Selective inhibition of inducible nitric oxide synthase exacerbates erosive joint disease.

NO is an essential cytotoxic agent in host defense, yet can be autotoxic if overproduced, as evidenced in inflammatory lesions and tissue destruction in experimental arthritis models. Treatment of streptococcal cell wal1-induced arthritis in rats with N:(G)-monomethyl-L-arginine (L-NMMA), a competitive nonspecific inhibitor of both constitutive and inducible isoforms of NO synthase (NOS), prevents intraarticular accumulation of leukocytes, joint swelling, and bone erosion. Because increased inducible NOS (iNOS) expression and NO generation are associated with pathogenesis of chronic inflammation, we investigated whether a selective inhibitor of iNOS, N:-iminoethyl-L-lysine (L-NIL), would have more directed anti-arthritic properties. Whereas both L-NMMA and L-NIL inhibited nitrite production by streptococcal cell wall-stimulated rat mononuclear cells in vitro and systemic treatment of arthritic rats with L-NMMA ablated synovitis, surprisingly L-NIL did not mediate resolution of inflammatory joint lesions. On the contrary, daily administration of L-NIL failed to reduce the acute response and exacerbated the chronic inflammatory response, as reflected by profound tissue destruction and loss of bone and cartilage. Although the number of iNOS-positive cells within the synovium decreased after treatment with L-NIL, immunohistochemical analyses revealed a distinct pattern of endothelial and neuronal NOS expression in the arthritic synovium that was unaffected by the isoform-specific L-NIL treatment. These studies uncover a contribution of the constitutive isoforms of NOS to the evolution of acute and chronic inflammation pathology which may be important in the design of therapeutic agents.

Mice lacking Smad3 show accelerated wound healing and an impaired local inflammatory response.

The generation of animals lacking SMAD proteins, which transduce signals from transforming growth factor-beta (TGF-beta), has made it possible to explore the contribution of the SMAD proteins to TGF-beta activity in vivo. Here we report that, in contrast to predictions made on the basis of the ability of exogenous TGF-beta to improve wound healing, Smad3-null (Smad3ex8/ex8) mice paradoxically show accelerated cutaneous wound healing compared with wild-type mice, characterized by an increased rate of re-epithelialization and significantly reduced local infiltration of monocytes. Smad3ex8/ex8 keratinocytes show altered patterns of growth and migration, and Smad3ex8/ex8 monocytes exhibit a selectively blunted chemotactic response to TGF-beta. These data are, to our knowledge, the first to implicate Smad3 in specific pathways of tissue repair and in the modulation of keratinocyte and monocyte function in vivo.

Secretory leukocyte protease inhibitor suppresses the inflammation and joint damage of bacterial cell wall-induced arthritis.

Disruption of the balance between proteases and protease inhibitors is often associated with pathologic tissue destruction. To explore the therapeutic potential of secretory leukocyte protease inhibitor (SLPI) in erosive joint diseases, we cloned, sequenced, and expressed active rat SLPI, which shares the protease-reactive site found in human SLPI. In a rat streptococcal cell wall (SCW)-induced model of inflammatory erosive polyarthritis, endogenous SLPI was unexpectedly upregulated at both mRNA and protein levels in inflamed joint tissues. Systemic delivery of purified recombinant rat SLPI inhibited joint inflammation and cartilage and bone destruction. Inflammatory pathways as reflected by circulating tumor necrosis factor alpha and nuclear factor kappaB activation and cartilage resorption detected by circulating levels of type II collagen collagenase-generated cleavage products were all diminished by SLPI treatment in acute and chronic arthritis, indicating that the action of SLPI may extend beyond inhibition of serine proteases.

Hemoglobin protects from streptococcal cell wall-induced arthritis.

OBJECTIVE: To investigate the ability of hemoglobin (Hgb), a nitric oxide (NO) scavenger, to deplete excess NO and reduce inflammation and injury in synovial tissue from joints with inflammatory arthritis. METHODS: The severity of streptococcal cell wall-induced arthritis was monitored following administration of Hgb. Plasma nitrite and nitrate levels were measured, and inducible NO synthase (iNOS) and cytokine messenger RNA (mRNA) expression in peripheral blood mononuclear cells (PBMC) and joint tissue were evaluated. RESULTS: Following systemic administration of Hgb to arthritic rats, plasma levels of nitrite and nitrate as well as iNOS mRNA expression in the joints and PBMC were significantly reduced. Moreover, inflammatory cell accumulation and disease pathology in the joint tissue were dramatically attenuated without obvious side effects. Consistent with this reduction in the inflammatory response, cytokine gene expression was decreased in the synovium of Hgb-treated rats. CONCLUSION: Modulation of NO levels through the use of a NO scavenger, Hgb, influences the development and severity of arthritis. These findings suggest that depletion of excess NO by NO scavengers provides a prototype for further exploration of potential treatments for chronic arthritis.

Lacrimal gland inflammation is responsible for ocular pathology in TGF-beta 1 null mice.

Mice homozygous for a nonfunctional transforming growth factor-beta 1 gene develop rampant inflammation in vital organs that contributes to a shortened life span. The presence of circulating anti-nuclear anti-bodies, immune deposits in tissues, leukocyte infiltration, and increased major histocompatibility complex antigen expression resembles an autoimmune-like syndrome. One of the overt symptoms that appears in these mice lacking transforming growth factor-beta 1 is the development of dry crusty eyes that close persistently as their health declines. Histologically, the eyes appear normal with little or no inflammation. However, inflammatory lesions, predominantly lymphocytic, develop in the lacrimal glands, disrupting their structure and function and severely limiting their ability to generate tears. This histopathology and aberrant function mimic that of Sjögren's syndrome, a human autoimmune disease characterized by dry eyes and dry mouth. Impeding the leukocyte infiltration into the glands with synthetic fibronectin peptides, which block adhesion, not only prevents the inflammatory pathology but also prevents the persistent eye closure characteristic of these mice.

Autoimmune Sjögren's-like lesions in salivary glands of TGF-beta1-deficient mice are inhibited by adhesion-blocking peptides.

The targeted disruption of the TGF-beta1 gene in mice (TGF-beta1 -/-) leads to extensive inflammation in vital organs, cachexia, and death within 3 to 4 wk. Significant inflammatory lesions develop initially in the periductal regions of the salivary glands and escalate as the animals become symptomatic. These inflammatory sites, characterized by lymphocytic infiltration and increased proliferation, cytokine mRNA expression, and IgG-positive cells, resemble lesions of Sjögren's syndrome. Moreover, the inflammatory pathology, enhanced MHC expression, and Ab production are consistent with an autoimmune-like etiology. Glandular atrophy and loss of acini with reduced saliva production appear to contribute to the wasting syndrome characteristic of the TGF-beta1 -/- mice. To determine whether the structural and functional defects were developmental due to the absence of TGF-beta1 or secondary to the inflammation, TGF-beta1 -/- mice were treated with synthetic fibronectin peptides, which block leukocyte infiltration. Daily systemic injections of RGD, CS-1, and/or peptides derived from the heparin-binding region of the A chain not only prevented leukocyte infiltration in the salivary glands of the TGF-beta1 -/- mice, but also reversed the acinar and ductal derangements. These data suggested that salivary gland development is not jeopardized in the absence of TGF-beta1, but that the extensive infiltration of inflammatory cells compromises glandular structure and function. The essential nature of TGF-beta1 in controlling inflammatory and immune processes is confirmed by these studies. Moreover, these TGF-beta1 -/- mice provide an important model of autoimmune disease that can be used in the design of therapeutic interventions.

Immune dysregulation in TGF-beta 1-deficient mice.

Approximately 2 wk after birth, mice having a TGF-beta 1 null mutation (TGF-beta 1(-/-)) exhibit a progressive wasting syndrome and death. Associated with this phenotype is a multifocal infiltration of lymphocytes and macrophages into target organs, especially the heart, lungs, and salivary glands. To explore the consequences of TGF-beta 1 deficiency on the immune system, lymphocyte phenotype and function were analyzed. Initially, lymphoid organ architecture seemed to be normal and, as symptoms developed, the thymus decreased in size, whereas lymph nodes were enlarged. Phenotypically, the TGF-beta 1(-/-) lymphoid cells seemed to be more differentiated in the thymus and activated in the lymph nodes, but remarkably unaffected in the spleen. Moreover, TGF-beta 1(-/-) spleen and lymph nodes displayed enhanced numbers of proliferating cells, as measured by proliferating cell nuclear Ag and/or cyclin-dependent kinase levels. Consistent with this hyperproliferative response, constitutive levels of IL-2 mRNA were elevated in the thymus and both IL-2 and IL-2R mRNA were increased in the lymph nodes. In contrast with the activation profile of TGF-beta 1(-/-) lymphoid cells in vivo, mitogen challenge of these cells in vitro revealed suppressed proliferation that was associated with a defect in inducible IL-2 mRNA expression and IL-2 secretion. Moreover, the addition of rIL-2 restored the deficient mitogen-induced proliferation. The mechanism leading to T cell anergy remains unclear; however, these data confirm the essential role for TGF-beta 1 in maintaining normal immune function.

Suppression of arthritis by an inhibitor of nitric oxide synthase.

Nitric oxide (NO), a toxic radical gas produced during the metabolism of L-arginine by NO synthase (NOS), has been implicated as a mediator of immune and inflammatory responses. A single injection of streptococcal cell wall fragments (SCW) induces the accumulation of inflammatory cells within the synovial tissue and a cell-mediated immune response that leads destructive lesions. We show here that NO production is elevated in the inflamed joints of SCW-treated rats. Administration of NG-monomethyl-L-arginine, an inhibitor of NOS, profoundly reduced the synovial inflammation and tissue damage as measured by an articular index and reflected in the histopathology. These studies implicate the NO pathway in the pathogenesis of an inflammatory arthritis and demonstrate the ability of a NOS inhibitor to modulate the disease.

Role of transforming growth factor beta in the pathophysiology of chronic inflammation.

Transforming growth factor beta (TGF-beta), a cytokine identified in acute and chronic inflammatory sites, mediates leukocyte recruitment and activation essential to the development of such lesions. Released by platelets upon aggregation and by leukocytes stimulated with bacterial products or inflammatory mediators, TGF-beta has potent chemotactic activity for blood neutrophils, monocytes, and lymphocytes. By augmenting integrin expression, TGF-beta facilitates leukocyte adhesion to the vessel wall and extracellular matrix at the site of inflammation. Once within the inflammatory site, mononuclear cells are stimulated by TGF-beta to release cytokines important in the network of molecules regulating the host response to microorganisms and immunologic challenge. Thus, bacteria and their products, in addition to directly recruiting and activating leukocytes at sites of infection, indirectly influence these events through the induction of cytokines such as TGF-beta. By antagonizing the activity of TGF-beta with neutralizing antibodies, a causal relationship between this cytokine, inflammation, and pathogenesis has been demonstrated. Administration of anti-TGF-beta to sites of chronic destructive inflammation not only blocked leukocyte recruitment and activation, but also inhibited the subsequent destruction of bone and cartilage characteristics of such lesions.