studies implicated a role for basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF-beta), granulocyte/macrophage colony-stimulating factor (GM-CSF), and interleukin 1 beta (IL-1 beta) in the increased proliferative activity of synovial fibroblast-conditioned media. Synovial cell synthesis of bFGF, TGF beta 1, GM-CSF, IL-1 beta, and IL-6 was confirmed by 35S-methionine labeling and immunoprecipitation. The constitutive production of inflammatory and mitogenic cytokines by synovial fibroblasts may represent the result of long-term, phenotypic changes that occurred in vivo. Persistent cytokine production by synovial fibroblasts may play an important role in the continued recruitment and activation of inflammatory cells in chronic arthritis and in the formation of rheumatoid pannus.     

Spontaneous production of fibroblast-activating factor(s) by synovial inflammatory cells. A potential mechanism for enhanced tissue destruction

A characteristic feature of rheumatoid arthritis is hyperplasia of the synovial lining cells and fibroblasts, the source of tissue-degrading mediators, in association with the appearance and persistence of lymphocytes in affected joints. Diseased synovial tissue obtained at arthroscopy from 10 of 12 rheumatoid arthritis patients was found to release a factor(s) that could stimulate quiescent fibroblasts to proliferate in vitro. Mononuclear cells isolated from this synovial tissue and from the synovial fluid spontaneously produced fibroblast-activating factor(s) (FAF). In contrast, synovial tissue from patients with noninflammatory joint disease did not release FAF. By gel filtration, FAF was detected in two peaks (40,000 and 15,000 mol wt) that were consistent with the previously described peripheral blood T lymphocyte- and monocyte-derived factors with identical activity. The mononuclear cells were predominantly OKT3+/Leu-1+ T lymphocytes and OKM1+ cells of monocyte/macrophage lineage that expressed HLA-DR antigens, suggesting prior activation of these cells. Mononuclear cells isolated from the peripheral blood of these patients did not spontaneously secrete FAF. Lymphocytes and monocytes from the site of synovial inflammation appear to be activated in situ to produce factors that may contribute to the hyperplasia and overgrowth of the synovial membrane in rheumatoid arthritis.     

Plasmid DNA encoding transforming growth factor-beta1 suppresses chronic disease in a streptococcal cell wall-induced arthritis model.

Transforming growth factor beta is a potent immunomodulator with both pro- and antiinflammatory activities. Based on its immunosuppressive actions, exogenous TGF-beta has been shown to inhibit autoimmune and chronic inflammatory diseases. To further explore the potential therapeutic role of TGF-beta, we administered a plasmid DNA encoding human TGF-beta1 intramuscularly to rats with streptococcal cell wall-induced arthritis. A single dose of 300 microg plasmid DNA encoding TGF-beta1, but not vector DNA, administered at the peak of the acute phase profoundly suppressed the subsequent evolution of chronic erosive disease typified by disabling joint swelling and deformity (articular index = 8.17+/-0. 17 vs. 1.25+/-0.76, n = 6, day 26, P < 0.01). Moreover, delivery of the TGF-beta1 DNA even as the chronic phase commenced virtually eliminated subsequent inflammation and arthritis. Both radiologic and histopathologic as well as molecular evidence supported the marked inhibitory effect of TGF-beta1 DNA on synovial pathology, with decreases in the inflammatory cell infiltration, pannus formation, cartilage and bone destruction, and the expression of proinflammatory cytokines that characterize this model. Increases in TGF-beta1 protein were detected in the circulation of TGF-beta1 DNA-treated animals, consistent with the observed therapeutic effects being TGF-beta1 dependent. These observations provide the first evidence that gene transfer of plasmid DNA encoding TGF-beta1 provides a mechanism to deliver this potent cytokine that effectively suppresses ongoing inflammatory pathology in arthritis.     

Synovial fluid ferritin in traumatic hemarthrosis, rheumatoid arthritis and osteoarthritis.

Synovial fluid ferritin levels in patients with traumatic hemarthrosis (HA), rheumatoid arthritis (RA), and osteoarthritis (OA) were measured by double antibody radioimmunoassay. Synovial fluid ferritin levels were significantly higher in 60 patients with HA (mean +/- S.D., 536 +/- 536 ng/ml) and 39 patients with RA (614 +/- 486 ng/ml) than in 20 patients with OA (130 +/- 119 ng/ml) (p < 0.01). Individual levels, however, considerably varied. In HA patients, the synovial fluid ferritin level correlated well with the duration of hemarthrosis, but not with hemoglobin, hematocrit, or an inflammatory synovial fluid index such as the leukocyte count. In RA patients, there was no significant correlation between the synovial fluid ferritin levels and any inflammatory parameter, such as catalase activity, synovial leukocyte counts (including polymorphs and monocytes) or the duration of arthritis. Our results indicate that the synovial fluid ferritin level reflects primarily hemoglobin degradation and appears unrelated to inflammation in joint diseases.     

The effects of bupivacaine and neostigmine on articular cartilage and synovium in the rabbit knee joint

We investigated the effects of intra-articular injections of bupivacaine and neostigmine on articular cartilage and the synovial membrane of rabbit knee joints. Saline, bupivacaine or neostigmine were each administered intra-articularly into 15 knee joints. Five joints per drug treatment were prepared for histopathological examination 24 h, 48 h and 10 days after injection. A pathologist examined the histological samples for inflammation of the articular cartilage, inflammatory cell infiltration, hypertrophy and hyperplasia of the synovial membrane, in a blinded manner. There no histopathological in the saline-treated control joints. Joints treated with bupivacaine and neostigmine showed significantly more histopathological changes than control joints. Joints treated with neostigmine showed significantly more histopathological changes than those treated with bupivacaine, except for articular cartilage inflammation on day 10. We conclude that intra-articular bupivacaine and neostigmine cause histopathological changes in rabbit knee joints, with neostigmine having a greater effect than bupivacaine.     

Cultured human synovial fibroblasts rapidly metabolize kinins and neuropeptides.

Kinins and substance P have been implicated in the pathogenesis of inflammatory arthritis by virtue of their abilities to induce vasodilation, edema, and pain. The relative biological potencies of these peptides in vivo would depend at least in part upon their rates of catabolism in the joint. We hypothesized that human synovial lining cells may regulate intraarticular levels of kinins and neuropeptides via degradation by cell surface-associated peptidases. We exposed intact human synovial fibroblasts to kinins and substance P, in the presence or absence of specific peptidase inhibitors, and measured the amount of intact substrate remaining and degradation product(s) generated over time. Aminopeptidase M (AmM; EC 3.4.11.2), neutral endopeptidase-24.11 (NEP-24.11; EC 3.4.24.11), and dipeptidyl(amino)peptidase IV (DAP IV; EC 3.4.14.5) were identified on the cell surface of synovial cells. Bradykinin degradation was due entirely to NEP-24.11 (1.39 +/- 0.29 nmol/min per well). Lysylbradykinin was also degraded by NEP-24.11 (0.80 +/- 0.19 nmol/min per well); however, in the presence of phosphoramidon, AmM-mediated conversion to bradykinin (3.74 +/- 0.46 nmol/min per well) could be demonstrated. The combined actions of NEP-24.11 (0.93 +/- 0.15 nmol/min per well) and DAP IV (0.84 +/- 0.18 nmol/min per well) were responsible for the degradation of substance P. AmM (2.44 +/- 0.33 nmol/min per well) and NEP-24.11 (1.30 +/- 0.45 nmol/min per well) were responsible for the degradation of the opioid peptide, [Leu5]enkephalin. The identity of each of the three peptidases was confirmed via synthetic substrate hydrolysis, inhibition profile, and immunological identification. The profiles of peptidase enzymes identified in cells derived from rheumatoid and osteoarthritic joints were identical. These data demonstrate the human synovial fibroblast to be a rich source of three specific peptidases and suggest that it may play a prominent role in regulating peptide levels in the joint.     

Effects of platelet-derived growth factor and other polypeptide mitogens on DNA synthesis and growth of cultured rat liver fat-storing cells.

In vitro and in vivo studies suggest that liver fat-storing cells (FSC) may play an important role in the development of liver fibrosis. We explored the effects of platelet-derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor (TGF)-alpha and TGF-beta, and basic fibroblast growth factor (bFGF) on DNA synthesis and growth of rat liver FSC. PDGF, EGF, TGF-alpha, and bFGF induced a dose-dependent increase in DNA synthesis with a peak effect at 24 h. PDGF produced the most striking effect with a maximum 18-fold increase over control. EGF, TGF-alpha, and bFGF elicited a maximum three- to fourfold increase in DNA synthesis. Analysis of growth curves revealed a similar pattern of potency of the growth factors. TGF-beta did not affect DNA synthesis of FSC; however, TGF-beta markedly potentiated the stimulatory effects of both EGF and PDGF. FSC showed high specific binding of 125I-PDGF and Scatchard analysis revealed high affinity receptors with an apparent Kd of 2.3 x 10(-10) M. Our data suggest that PDGF is a key mitogen for FSC and that the coordinate release of other growth factors together with PDGF by inflammatory cells represents a potent potential stimulus for FSC proliferation in conditions of chronic self-perpetuating liver inflammation.     

Human Alveolar Macrophage Growth Factor for Fibroblasts: REGULATION AND PARTIAL CHARACTERIZATION

The number of fibroblasts composing the alveolar structures in controlled within narrow limits by a strictly modulated rate of fibroblast replication. One possible source of growth-modulating signals for alveolar fibroblasts is the alveolar macrophage, a member of the mononuclear phagocyte family of cells, which collectively are known to be important sources of growth factors for a variety of target cells. To evaluate the role of alveolar macrophages in the control of alveolar fibroblast replication, macrophages from normal individuals obtained by bronchoalveolar lavage were maintained in suspension culture with and without added stimuli, and supernates were evaluated for fibroblast growth-promoting effect. Supernates from unstimulated macrophages contained no growth factor activity. In marked contrast, supernates from macrophages stimulated with particulates and immune complexes contained a growth factor that caused a significant increase in fibroblast replication rate. Maximum growth factor activity was observed 3-4 h after macrophage stimulation, at a concentration of 1-2 × 10⁶ macrophages/ml. The alveolar macrophagederived growth factor eluted from DEAE-cellulose at 0.27 M NaCl at neutral pH had an apparent molecular weight of 18,000, and appeared to be distinct from other characterized growth factors. The alveolar macrophage-derived growth factor stimulated lung fibroblast DNA synthesis within 12 h, with cell division apparent within 48 h. In serum-free culture, the alveolar macrophage-derived growth factor by itself did not promote fibroblast replication, but rather acted as a progression factor causing a synergistic increase in fibroblast replication rate in the presence of competence factors such as fibroblast growth factor or platelet-derived growth factor. These studies suggest that when stimulated, human alveolar macrophages may modulate, in part, the replication rate of alveolar fibroblasts by releasing a growth factor within the alveolar microenvironment.     

Ⅱ型遗传性出血性毛细血管扩张症血管生长发育相关蛋白质分析及意义

目的分析和检测1个Ⅱ型遗传性出血性毛细血管扩张症(HHT-2)家系成员血浆及外周血白细胞转化生长因子(TGF)-β1、TGF-β2、血管内皮生长因子(VFGF)、血小板源性生长因子受体α(PDGFRα)等蛋白质浓度,探讨这些血管生长发育相关蛋白在 HHT 发病机制中的变化及意义。方法根据鼻出血、毛细血管扩张及家族史进行 HHT 的临床诊断,并经基因筛查,以证实 HHT 的诊断。对该家系中5例新成员进行 HHT 的诊断和排除诊断。用 ELSA 方法检测该家系成员血浆 TGF-β1、TGF-β2及 VEGF 浓度;用流式细胞术结合直接按或间接免疫荧比技术分析该家系成员外周血白细胞TGF-β1、VEGF、及 PDGFRα蛋白表达。结果基因筛查的5例新家系成员 ALK1基因8号外显子不存在 C1231T 突变。该家系成员中3例 HHT 患者血浆 TGF-β1和 TGF-β2浓度分别为(16 954±3 709)ng/L和(11 548±2 611)ng/L;与正常对照相比差异无统计学意义(P>0.05);3例 HHT 患者、6名未受累家系成员、6名正常对照血浆 VEGF 浓度值分别为(179.2±22.0)μg/L,(149.8±22.7)μg/L,(132.9±21.0)μg/L;HHT 患告血浆 VEGF 浓度明显高于正常对照组(P<0.05)。HHT 家系成员外周血白细胞 PDGFRα和 VEGF 蛋白表达均上调(P 值均<0.05);TGF-β1蛋白表达与正常对照比较差异无统计学意义。结论 HHT-2患者血浆 TGF-β1浓度、单核细胞及粒细胞 TGF-β1蛋白表达与正常对照比较差异无统计学意义;而血浆 VEGF 浓度及外周血白细胞 VEGF 表达均明显高于正常对照;HHT-2患者外周血白细胞存在 PDGFRα高表达,提示存在血管生长发育相关蛋白的代偿机制。     

Hypoxia-reoxygenation-induced apoptosis in cultured adult rat myocytes and the protective effect of platelets and transforming growth factor-beta(1).

The outcome of myocardial ischemia-reperfusion has been partially attributed to the degree of apoptosis in cardiomyocytes. Aggregating platelets by release of transforming growth factor-beta(1) (TGF-beta(1)) protect the isolated heart against ischemia-reperfusion injury and preserve myocardial TGF-beta(1) content. To gain more insight into the modulation of hypoxia-reoxygenation-induced injury (apoptosis and necrosis) to myocytes by TGF-beta(1) and aggregating platelets, cultured adult rat myocytes were exposed for 48 or 72 h to hypoxia alone, or to hypoxia followed by 3 h of reoxygenation. Apoptosis in the cells was determined by in situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining and DNA fragmentation on gel electrophoresis. Hypoxia alone caused a time-dependent increase in myocyte apoptosis (number of apoptotic cells: 19+/-3% at 48 h and 39+/-5% at 72 h compared with 5+/-1% in control cells, based on a 500-cell count). Three hours of reoxygenation after 48 h of hypoxia further increased the number of apoptotic cells (34+/-8 versus 19+/-3% in hypoxia for 48 h), but reoxygenation after 72 h of hypoxia did not additionally increase the number of apoptotic cells, perhaps because of extensive cell necrosis on prolonged hypoxia. Forty-eight hours of hypoxia followed by 3 h of reoxygenation also resulted in a decrease in Bcl-2 and an increase in Fas protein level. Incubation of myocytes with either recombinant TGF-beta(1) (0.5-5 ng/ml) or aggregated platelet supernatant (from 2-3 x10(7) platelets/ml, containing approximately 0.5 ng/ml of TGF-beta(1)) markedly (P<.01) decreased the number of apoptotic cells after hypoxia-reoxygenation. Incubation with TGF-beta(1) also reduced myocyte necrosis as evident from lactate dehydrogenase release and trypan blue dye exclusion. These data demonstrate that hypoxia-reoxygenation results in apoptosis and necrosis in cultured adult rat myocytes; this can be attenuated by TGF-beta(1). Similarity of data with TGF-beta(1) and aggregated platelet supernatant suggests that platelet-mediated cardioprotection during hypoxia-reoxygenation may relate in part to the release of TGF-beta(1).     

Anchorage-independent growth of synoviocytes from arthritic and normal joints. Stimulation by exogenous platelet-derived growth factor and inhibition by transforming growth factor-beta and retinoids.

Exuberant tumor-like synovial cell proliferation with invasion of periarticular bone is a feature of rheumatoid arthritis in humans and of streptococcal cell wall (SCW)-induced arthritis in rats. These histologic observations prompted us to examine synoviocytes from arthritic joints for phenotypic characteristics of transformed cells. The capacity to grow in vitro under anchorage-independent conditions is a characteristic that correlates closely with potential in vivo tumorigenicity. In medium supplemented with 20% serum or in basal media supplemented with platelet-derived growth factor (PDGF), early passage synoviocytes from both SCW-induced and rheumatoid arthritic joints formed colonies in soft agarose. Epidermal growth factor (EGF), interleukin 1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), and transforming growth factor-beta (TGF-beta) did not support growth, although EGF enhanced PDGF-dependent growth. On the other hand, TGF-beta, as well as all-trans-retinoic acid, inhibited colony growth. Early passage normal rat and human synoviocytes also grew under the same conditions, but lung, skin, and late-gestation embryonic fibroblast-like cells did not. Considered in the context of other published data our findings provide cogent evidence that synoviocytes, but not other types of fibroblast-like cells, readily acquire phenotypic characteristics commonly associated with transformed cells. Expression of the transformed phenotype in the inflammatory site is likely regulated by paracrine growth factors, such as PDGF and TGF-beta.     

Synthetic fibronectin peptides suppress arthritis in rats by interrupting leukocyte adhesion and recruitment.

In an experimental model of arthritis, increased leukocyte adhesion is associated with the evolution of acute and chronic synovial inflammation. Whereas peripheral blood mononuclear cells (PBMC) from control animals bind minimally to fibronectin matrices, PBMC from animals receiving arthropathic doses of bacterial cell walls demonstrate increased integrin mRNA expression and enhanced adhesion. To determine whether this augmented adhesion was causal in the development of synovial pathology, peptides synthesized from several fibronectin domains which inhibited leukocyte adhesion in vitro were administered to arthritic animals either as free peptides or coupled to a carrier molecule. Not only were peptides containing either the RGD or CS-1 cell-binding domains inhibitory to chronic synovial pathology (articular index = 10.5 +/- 0.3 for untreated animals compared to 1.25 +/- 0.25 for RGD and 2.5 +/- 0.7 for CS-1), but three peptides synthesized from the carboxy-terminal 33-kD heparin-binding domain of fibronectin were also found to significantly inhibit leukocyte recruitment and the evolution of arthritis. Based on these data, which are the first to explore the therapeutic potential of heparin-binding fibronectin peptides in chronic inflammation, it appears that antagonism of cellular adhesion and recruitment by fibronectin peptides may provide an important mechanism for modulating the multi-step adhesion process and attenuating aberrant inflammatory responses.     

leflunomide对IgA肾病大鼠肾脏保护作用的机制研究

目的 观察leflunomide对实验性IgA肾病(IgAN)大鼠肾脏病理及肾组织转化生长因子-β1(TGF-β1)、单核细胞趋化因子-1(MCP-1)表达的影响.方法 建立IgAN大鼠模型,随机分成模型组、泼尼松组、leflunomide组,同时设立正常对照组.用免疫荧光和光镜观察免疫复合物在肾脏的沉积及系膜区基质增生程度;用免疫组化和逆转录-聚合酶链反应(RT-PCR)方法分别检测肾组织TGF-β1、MCP-1的蛋白和基因表达水平.结果 与模型组比较,leflunomide组免疫复合物在肾脏的沉积明显减少,系膜区基质增生程度显著减轻(P均＜0.01);leflunomide在基因和蛋白水平均能够有效抑制TGF-β1和MCP-1在肾组织中的表达(P＜0.05或P＜0.01).结论 leflunomide能减少免疫复合物在肾脏的沉积,并且下调TGF-β1、MCP-1在肾脏的表达,减少局部炎症反应,减轻系膜区基质增生,延缓肾脏纤维化的进程,保护肾脏.     

Monocyte activation in angiogenesis and collateral growth in the rabbit hindlimb.

We have previously shown that monocytes adhere to the vascular wall during collateral vessel growth (arteriogenesis) and capillary sprouting (angiogenesis). In this study we investigated the association of monocyte accumulation with both the production of the cytokines-basic fibroblast growth factor (bFGF) and TNF-alpha-and vessel proliferation in the rabbit after femoral artery occlusion. In particular, we studied the effects of an increase in monocyte recruitment by LPS on capillary density as well as collateral and peripheral conductance after 7 d of occlusion. Monocytes accumulated around day 3 in collateral arteries when maximal proliferation was observed, and stained strongly for bFGF and TNF-alpha. In the lower limb where angiogenesis was shown to be predominant, macrophage accumulation was also closely associated with maximal proliferation (around day 7). LPS treatment significantly increased capillary density (424+/-26.1 n/mm2 vs. 312+/-20.7 n/mm2; P < 0.05) and peripheral conductance (109+/-33.8 ml/min/100 mmHg vs. 45+/-6.8 ml/min/100 mmHg; P < 0.05) as compared with untreated animals after 7 d of occlusion. These results indicate that monocyte activation plays a major role in angiogenesis and collateral artery growth.     

Macrophage production of transforming growth factor beta and fibroblast collagen synthesis in chronic pulmonary inflammation

A rat model of bleomycin-induced pulmonary inflammation and fibrosis was used to examine the relationship between collagen synthesis and transforming growth factor beta (TGF-beta) production, and cellular distribution. Total lung TGF-beta was elevated within 2 h of intratracheal bleomycin administration and peaked 7 d later at levels 30-fold higher than controls. This was followed by a gradual decline with lower but persistent levels of production in the late phase of the response between 21 and 28 d later. The peak TGF-beta levels preceded the maximum collagen and noncollagen protein synthesis measured by [3H]proline incorporation into lung fibroblast explants of bleomycin-treated rats. The pattern of immunohistochemical staining localized TGF-beta initially in the cytoplasm of bronchiolar epithelium cells and subepithelial extracellular matrix. The peak of lung TGF-beta levels at 7 d coincided with intense TGF-beta staining of macrophages dispersed in the alveolar interstitium and in organized clusters. Later in the course of the response. TGF-beta was primarily associated with extracellular matrix in regions of increased cellularity and tissue repair, and coincided with the maximum fibroblast collagen synthesis. This temporal and spatial relationship between collagen production and TGF-beta production by macrophages suggests an important if not primary role for TGF-beta in the pathogenesis of the pulmonary fibrosis.     

TGF-beta promotes proliferation of cultured SMC via both PDGF-AA-dependent and PDGF-AA-independent mechanisms.

Transforming growth factor-beta 1 (TGF-beta 1) has been implicated in mediating smooth muscle cell (SMC) growth after vascular injury. Studies examining TGF-beta-induced growth of cultured SMC have identified only modest mitogenic effects which are largely dependent on autocrine production of platelet-derived growth factor-AA (PDGF-AA). Recent studies have suggested, however, that TGF-beta also may have delayed growth effects independent of PDGF-AA. The aims of the present studies were to examine the effects of TGF-beta on chronic growth responses of cultured SMC. Results demonstrated that TGF-beta elicited a delayed growth response (24 fold increase in 3H-TdR incorp. from 48-72 h) and enhanced SMC production of PDGF-AA (eightfold increase at 24 h). Neutralizing antibodies to PDGF-AA, however, inhibited only 10-40% of delayed TGF-beta-induced growth. Co-treatment with TGF-beta transiently delayed epidermal growth factor (EGF)-, basic fibroblast growth factor (bFGF)-, or PDGF-BB-induced entry into S phase but enhanced the delayed growth responses to these growth factors by 16.0-, 5.8-, or 4.2-fold, respectively. Neutralizing antibodies to PDGF-AA had no effect on these synergistic responses and exogenous PDGF-AA did not increase growth responses to EGF, bFGF, or PDGF-BB. In summary, TGF-beta induces marked delayed growth responses, alone and in combination with EGF, bFGF or PDGF-BB, that are largely independent of PDGF-AA.     

Fibrin(ogen) exacerbates inflammatory joint disease through a mechanism linked to the integrin alphaMbeta2 binding motif

Fibrin deposition within joints is a prominent feature of arthritis, but the precise contribution of fibrin(ogen) to inflammatory events that cause debilitating joint damage remains unknown. To determine the importance of fibrin(ogen) in arthritis, gene-targeted mice either deficient in fibrinogen (Fib-) or expressing mutant forms of fibrinogen, lacking the leukocyte receptor integrin alphaMbeta2 binding motif (Fibgamma390-396A) or the alphaIIbbeta3 platelet integrin-binding motif (FibgammaDelta5), were challenged with collagen-induced arthritis (CIA). Fib- mice exhibited fewer affected joints and reduced disease severity relative to controls. Similarly, diminished arthritis was observed in Fibgamma390-396A mice, which retain full clotting function. In contrast, arthritis in FibgammaDelta5 mice was indistinguishable from that of controls. Fibrin(ogen) was not essential for leukocyte trafficking to joints, but appeared to be involved in leukocyte activation events. Fib- and Fibgamma390-396A mice with CIA displayed reduced local expression of TNF-alpha, IL-1beta, and IL-6, which suggests that alphaMbeta2-mediated leukocyte engagement of fibrin is mechanistically upstream of the production of proinflammatory mediators. Supporting this hypothesis, arthritic disease driven by exuberant TNF-alpha expression was not impeded by fibrinogen deficiency. Thus, fibrin(ogen) is an important, but context-dependent, determinant of arthritis, and one mechanism linking fibrin(ogen) to joint disease is coupled to alphaMbeta2-mediated inflammatory processes.