Mice overexpressing chemokine ligand 2 (CCL2) in astrocytes display enhanced nociceptive responses.

Recent findings demonstrate that chemokines, and more specifically CC chemokine ligand 2 (CCL2 or monocyte chemoattractant protein-1), play a major role in pain processing. In the present study, we assess nociceptive responses of mice that overexpressed CCL2 under control of glial fibrillary acidic protein promoter (CCL2 tg). In models of acute nociception CCL2 tg mice demonstrated significantly enhanced nociceptive behavior relative to wild-type controls in responses to both thermal (hot plate) and chemical (formalin test) stimulus modalities. There were no differences in mechanical allodynia in the partial sciatic nerve ligation model, in terms of either magnitude or duration of the allodynic response; however, both groups responded to the maximal extent measurable. In a model of inflammatory pain, elicited by intraplantar administration of complete Freund's adjuvant (CFA), CCL2 tg mice displayed both greater edema and thermal hyperalgesia compared with control mice. In control mice, edema and hyperalgesia returned to baseline values 5-7 days post CFA. However, in CCL2 tg mice, thermal hyperalgesia was significantly different from baseline up to 3 weeks post CFA. Parallel to these enhanced behavioral responses CCL2 serum levels were significantly greater in CCL2 overexpressing mice and remained elevated 7 days post CFA. Consequently, proinflammatory cytokine mRNA expression (IL-1beta, IL-6, and TNFalpha) levels were greater in skin, dorsal root ganglia (DRG), and spinal cord, whereas the anti-inflammatory cytokine (IL-10) level was lower in skin and DRG in CCL2 overexpressing mice than in control mice. Taken together with data from CCR2-deficient mice, these present data confirm a key role of CCL2/CCR2 axis in pain pathways and suggest that inhibiting this axis may result in novel pain therapies.

Stromelysin-1 regulates adipogenesis during mammary gland involution.

The matrix metalloproteinase MMP-3/stromelysin-1 (Str1) is highly expressed during mammary gland involution induced by weaning. During involution, programmed cell death of the secretory epithelium takes place concomitant with the repopulation of the mammary fat pad with adipocytes. In this study, we have used a genetic approach to determine the role of Str1 during mammary involution. Although Str1 has been shown to induce unscheduled apoptosis when expressed ectopically during late pregnancy (Alexander, C.M., E.W. Howard, M.J. Bissell, and Z. Werb. 1996. J. Cell Biol. 135:1669-1677), we found that during post-lactational involution, mammary glands from transgenic mice that overexpress the tissue inhibitor of metalloproteinases, TIMP-1 (TO), or mice carrying a targeted mutation in Str1 showed accelerated differentiation and hypertrophy of adipocytes, while epithelial apoptosis was unaffected. These data suggest that matrix metalloproteinases (MMPs) do not induce unscheduled epithelial cell death after weaning, but instead alter the stromal microenvironment. We used adipogenic 3T3-L1 cells as a cell culture model to test the function of MMPs during adipocyte differentiation. Fibroblastic 3T3-L1 progenitor cells expressed very low levels of MMPs or TIMPs. The transcription of a number of MMP and TIMP mRNAs [Str1, MT1-MMP, (MMP-14) collagenase-3 (MMP-13), gelatinase A (MMP-2), and TIMP-1, -2 and -3] was induced in committed preadipocytes, but only differentiated adipocytes expressed an activated MMP, gelatinase A. The addition of MMP inhibitors (GM 6001 and TIMP-1) dramatically accelerated the accumulation of lipid during differentiation. We conclude that MMPs, especially Str1, determine the rate of adipocyte differentiation during involutive mammary gland remodeling.

Deficiency in inducible nitric oxide synthase results in reduced atherosclerosis in apolipoprotein E-deficient mice.

Inducible NO synthase (iNOS) present in human atherosclerotic plaques could contribute to the inflammatory process of plaque development. The role of iNOS in atherosclerosis was tested directly by evaluating the development of lesions in atherosclerosis-susceptible apolipoprotein E (apoE)-/- mice that were also deficient in iNOS. ApoE-/- and iNOS-/- mice were cross-bred to produce apoE-/-/iNOS-/- mice and apoE-/-/iNOS+/+ controls. Males and females were placed on a high fat diet at the time of weaning, and atherosclerosis was evaluated at two time points by different methods. The deficiency in iNOS had no effect on plasma cholesterol, triglyceride, or nitrate levels. Morphometric measurement of lesion area in the aortic root at 16 wk showed a 30-50% reduction in apoE-/-/iNOS-/- mice compared with apoE-/-/iNOS+/+ mice. Although the size of the lesions in apoE-/-/iNOS-/- mice was reduced, the lesions maintained a ratio of fibrotic:foam cell-rich:necrotic areas that was similar to controls. Biochemical measurements of aortic cholesterol in additional groups of mice at 22 wk revealed significant 45-70% reductions in both male and female apoE-/-/iNOS-/- mice compared with control mice. The results indicate that iNOS contributes to the size of atherosclerotic lesions in apoE-deficient mice, perhaps through a direct effect at the site of the lesion.

Essential role for p38alpha mitogen-activated protein kinase in placental angiogenesis.

The p38 family of mitogen-activated protein kinases (MAPKs) mediates signaling in response to environmental stresses and inflammatory cytokines, but the requirements for the p38 MAPK pathway in normal mammalian development have not been elucidated. Here, we show that targeted disruption of the p38alpha MAPK gene results in homozygous embryonic lethality because of severe defects in placental development. Although chorioallantoic placentation is initiated appropriately in p38alpha null homozygotes, placental defects are manifest at 10.5 days postcoitum as nearly complete loss of the labyrinth layer and significant reduction of the spongiotrophoblast. In particular, p38alpha mutant placentas display lack of vascularization of the labyrinth layer as well as increased rates of apoptosis, consistent with a defect in placental angiogenesis. Furthermore, p38alpha mutants display abnormal angiogenesis in the embryo proper as well as in the visceral yolk sac. Thus, our results indicate a requirement for p38alpha MAPK in diploid trophoblast development and placental vascularization and suggest a more general role for p38 MAPK signaling in embryonic angiogenesis.

Cleavage of aggrecan at the Asn341-Phe342 site coincides with the initiation of collagen damage in murine antigen-induced arthritis: a pivotal role for stromelysin 1 in matrix metalloproteinase activity.

OBJECTIVE: The destruction of articular cartilage during arthritis is due to proteolytic cleavage of the extracellular matrix components. This study investigates the kinetic involvement of metalloproteinases (MMPs) in the degradation of the 2 major cartilage components, aggrecan and type II collagen, during murine antigen-induced arthritis (AIA). In addition, the role of stromelysin 1 (SLN-1) induction of MMP-induced neoepitopes was studied. METHODS: VDIPEN neoepitopes in aggrecan and collagenase-induced COL2-3/4C neoepitopes in type II collagen were identified by immunolocalization. Stromelysin 1-deficient knockout (SLN1-KO) mice were used to study SLN-1 involvement. RESULTS: In AIA, the VDIPEN epitopes in aggrecan appeared after initial proteoglycan (PG) depletion. The collagenase-induced type II collagen neoepitopes colocalized with VDIPEN epitopes. Remarkably, cartilage from arthritic SLN1-KO mice showed neither the induction of VDIPEN nor collagen cleavage-site neoepitopes during AIA, suggesting that stromelysin is a pivotal mediator in this process. PG depletion, as measured by the loss of Safranin O staining, was similar in SLN1-KO mice and wild-type strains. Furthermore, in vitro induction of VDIPEN epitopes in aggrecan and COL2-3/4C epitopes in type II collagen, on exposure of cartilage to interleukin-1, could not be accomplished in SLN1-KO mice, whereas intense staining was achieved for both epitopes in cartilage of wild-type strains. CONCLUSION: This study emphasizes that SLN-1 is essential in the induction of MMP-specific aggrecan and collagen cleavage sites during AIA. It suggests that SLN-1 is not a dominant enzyme in PG breakdown, but that it activates procollagenases and is crucial in the initiation of collagen damage.

Impaired wound contraction in stromelysin-1-deficient mice.

OBJECTIVE: To determine whether the deletion of stromelysin-1, a single metalloproteinase gene product, will alter the time course and quality of dermal wound repair in mice. SUMMARY BACKGROUND DATA: After dermal injury, a highly coordinated program of events is initiated by formation of a fibrin clot, followed by migration of keratinocytes, contraction of the dermis, recruitment of inflammatory macrophages, formation of granulation tissue with angiogenesis, and finally tissue remodeling. Matrix metalloproteinases are rapidly induced in the dermis and granulation tissue and at the leading edge of the epidermis in the healing wounds. METHODS: Incisional and circular full-thickness wounds 2 to 10 mm were made in the dermis of stromelysin-1-deficient and wild-type mice. The wounds were analyzed for rate of cellular migration and epithelialization. The wound contraction was examined by immunohistochemical staining for alpha-smooth muscle actin and fluorescent staining for fibrillar actin. RESULTS: Independent of the age of the animal, excisional wounds in stromelysin-1-deficient mice failed to contract and healed more slowly than those in wild-type mice. Cellular migration and epithelialization were unaffected in the stromelysin-1-deficient animals. The functional defect in these mice is failure of contraction during the first phase of healing because of inadequate organization of actin-rich stromal fibroblasts. CONCLUSIONS: Excisional dermal wound healing is impaired in mice with a targeted deletion in the stromelysin-1 gene. Incisional wound healing is not affected. These data implicate stromelysin-1 proteolysis during early wound contraction and indicate that stromelysin-1 is crucial for the organization of a multicellular actin network.

Matrix metalloproteinase deficiencies affect contact hypersensitivity: stromelysin-1 deficiency prevents the response and gelatinase B deficiency prolongs the response.

Matrix metalloproteinases (MMPs) are expressed by T cells and macrophages, but there is a paucity of evidence for their role in immune responses. We have studied mice with deficiencies of stromelysin-1 (MMP-3) or gelatinase B (MMP-9) in a dinitrofluorobenzene (DNFB)-induced model of contact hypersensitivity (CHS). Stromelysin-1-deficient mice showed a markedly impaired CHS response to topical DNFB, although they responded normally to cutaneously applied phenol, an acute irritant. Lymphocytes from lymph nodes of DNFB-sensitized stromelysin-1-deficient mice did not proliferate in response to specific soluble antigen dinitrobenzenesulfonic acid, but did proliferate identically to lymph node lymphocytes from wild-type mice when presented with the mitogen Con A. An intradermal injection of stromelysin-1 immediately before DNFB sensitization rescued the impaired CHS response to DNFB in stromelysin-1-deficient mice. Unlike stromelysin-1-deficient mice, gelatinase B-deficient mice exhibited a CHS response comparable to wild-type controls at 1 day postchallenge, but the response persisted beyond 7 days in contrast to the complete resolution observed in wild-type mice by 7 days. However, gelatinase B-deficient mice had a normal rate of resolution of acute inflammation elicited by cutaneous phenol. Gelatinase B-deficient mice failed to show IL-10 production at the site of CHS, an essential feature of resolution in control mice. These results indicate that stromelysin-1 and gelatinase B serve important functions in CHS. Stromelysin-1 is required for initiation of the response, whereas gelatinase B plays a critical role in its resolution.

Stromelysin-1-deficient fibroblasts display impaired contraction in vitro.

Targeted disruption of the stromelysin-1 gene in mice causes a delay in excisional wound healing due to a failure in wound contraction. Therefore, we postulated that stromelysin-1 activity is responsible for initiating contraction. To test this hypothesis, we compared the contractile capacity of fibroblasts from stromelysin-1 knockout mice (strom-1 KO) with that of normal fibroblasts using a collagen gel contraction model. Fibroblast cultures were established from explants of skin and lung parenchyma from strom-1 KO and wild-type mice, then transferred to the surface of collagen gels. The extent of contraction was determined by measuring greatest gel diameter. Results demonstrated that (1) all fibroblasts contracted collagen gels in a uniform concentric fashion, (2) skin fibroblasts from both sets of mice exhibited greater gel contraction than did lung fibroblasts, and (3) strom-1 KO fibroblasts demonstrated significantly less contraction (21-23%) than wild-type fibroblasts. These data support the hypothesis that absence of stromelysin-1 results in defective fibroblast contraction that may contribute to delayed wound healing.

Transcriptional activation following cerebral ischemia in mice of a promoter-deleted nitric oxide synthase-2 gene.

Nitric oxide synthase (NOS)-2 is transcriptionally activated in a wide variety of injurious conditions, including cerebral ischemia, and the resulting nitric oxide is implicated both in tissue damage and recovery. Studies in vitro suggest that the proximal region of the NOS-2 promoter is obligatory for gene activation by proinflammatory cytokines. However, following cerebral ischemia in a NOS-2 gene-deficient mouse in which this region and exons 1-4 have been deleted, we find temporal and spatial expression, identical to wild-type, from a previously unidentified promoter region. The resulting protein is predicted to lack the first 113 amino acids and is NOS-2-incompetent. Fortuitously, this gene-deficient mouse presents a unique opportunity to determine more about the mechanisms of NOS-2 gene regulation in vivo.

Attenuated acute cardiac rejection in NOS2 -/- recipients correlates with reduced apoptosis.

BACKGROUND: The mechanisms through which NOS2-mediated pathways regulate graft failure in acute cardiac rejection are ill defined. To determine whether apoptosis promoted by NOS2 may contribute, we used a heterotopic transplant model to study mouse cardiac allografts placed in recipients with targeted gene deletion of NOS2. METHODS AND RESULTS: Using 5 different indexes of apoptosis, we showed that mouse cardiac allografts placed in NOS2 -/- recipients (n=7) had reduced apoptotic activity compared with those in NOS2 +/+ controls (n=8). There were significantly fewer TUNEL-positive nuclei per high-powered field (P<0.01), less DNA fragmentation (antinucleosome ELISA; P<0.05), lower corrected transcript levels for caspase-1 and -3 (32P reverse transcriptase-polymerase chain reaction; P<0.01), and reduced caspase-3 activity (cleavage of DEVD-pNA [P<0.001] and poly [ADP-ribose] polymerase) in grafts from NOS2 -/- recipients. This concordant reduction in apoptotic indexes paralleled the improved histological outcome of grafts transplanted into NOS2 -/- recipients (assessed as rejection scores; P=0.012). To identify pathways controlled by NOS2, we compared intragraft transcript levels of potential triggers and regulators. Whereas Fas ligand/Fas and tumor necrosis factor (TNF)-alpha/TNF receptor-1 levels were not altered by NOS2 deficiency, transcript levels for p53 were significantly lower in grafts from NOS2 -/- recipients, coinciding with a significant increase in the antiapoptotic Bcl-2/Bax balance and decrease in Bcl-Xl levels. CONCLUSIONS: Using NOS2 knockout mice, we demonstrated that NOS2-mediated pathways can promote acute rejection, at least in part, by inducing apoptotic cell death. When NOS2 is present, p53 might control NOS2-mediated apoptosis by stimulating Bax and repressing Bcl-2 and Bcl-Xl expression, which may activate the cell death program in the rejecting heart.

NOS2 mediates opposing effects in models of acute and chronic cardiac rejection: insights from NOS2-knockout mice.

To compare regulatory effects of NOS2 in acute and chronic cardiac allograft rejection, we used NOS2 knockout mice as recipients in a cardiac transplant model. To study acute and chronic rejection separately but within the same genetic strain combination, we compared allografts placed into recipients without or with immunosuppression (anti-CD4/8 for 28 days). NOS2 mRNA and protein expression were compared using 32P-RT-PCR and immunohistochemistry. In our acute rejection model, NOS2 was predominately localized to graft-infiltrating immune cells. At day 7, grafts in NOS2-deficient recipients (n = 7) showed reduced inflammatory infiltrates and myocyte damage resulting in significantly lower rejection scores (1.6 +/- 0.4) compared to wild-type controls (n = 18; 2.8 +/- 0.2, P = 0.002). In contrast, in our chronic rejection model, additional NOS2 expression was localized to graft-parenchymal cells. At day 55, grafts in NOS2-deficient recipients (n = 12) showed more parenchymal infiltration and parenchymal destruction (rejection score 3.8 +/- 0.1) than wild-type controls (n = 15; 1.6 +/- 0.2, P < 0.0001). This was associated with a significant decrease in ventricular contractility (palpation score 0.3 +/- 0.1 compared to 2.3 +/- 0.3 in wild-type, P < 0.0001). Hence, NOS2 promotes acute but prevents chronic rejection. These opposing effects during acute and chronic cardiac allograft rejection are dependent on the temporal and spatial expression pattern of NOS2 during both forms of rejection.

Exacerbated transplant arteriosclerosis in inducible nitric oxide-deficient mice.

BACKGROUND: Inducible NO synthase (NOS2, or iNOS) is upregulated in grafts with transplant arteriosclerosis. However, the functional role of NOS2 in the pathogenesis of transplant arteriosclerosis remains unclear. NOS2 may regulate lesion development by modulating the early alloimmune response and/or late myointimal thickening. METHODS AND RESULTS: To determine whether NOS2-mediated pathways protect against or promote transplant arteriosclerosis, we used NOS2-deficient mice as recipients in our vascularized chronic cardiac rejection model. The severity of vascular thickening in 55-day grafts placed into NOS2 -/- recipients (n=13) was compared with that in wild-type recipients (n=15). Computer-assisted analysis of all elastin-stained vessels (n=283) showed significantly increased luminal occlusion (77.11+/-9.4% versus 40.8+/-13.6%, P<.0001) and intima/media ratios in allografts from NOS2 -/- recipients (1.9+/-1.3 versus 0.4+/-0.3, P=.0002). To elucidate potential mechanisms, we studied NOS2 effects on T-cell differentiation (Th1/Th2) and neointimal smooth muscle cell accumulation. Normalized mRNA levels for Th1- (signal transducer and activator of transcription [STAT] 4, interleukin [IL]-2, interferon-gamma) and Th2- (STAT 6, IL-4, and IL-5) associated factors were comparable in both groups. In contrast, quantitative analysis of the alpha-actin-positive area showed a significant increase in the contribution of smooth muscle cells within the neointima in allografts from NOS2 -/- recipients (28.2+/-2.0%) compared with wild-type controls (13.2+/-2.3%; P<.0001). CONCLUSIONS: NOS2 plays a protective role in the development of transplant arteriosclerosis, suppressing neointimal smooth muscle cell accumulation.

Experimental autoimmune encephalomyelitis is exacerbated in mice lacking the NOS2 gene.

Nitric oxide is believed to be a prominent mediator of inflammation based in part on the correlative expression of the inducible nitric oxide synthase (iNOS) gene in various pathologies. The resulting high output of the highly reactive molecule nitric oxide is then believed to play an important role in the evolving inflammatory response. Studies have shown that iNOS and nitric oxide are present in the tissues of patients with multiple sclerosis (MS). In rodent models of MS, experimental autoimmune encephalomyelitis (EAE), it has been shown that nonspecific NOS inhibitors partially ameliorate the disease. To determine the importance of iNOS in this model of MS, we induced EAE in mice containing a disrupted iNOS (NOS2) gene. Surprisingly, by day 24, the NOS2 knockout mice had a greater incidence of EAE than wild-type control mice (75 vs 12%), and had a higher average severity score (2.42 vs 0.44). These differences appear to result largely from the failure of the disease to remit in NOS2 KO mice. Wild-type mice have a profound ability to reverse EAE (82%) compared with the knockout mice (19%). This result implies that iNOS may in some instances play a protective role in autoimmune-mediated tissue destruction.

Susceptibility of stromelysin 1-deficient mice to collagen-induced arthritis and cartilage destruction.

OBJECTIVE: It has long been proposed that stromelysin is one of the major degradative matrix metalloproteinases responsible for the loss of cartilage in rheumatoid arthritis (RA) and osteoarthritis (OA). This hypothesis was tested by examining the arthritic paws of stromelysin 1 (SLN1)-deficient mice for loss of cartilage and for generation of neoepitopes that would be indicative of aggrecan cleavage. METHODS: The SLN1 gene was inactivated in murine embryonic stem cells, and knockout mice deficient in SLN1 activity were bred onto the B10.RIII background. The incidence and severity of collagen-induced arthritis (CIA) were compared in wild-type and knockout mice. Paws from mice with CIA were examined for loss of cartilage and for proteoglycan staining, as well as for the generation of the neoepitope FVDIPEN341. RESULTS: SLN1-deficient mice developed CIA, as did the wild-type N2 mice. Histologic analyses demonstrated no significant differences among the B10.RIII, wild-type, and knockout mice in loss of articular cartilage and proteoglycan staining. No decrease in the FVDIPEN341 epitope was observed in the SLN1-deficient mice. CONCLUSION: Disruption of the SLN1 gene neither prevents nor reduces the cartilage destruction associated with CIA. Moreover, SLN1 depletion does not prevent the cleavage of the aggrecan Asn341-Phe342 bond.

Coordinate expression of matrix metalloproteinase family members in the uterus of normal, matrilysin-deficient, and stromelysin-1-deficient mice.

The expression patterns of matrix metalloproteinase (MMP) family members during the murine estrous cycle and postpartum uterine involution were analyzed, and the consequence of removing specific MMPs during uterine functions was determined using mice deficient in either matrilysin (MAT) or stromelysin-1 (STR-1). In wild-type animals, MAT, STR-1, STR-2, STR-3, and gelatinase A were consistently expressed during the most active phases of the estrous cycle, estrus and proestrus. The messenger RNA for these MMPs as well as collagenase-3 and the tissue inhibitors of metalloproteinases were also expressed during uterine involution, as determined by Northern analysis and in situ hybridization. Notably, MAT, STR-2, and collagenase-3 messenger RNA levels were elevated at early times of involution and rapidly decreased with time, whereas the transcripts for other MMPs remained elevated throughout the involution process. Involution proceeded normally in mice lacking MAT or STR-1; however, the expression of STR-1 and STR-2 was dramatically up-regulated in MAT nullizygous mice, and the expression of MAT and STR-2 was moderately up-regulated in STR-1-deficient animals. We conclude that the concerted action of several MMPs is likely to play an important role in the remodeling of the postpartum uterus, and that mechanisms that compensate for the loss of a specific MMP during this process appear to exist.

Clinical and serologic manifestations of autoimmune disease in MRL-lpr/lpr mice lacking nitric oxide synthase type 2.

Nitric oxide (NO) is an important mediator of the inflammatory response. MRL-lpr/lpr mice overexpress inducible nitric oxide synthase (NOS2) and overproduce NO in parallel with the development of an autoimmune syndrome with a variety of inflammatory manifestations. In previous studies, we showed that inhibiting NO production with the nonselective nitric oxide synthase (NOS) inhibitor NG-monomethyl-arginine reduced glomerulonephritis, arthritis, and vasculitis in MRL-lpr/lpr mice. To define further the role of NO and NOS2 in disease in MRL-lpr/lpr mice, mice with targeted disruption of NOS2 were produced by homologous recombination and bred to MRL-lpr/lpr mice to the N4 generation. MRL-lpr/lpr littermates homozygous for disrupted NOS2 (-/-), heterozygous for disrupted NOS2 (+/-), or wildtype (+/+) were derived for this study. Measures of NO production were markedly decreased in the MRL-lpr/lpr (-/-) mice compared with MRL-lpr/lpr (+/+) mice, with intermediate production by the MRL-lpr/lpr (+/-) mice. There was no detectable NOS2 protein by immunoblot analysis of the spleen, liver, kidney, and peritoneal macrophages of the (-/-) animals, whereas that of (+/+) was high and (+/-) intermediate. The (-/-) mice developed glomerular and synovial pathology similar to that of the (+/-) and (+/+) mice. However, (-/-) mice and (+/-) mice had significantly less vasculitis of medium-sized renal vessels than (+/+) mice. IgG rheumatoid factor levels were significantly lower in the (-/-) mice as compared with (+/+) mice, but levels of anti-DNA antibodies were comparable in all groups. Our findings show that NO derived from NOS2 has a variable impact on disease manifestations in MRL-lpr/lpr mice, suggesting heterogeneity in disease mechanisms.

Identification of nitric oxide synthase as a protective locus against tuberculosis.

Mutagenesis of the host immune system has helped identify response pathways necessary to combat tuberculosis. Several such pathways may function as activators of a common protective gene: inducible nitric oxide synthase (NOS2). Here we provide direct evidence for this gene controlling primary Mycobacterium tuberculosis infection using mice homozygous for a disrupted NOS2 allele. NOS2(-/-) mice proved highly susceptible, resembling wild-type littermates immunosuppressed by high-dose glucocorticoids, and allowed Mycobacterium tuberculosis to replicate faster in the lungs than reported for other gene-deficient hosts. Susceptibility appeared to be independent of the only known naturally inherited antimicrobial locus, NRAMP1. Progression of chronic tuberculosis in wild-type mice was accelerated by specifically inhibiting NOS2 via administration of N6-(1-iminoethyl)-L-lysine. Together these findings identify NOS2 as a critical host gene for tuberculostasis.

Inducible nitric oxide synthase plays a critical role in resolving intestinal inflammation.

BACKGROUND & AIMS: Overproduction of nitric oxide by inducible nitric oxide synthase (iNOS) has been proposed as a pathogenic factor in colitis. The objective of this study was to examine the role of iNOS using iNOS-deficient mice in experimental colitis. METHODS: Colitis was induced by intrarectal instillation of 3% acetic acid and assessed for neutrophilic infiltration and intestinal injury over 7 days. iNOS messenger RNA expression was also measured. RESULTS: At 24 hours, acetic acid induced a mild colitis in wild-type mice. An increase in neutrophil infiltration and tissue edema was also observed. In the iNOS-deficient mice, a twofold increase in macroscopic damage was observed. Neutrophil infiltration and tissue edema were similar to those in wild-type animals at this time point. Although inflammation in wild-type mice had resolved by 7 days, a sevenfold increase in damage score and elevated myeloperoxidase level were still evident in iNOS-deficient mice. A striking increase in the message for iNOS was observed in inflamed wild-type mice at 24 hours and was still present at 72 hours. No message was found in iNOS-deficient mice. CONCLUSIONS: Induction of iNOS seems to be a critical protective response to injury in intestinal inflammation possibly by reducing leukocytic infiltration.

Aggrecanase and metalloproteinase-specific aggrecan neo-epitopes are induced in the articular cartilage of mice with collagen II-induced arthritis.

OBJECTIVE: To analyze the roles of two classes of proteinases, 'aggrecanase', and matrix metalloproteinases (MMPs), in chondrodestruction during murine collagen-induced arthritis (CIA). METHODS: Generation of the 'aggrecanase' neo-epitope (NITEGE373), and the MMP neo-epitope (VDIPEN341) within aggrecan was studied by immunoperoxidase microscopy using specific anti-peptide antibodies in normal and stromelysin-1 (SLN-1) deficient knockout mice with CIA. RESULTS: High levels of NITEGE373 and VDIPEN341 neo-epitopes were observed in foci within CIA paw articular cartilage exhibiting depletion of glycosaminoglycans, in advance of significant cartilage erosion. The highest concentrations of NITEGE373 and VDIPEN341 labeling were observed and often co-distributed in the chondrocyte pericellular matrix, suggesting that stimulated chondrocytes can synthesize and/or activate both enzymes. Other regions of the cartilage frequently exhibited either NITEGE373 or VDIPEN341 labeling, but not both neo-epitopes simultaneously, suggesting that 'aggrecanase' and MMP cleavages of aggrecan may be generated independently. No detectable differences were observed in expression or distribution of either neo-epitope in SLN-1 knockout versus wild-type mice. In addition, in vitro digestion of joint sections with SLN-1 did not alter the expression of cartilage NITEGE373, while markedly increasing VDIPEN341 labeling. Peripheral nerves and brains of naive mice also exhibited intense anti-NITEGE373 labeling. CONCLUSIONS: These data indicate that NITEGE373 and VDIPEN341 aggrecan neo-epitopes are sensitive and specific markers of early joint pathology, and are consistent with the hypothesis that SLN-1 does not have 'aggrecanase' activity, and that 'aggrecanase' is distinct from the MMPs which cleave aggrecan at the MMP site.

Molecular cloning and structural analysis of the functional mouse genomic XPG gene.

The mouse XPG gene is a homolog of the human DNA excision repair gene known to be defective in the hereditary sun-sensitive disorder xeroderma pigmentosum (group-G). Defects in mouse XPG have been shown to directly affect the sensitivity of cultured cells to chemotherapy agents and may play a role in tumor cell drug resistance in vivo. A full-length cosmid clone of mouse XPG was isolated by complementation of the UV sensitivity and repair defect in CHO-UV135 cells. Exon mapping determined that the gene consisted of 15 exons within 32 kb of genomic DNA. Sequencing of intron-exon boundaries revealed that mouse XPG possesses a rare class of intron previously identified in only four other eukaryotic genes; it utilizes AT and AC dinucleotides instead of the expected GT and AG within the splice junctions. Promoter analysis determined that mouse XPG is expressed constitutively and probably initiates transcription from multiple start sites, yet, unlike the yeast homolog RAD2, we found no evidence that it is UVC inducible in cultured cells. Amino acid comparison with human XPG identified a highly conserved acidic region of homology not previously described.