Pivotal role of matrix metalloproteinase 13 in extracellular matrix turnover in idiopathic pulmonary fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a fatal disease characterized by excessive deposition of extracellular matrix (ECM). OBJECTIVES: We investigated the regulation of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) in lung fibrosis. METHODS: MMP and TIMP expression, collagenolytic activity and collagen content was assessed in IPF (n=16) versus donor (n=6) lung homogenates and accomplished by in-situ-zymography for gelatinolytic and collagenolytic activities, combined with MMP antigen detection. Role of MMP13 was assessed employing the bleomycin model of lung fibrosis in MMP-13(-/-) versus wild-type mice. MEASUREMENTS AND MAIN RESULTS: In IPF, MMPs-1, 2, 7, 9 and 13, but not MMP-8, were significantly upregulated, whereas none of the TIMPs (1-4) were significantly altered. Collagen content was slightly increased and collagenolytic activity was most prominent in the airways and co-localized with MMP-13. We observed an exaggerated early inflammatory response and an augmented lung fibrosis in bleomycin-challenged MMP-13(-/-) versus wild-type mice, with elevated lung collagen content 28d after bleomycin challenge in the MMP-13(-/-) mice. CONCLUSIONS: Our data suggest that i) collagen deposition in IPF lungs is not primarily due to excessive TIMP production, but rather due to overwhelming ECM production in face of an overall increased, but spatially imbalanced collagenolytic activity, ii) preferential distribution of collagenolytic activity, largely MMP-13, in the airways offers an explanation for the development of honeycomb cysts and iii) despite an overall increase in inflammatory cell content the presence of MMP-13 seems to limit the overall extent of ECM deposition in lung fibrosis.

Neuroimmune semaphorin 4A as a drug and drug target for asthma.

Neuroimmune semaphorin 4A (Sema4A) has been shown to play an important costimulatory role in T cell activation and regulation of Th1-mediated diseases such as multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE), and experimental autoimmune myocarditis (EAM). Sema4A has three functional receptors, Tim-2 expressed on CD4+ T cells, Th2 cells in particular, and Plexin B1 and D1 predominantly expressed on epithelial and endothelial cells, correspondingly. We recently showed that Sema4A has a complex expression pattern in lung tissue in a mouse model of asthma. We and others have shown that corresponding Plexin expression can be found on immune cells as well. Moreover, we demonstrated that Sema4A-deficient mice displayed significantly higher lung local and systemic allergic responses pointing to its critical regulatory role in the disease. To determine the utility of Sema4A as a novel immunotherapeutic, we introduced recombinant Sema4A protein to the allergen-sensitized WT and Sema4A(-/-) mice before allergen challenge. We observed significant reductions in the allergic inflammatory lung response in Sema4A-treated mice as judged by tissue inflammation including eosinophilia and mucus production. Furthermore, we demonstrated that in vivo administration of anti-Tim2 Ab led to a substantial upregulation of allergic inflammation in WT mouse lungs. These data highlight the potential to develop Sema4A as a new therapeutic for allergic airway disease.

Treatment of experimental asthma using a single small molecule with anti-inflammatory and BK channel-activating properties.

Large conductance voltage- and calcium-activated potassium (BK) channels are highly expressed in airway smooth muscle (ASM). Utilizing the ovalbumin (OVA) and house dust mite (HDM) models of asthma in C57BL/6 mice, we demonstrate that systemic administration of the BK channel agonist rottlerin (5 µg/g) during the challenge period reduced methacholine-induced airway hyperreactivity (AHR) in OVA- and HDM-sensitized mice (47% decrease in peak airway resistance in OVA-asthma animals, P<0.01; 54% decrease in HDM-asthma animals, P<0.01) with a 35-40% reduction in inflammatory cells and 20-35% reduction in Th2 cytokines in bronchoalveolar lavage fluid. Intravenous rottlerin (5 µg/g) reduced AHR within 5 min in the OVA-asthma mice by 45% (P<0.01). With the use of an ex vivo lung slice technique, rottlerin relaxed acetylcholine-stimulated murine airway lumen area to 87 ± 4% of the precontracted area (P<0.01 vs. DMSO control). Rottlerin increased BK channel activity in human ASM cells (V50 shifted by 73.5±13.5 and 71.8±14.6 mV in control and asthmatic cells, respectively, both P<0.05 as compared with pretreatment) and reduced the frequency of acetylcholine-induced Ca(2+) oscillations in murine ex vivo lung slices. These findings suggest that rottlerin, with both anti-inflammatory and ASM relaxation properties, may have benefit in treating asthma.

Neuroimmune semaphorin 4D is necessary for optimal lung allergic inflammation.

Neuroimmune semaphorin 4D (Sema4D) was found to be expressed and function in the nervous and immune systems. In the immune system, Sema4D is constitutively expressed on T cells and regulates T cell priming. In addition, it displays a stimulatory function on macrophages, DC, NK cells, and neutrophils. As all these cells are deeply involved in asthma pathology, we hypothesized that Sema4D plays a critical non-redundant regulatory role in allergic airway response. To test our hypothesis, we exposed Sema4D(-/-) and WT mice to OVA injections and challenges in the well-defined mouse model of OVA-induced experimental asthma. We observed a significant decrease in eosinophilic airway infiltration in allergen-treated Sema4D(-/-) mice relative to WT mice. This reduced allergic inflammatory response was associated with decreased BAL IL-5, IL-13, TGFß1, IL-6, and IL-17A levels. In addition, T cell proliferation in OVA323₋339-restimulated Sema4D(-/-) cell cultures was downregulated. We also found increased Treg numbers in spleens of Sema4D(-/-) mice. However, airway hyperreactivity (AHR) to methacholine challenges was not affected by Sema4D deficiency in either acute or chronic experimental disease setting. Surprisingly, lung DC number and activation were not affected by Sema4D deficiency. These data provide a new insight into Sema4D biology and define Sema4D as an important regulator of Th2-driven lung pathophysiology and as a potential target for a combinatory disease immunotherapy.

Neuroimmune semaphorin 4A downregulates the severity of allergic response.

To define the role of semaphorin 4A (Sema4A) in allergic response, we employed Sema4A⁻/⁻ and wild-type (WT) mice in the experimental model of ovalbumin (OVA)-induced allergic airway inflammation. We observed a selective increase in eosinophilic airway infiltration accompanied by bronchial epithelial cell hyperplasia in allergen-treated Sema4A⁻/⁻ mice relative to WT mice. This enhanced inflammatory response was associated with a selective increase in bronchoalveolar lavage (BAL) interleukin 13 (IL-13) content, augmented airway hyperreactivity, and lower regulatory T cell (Treg) numbers. In vivo allergen-primed Sema4A⁻/⁻ CD4+ T cells were more effective in transferring T helper type 2 (Th2) response to naive mice as compared with WT CD4+ T cells. T-cell proliferation and IL-13 productions in OVA323₋339-restimulated Sema4A⁻/⁻ cell cultures were upregulated. Generated bone marrow chimeras showed an equal importance of both lung-resident cell and inflammatory cell Sema4A expression in optimal disease regulation. These data provide a new insight into Sema4A biology and define Sema4A as an important regulator of Th2-driven lung pathophysiology.

Biology and function of neuroimmune semaphorins 4A and 4D.

Semaphorins belong to a family of membrane-bound and secreted molecules that regulate the functional activity of axons in the nervous system. Sema4A and Sema4D were the first semaphorins also found to be expressed in immune cells and were, therefore, termed "immune semaphorins". It is known that Sema4A has three functional receptors, namely Plexin D1, Plexin B1, and Tim-2, whereas Sema4D binds to Plexin B1 and CD72. Recent studies suggest that immune semaphorins play critical roles in many physiological and pathological processes and such. In this review, we summarize the current knowledge on the biology of neuroimmune semaphorins and their corresponding receptors, their distribution in organs and tissues, function in the immune response, and critical regulatory roles in various diseases.

Altered lymphocyte trafficking and diminished airway reactivity in transgenic mice expressing human MMP-9 in a mouse model of asthma.

Matrix metalloproteinase-9 (MMP-9) is hypothesized to facilitate leukocyte extravasation and extracellular remodeling in asthmatic airways. Careful descriptive studies have shown that MMP-9 levels are higher in the sputum of asthmatics; however, the consequence of increased MMP-9 activity has not been determined in this disease. We induced asthma in transgenic mice that express human MMP-9 in the murine lung tissue macrophage to determine the direct effect of human MMP-9 expression on airway inflammation. Transgenic (TG) and wild-type (WT) mice were immunized and challenged with ovalbumin. Forty-eight hours after the ovalbumin challenge, airway hyperresponsiveness (AHR) was measured, and inflammatory cell infiltration was evaluated in bronchoalveolar lavage fluid (BALF) and lung tissue. Baseline levels of inflammation were similar in the TG and WT groups of mice, and pulmonary eosinophilia was established in both groups by sensitization and challenge with ovalbumin. There was a significant reduction in AHR in sensitized and challenged trangenics compared with WT controls. Although total BALF cell counts were similar in both groups, the lymphocyte number in the lavage of the TG group was significantly diminished compared with the WT group (0.25 +/- 0.08 vs. 0.89 +/- 0.53; P = 0.0032). In addition, the draining lymphocytes were found to be larger in the TG animals compared with the WT mice. Equal numbers of macrophages, eosinophils, and neutrophils were seen in both groups. IL-13 levels were found to be lower in the sensitized TG compared with the WT mice. These results demonstrate an inverse relationship between human MMP-9 and AHR and suggest that MMP-9 expression alters leukocyte extravasation by reducing lymphocyte accumulation in the walls of asthmatic airways.

Transgenic expression of matrix metalloproteinase-9 causes adult-onset emphysema in mice associated with the loss of alveolar elastin.

Matrix metalloproteinase (MMP)-9 has been consistently identified in the lungs of patients with chronic obstructive pulmonary disease (COPD). However, its role in the development of the disease remains undefined. Mice that specifically express human MMP-9 in their macrophages were generated, and morphometric, biochemical, and histological analyses were conducted on the transgenic and littermate control mice over 1 yr to determine the effect of macrophage MMP-9 expression on emphysema formation and lung matrix content. Lung morphometry was normal in transgenic mice at 2 mo of age (mean linear intercept = 50+/-3 littermate mice vs. 51+/-2 transgenic mice). However, after 12 mo of age, the MMP-9 transgenic mice developed significant air space enlargement (mean linear intercept = 53+/-3 littermate mice vs. 61+/-2 MMP-9 transgenic mice; P<0.04). Lung hydroxyproline content was not significantly different between wild-type and transgenic mice, but MMP-9 did significantly decrease alveolar wall elastin at 1 yr of age (4.9+/-0.3% area of alveolar wall in the littermate mice vs. 3.3+/-0.3% area of alveolar wall in the MMP-9 mice; P<0.004). Thus these results establish a central role for MMP-9 in the pathogenesis of this disease by demonstrating that expression of this protease in macrophages can alter the extracellular matrix and induce progressive air space enlargement in mice.