CXCR3 determines strain susceptibility to murine cerebral malaria by mediating T lymphocyte migration toward IFN-gamma-induced chemokines

Cerebral malaria (CM) results from the binding of infected erythrocytes and leukocytes to brain endothelia. The precise mechanisms underlying lymphocyte recruitment and activation in CM remain unclear. Therefore, the expression of various chemokines was quantified in brains of mice infected with Plasmodium berghei ANKA (PbA). Several chemokines attracting monocytes and activated T-lymphocytes were expressed at high levels. Their expression was almost completely abrogated in IFN-gamma ligand and receptor KO mice, indicating that IFN-gamma is an essential chemokine inducer in vivo. Surprisingly, the expression levels of chemokines, IFN-gamma and also adhesion molecules in the brain were not lower in CM-resistant Balb/c and DBA/2 mice compared to CM-sensitive C57BL/6 and DBA/1 mice, although T lymphocyte sequestration in the brain was significantly less in CM-resistant than in CM-sensitive mice. This difference correlated with a higher up-regulation of the CXC chemokine receptor (CXCR)-3 on splenic T cells and a higher chemotactic response to IFN-gamma-inducible protein-10 (IP-10) in C57BL/6 compared to Balb/c mice. In conclusion, parasite-induced IFN-gamma in the brain results in high local expression levels of specific chemokines for monocytes and lymphocytes. The strain-dependent susceptibility to develop CM is more related to the expression of CXCR3 in circulating leukocytes than to the chemokine expression levels in the brain.     

Matrix metalloproteinases promote inflammation and fibrosis in asbestos-induced lung injury in mice

Inhalation of asbestos fibers causes pulmonary inflammation and eventual pulmonary fibrosis (asbestosis). Although the underlying molecular events are poorly understood, protease/antiprotease and oxidant/antioxidant imbalances are believed to contribute to the disease. Implicated in other forms of pulmonary fibrosis, the matrix metalloproteinases (MMPs) have not been examined in asbestosis. We therefore hypothesized that MMPs play a pathogenic role in asbestosis development. Wild-type C57BL/6 mice were intratracheally instilled with 0.1 mg crocidolite asbestos, causing an inflammatory response at 1 d and a developing fibrotic response at 7, 14, and 28 d. Gelatin zymography demonstrated an increase in MMP-9 (gelatinase B) during the inflammatory phase, while MMP-2 (gelatinase A) was profoundly increased in the fibrotic phase. Immunohistochemistry revealed MMP-9 in and around bronchiolar and airspace neutrophils that were often associated with visible asbestos fibers. MMP-2 was found in fibrotic regions at 7, 14, and 28 d. No increases in RNA levels of MMP-2, MMP-9, or MMP-8 were found, but levels of MMP-7, MMP-12, and MMP-13 RNA did increase at 14 d. The MMP inhibitors, TIMP-1 and TIMP-2, were also increased at 7-28 d after asbestos exposure. To confirm the importance of MMP activity in disease progression, mice exposed to asbestos were given daily injections of the MMP inhibitor, GM6001. MMP inhibition reduced inflammation and fibrosis in asbestos-treated mice. Collectively, these data suggest that MMPs contribute to the pathogenesis of asbestosis through effects on inflammation and fibrosis development.     

CNS hypoxia is more pronounced in murine cerebral than noncerebral malaria and is reversed by erythropoietin

Cerebral malaria (CM) is associated with high mortality and risk of sequelae, and development of adjunct therapies is hampered by limited knowledge of its pathogenesis. To assess the role of cerebral hypoxia, we used two experimental models of CM, Plasmodium berghei ANKA in CBA and C57BL/6 mice, and two models of malaria without neurologic signs, P. berghei K173 in CBA mice and P. berghei ANKA in BALB/c mice. Hypoxia was demonstrated in brain sections using intravenous pimonidazole and staining with hypoxia-inducible factor-1α-specific antibody. Cytopathic hypoxia was studied using poly (ADP-ribose) polymerase-1 (PARP-1) gene knockout mice. The effect of erythropoietin, an oxygen-sensitive cytokine that mediates protection against CM, on cerebral hypoxia was studied in C57BL/6 mice. Numerous hypoxic foci of neurons and glial cells were observed in mice with CM. Substantially fewer and smaller foci were observed in mice without CM, and hypoxia seemed to be confined to neuronal cell somas. PARP-1-deficient mice were not protected against CM, which argues against a role for cytopathic hypoxia. Erythropoietin therapy reversed the development of CM and substantially reduced the degree of neural hypoxia. These findings demonstrate cerebral hypoxia in malaria, strongly associated with cerebral dysfunction and a possible target for adjunctive therapy.     

不同种鼠对实验脑型疟发生的影响研究

目的比较昆明小鼠和C57BL/6小鼠作为种鼠对实验脑型疟模型的影响。方法分别以伯氏疟原虫ANKA株感染C57BL/6小鼠和昆明小鼠作为传代用种鼠,当种鼠原虫率为5%～15%时接种子代C57BL/6小鼠,观察两组小鼠原虫率、脑型疟发生率以及死亡率。同时,通过脑组织切片和脑部淋巴细胞的流式检测,观察两组发生脑型疟小鼠的脑部微血管中感染疟原虫红细胞和CD8＋T细胞的粘附情况,另外,通过感染CD8＋TKO小鼠,证实CD8＋T细胞在两组小鼠发生脑型疟中的作用。结果用昆明小鼠作为种鼠的实验组的原虫率和脑型疟发生率均明显高于用C57BL/6小鼠作为种鼠的实验组,发生脑型疟小鼠的脑部组织切片发现,脑部微血管可见明显的感染疟原虫红细胞的粘附和CD8＋T淋巴细胞浸润;而用昆明小鼠作为种鼠感染CD8＋T细胞缺失的C57BL/6小鼠并不能诱导实验脑型疟的发生。结论与C57BL/6小鼠相比,昆明小鼠作为种鼠的实验组的脑型疟发病率更高,而且感染疟原虫红细胞和CD8＋T细胞在脑部微血管内的粘附也是该脑型疟发生的主要因素,因此,更适合用于实验脑型疟模型的建立及其机制的探讨。     

Metalloproteinases in juvenile angiofibroma--a collagen rich tumor

Matrix metalloproteinases (MMPs) act in diverse physiological and pathological conditions such as tumor growth and angiogenesis by cleaving extracellular matrix and nonmatrix substrates. MMPs with gelatinase/collagenase activity have not yet been studied in juvenile angiofibroma, a unique fibrovascular tumor with prominent collagen expression. Quantitative real-time polymerase chain reaction studies, Western blot analysis, immunofluorescence studies, gel zymography, and in situ zymography were used to analyze MMP-1, MMP-2, MMP-9, MMP-13, MMP-14, TIMP-1, and TIMP-2 in 9 juvenile angiofibromas and 2 inferior nasal turbinate specimens. Quantitative real-time polymerase chain reaction found significantly elevated expression of MMP-2, MMP-9, and MMP-14 (P < .05) in tumor tissue compared with the inferior nasal turbinate specimens. Western blot analysis detected more prominent MMP-1, MMP-2, and MMP-9 protein levels in juvenile angiofibromas compared with inferior nasal turbinates, but not MMP-13, MMP-14, TIMP-1, and TIMP-2. Immunofluorescent staining proved a mainly stromal localization of the analyzed MMPs. Only MMP-9 and MMP-14 were also detected in vessel walls. MMP-1, MMP-2, and MMP-13 also stained mast cells. Gel zymography indicated increased MMP-2 and MMP-9 gelatinase activity in juvenile angiofibromas compared with inferior nasal turbinates. Finally, in situ zymography detected very high stromal gelatinase/collagenase activity. This study indicates significant expression of MMPs with gelatinase/collagenase activity in juvenile angiofibromas with evidence of a disturbed balance of MMPs to TIMPs toward enhanced MMP activity. These MMPs are assumed to be involved in tumor pathology with an influence on tumor growth and angiogenesis.     

Expression and implications of tissue inhibitor of metalloproteinases-4 in mouse embryo

Matrix metalloproteinases (MMP), tissue inhibitors of metalloproteinases (TIMP), and MMP-TIMP interactions may contribute to the highly programmed process of embryo implantation. The loss of the delicate MMP-TIMP balance may lead to abnormal implantation. The role of TIMP-4 in mouse implantation has not been reported. This study examined mRNA and protein expression levels of TIMP-4 in the blastocyst and uteri of pregnant mice. We also investigated the effects of a specific TIMP-4 antibody on embryo outgrowth and on the gene and protein expression levels of two gelatinases. High levels of TIMP-4 mRNA and protein were detected in day 3-5 embryos and in the trophoblast cells of mice blastocysts, suggesting that TIMP-4 may be involved in embryo implantation. Furthermore, TIMP-4 antibody promoted blastocyst outgrowth in a dose-dependent manner, but had no effect on blastocyst adhesion to extracellular matrix. A specific TIMP-4 antibody also increased mRNA and protein expression levels and the enzymatic activities of gelatinase A (MMP-2) and gelatinase B (MMP-9). This study suggests that TIMP-4 may restrict mouse blastocyst outgrowth and embryo implantation by inhibiting the activities of MMP-2 and -9.     

Gelatinase B/matrix metalloproteinase-9 contributes to cellular infiltration in a murine model of zymosan peritonitis

Murine zymosan-induced peritonitis represents a well-defined model of acute inflammation. However, the molecular mechanisms by which leukocytes degrade basement membranes during extravasation into the peritoneum are not clear. Gelatinase B (MMP-9) is thought to participate in cellular migration, yet its role in leukocyte transmigration through endothelia during inflammation remains controversial. The aim of the present study was to evaluate the role of MMP-9 in the cell influx during zymosan-induced experimental peritonitis. In zymosan-treated Balb/c mice MMP-9 and its natural inhibitor (tissue inhibitor of metalloproteinase 1 - TIMP-1) were present in the peritoneal fluid and plasma at the time of peritoneal neutrophil (polymorphonuclear leukocyte - PMN) infiltration and persisted there until the time of monocytes/macrophages influx. To probe the function of gelatinases, gelatinase B-deficient mice (MMP-9(-/-)) were used as well as Balb/c mice treated with cyclic CTTHWGFTLC (INH), a specific peptide inhibitor of gelatinases. The studies revealed that in either group of mice deprived of MMP-9 activity, PMN infiltration was impaired at the time of their maximal extravasation (6h) while tumor necrosis factor alpha (TNF-alpha), cytokine-induced neutrophil chemoattractant (KC) and interleukin 10 (IL-10) levels were not changed. At later stages (24 h post-zymosan) a significant increase in PMNs was observed in MMP-9(-/-) mice, but not in the inhibitor-treated mice, in comparison to their respective controls. Moreover, intraperitoneal (i.p.) injection of recombinant mouse pro-MMP-9 induced leukocyte accumulation in peritoneum. Collectively, the findings indicate that gelatinase B participates in leukocyte transmigration; however, its function can be compensated by other mechanisms.     

Distribution of matrix metalloproteinases and their inhibitor, TIMP-1, in developing human osteophytic bone

Connective tissues synthesise and secrete a family of matrix metalloproteinases (MMPs) which are capable of degrading most components of the extracellular matrix. Animal studies suggest that the MMPs play a role in bone turnover. Using specific polyclonal antisera, immunohistochemistry was used to determine the patterns of synthesis and distribution of collagenase (MMP-1), stromelysin (MMP-3), gelatinase A (MMP-2) and gelatinase B (MMP-9) and of the tissue inhibitor of metalloproteinases-1 (TIMP-1) within developing human osteophytic bone. The different MMPs and TIMP showed distinct patterns of localisation. Collagenase expression was seen at sites of vascular invasion, in osteoblasts synthesising new matrix and in some osteoclasts at sites of resorption. Chondrocytes demonstrated variable levels of collagenase and stromelysin expression throughout the proliferative and hypertrophic regions, stromelysin showing both cell-associated and strong matrix staining. Intense gelatinase B expression was observed at sites of bone resorption in osteoclasts and mononuclear cells. Gelatinase A was only weakly expressed in the fibrocartilage adjacent to areas of endochondral ossification. There was widespread but variable expression of TIMP-1 throughout the fibrous tissue, cartilage and bone. These results indicate that MMPs play a role in the development of human bone from cartilage and fibrous tissue and are likely to have multiple functions.     

Expression of matrix metalloproteinases 2 and 7 in tumor cells correlates with the World Health Organization classification subtype and clinical stage of thymic epithelial tumors

To investigate the roles of matrix metalloproteinases (MMPs) in thymic epithelial tumors, we examined the expression of MMP-2, -7, and -9; membrane-type 1 (MT1)-MMP; and tissue inhibitor of metalloproteinase-2 (TIMP-2) in 57 tumors by immunohistochemistry and in selected 15 cases by in situ hybridization. The tumors consisted of 5 type A, 12 type AB, 11 type B1, 11 type B2, 9 type B3, and 9 type C thymomas according to the World Health Organization histologic classification system and of 22 stage I, 13 stage II, 8 stage III, and 14 stage IV thymomas according to the Masaoka staging system. In the positive cases, MMPs and TIMP-2 were expressed in both tumor cells and stromal cells. The cellular localization of MMPs detected by immunohistochemistry was almost identical with that of the mRNA signals detected by in situ hybridization. MMP-2 and MMP-7 were predominantly expressed in type B3 thymoma and type C thymoma, respectively. Expression of MT1-MMP and TIMP-2 correlated with that of MMP-2, indicating a proteolytic activation of the latter. MMP-9 was prominent in type B2 thymoma. Expression in tumor cells of MMP-2 or MMP-7 was also correlated with clinical stage. The present study suggests that certain MMPs may play an important role in the tumor progression of different subtypes of thymic epithelial tumors and that MMP-2 and MMP-7 may contribute to the tumor aggressiveness and malignant potential.     

Comparative study of brain CD8+ T cells induced by sporozoites and those induced by blood-stage Plasmodium berghei ANKA involved in the development of cerebral malaria

To obtain insight into the mechanisms that contribute to the pathogenesis of Plasmodium infections, we developed an improved rodent model that mimics human malaria closely by inducing cerebral malaria (CM) through sporozoite infection. We used this model to carry out a detailed study on isolated T cells recruited from the brains of mice during the development of CM. We compared several aspects of the immune response related to the experimental model of Plasmodium berghei ANKA infection induced by sporozoites in C57BL/6 mice and those related to a blood-stage infection. Our data show that in both models, oligoclonal TCRVbeta4(+), TCRVbeta6(+), TCRVbeta8.1(+), and TCRVbeta11(+) major histocompatibility complex class I-restricted CD8 T cells were present in the brains of CM(+) mice. These CD8(+) T cells display an activated phenotype, do not undergo apoptosis, secrete gamma interferon or tumor necrosis factor alpha, and are associated with the development of the neurological syndrome.     

Matrix metalloproteinase 9 (gelatinase B) is expressed in multinucleated giant cells of human giant cell tumor of bone and is associated with vascular invasion.

Human giant cell tumor (GCT) consists of multinucleated giant cells and mononuclear stromal cells, and is characterized by frequent vascular invasion without distant metastases. To study the role of matrix metalloproteinases (MMPs) in the vascular invasion, we examined production of MMP-1 (tissue collagenase), -2 (gelatinase A), -3 (stromelysin-1), -9 (gelatinase B), and tissue inhibitors of metalloproteinases (TIMP-1 and -2) in GCT. MMP-9 was highly and predominantly expressed in giant cells by both immunohistochemistry and in situ hybridization. Expression of other MMPs was also observed in some cases but was inconstant. Sandwich enzyme immunoassays demonstrated that MMP-9 is the predominant MMP secreted by GCT. There was a definite imbalance between the amounts of MMP-9 and those of TIMPs in the culture media of GCT, leading to detectable gelatinolytic activity in an assay using 14C-gelatin. Gelatin zymography demonstrated the main activity at about 90 kd, which was identified as the zymogen of MMP-9 by immunoblotting. Immunohistochemistry for type IV collagen and laminin, major basement membrane components, showed that disappearance of the proteins is closely associated with MMP-9-positive giant cells. These results indicate the production of MMP-9 by multinucleated giant cells and suggest that the metalloproteinase may contribute to proteolysis associated with vascular invasion and local bone resorption in human GCT.     

Quantitation of brain edema and localisation of aquaporin 4 expression in relation to susceptibility to experimental cerebral malaria

The pathogenic mechanisms underlying the occurrence of cerebral malaria (CM) are still incompletely understood but, clearly, cerebral complications may result from concomitant microvessel obstruction and inflammation. The extent to which brain edema contributes to pathology has not been investigated. Using the model of P. berghei ANKA infection, we compared brain microvessel morphology of CM-susceptible and CM-resistant mice. By quantitative planimetry, we provide evidence that CM is characterized by enlarged perivascular spaces (PVS). We show a dramatic aquaporin 4 (AQP4) upregulation, selectively at the level of astrocytic foot processes, in both CM and non-CM disease, but significantly more pronounced in mice with malarial-induced neurological syndrome. This suggests that a threshold of AQP4 expression is needed to lead to neurovascular pathology, a view that is supported by significantly higher levels in mice with clinically overt CM. Numbers of intravascular leukocytes significantly correlated with both PVS enlargement and AQP4 overexpression. Thus, brain edema could be a contributing factor in CM pathogenesis and AQP4, specifically in its astrocytic location, a key molecule in this mechanism. Since experimental CM is associated with substantial brain edema, it models paediatric CM better than the adult syndrome and it is tempting to evaluate AQP4 in the former context. If AQP4 changes are confirmed in human CM, it may represent a novel target for therapeutic intervention.     

Role of stromelysin 1 and gelatinase B in experimental acute lung injury

Matrix metalloproteinases (MMPs) are upregulated locally in sites of inflammation, including the lung. Several MMP activities are upregulated in acute lung injury models but the exact role that these MMPs play in the development of the lung injury is unclear due to the absence of specific inhibitors. To determine the involvement of individual MMPs in the development of lung injury, mice genetically deficient in gelatinase B (MMP-9) and stromelysin 1 (MMP-3) were acutely injured with immunoglobulin G immune complexes and the intensity of the lung injury was compared with genetically identical wild-type (WT) mice with normal MMP activities. In the WT mice there was upregulation of gelatinase B and stromelysin 1 in the injured lungs which, as expected, was absent in the genetically deficient gelatinase B- and stromelysin 1-deficient mice, respectively. In the deficient mice there was little in the way of compensatory upregulation of other MMPs. The gelatinase B- and the stromelysin 1-deficient mice had less severe lung injury than did the WT controls, suggesting that both MMPs are involved in the pathogenesis of the lung injury. Further, the mechanism of their involvement in the lung injury appears to be different, with the stromelysin 1-deficient mice having a reduction in the numbers of neutrophils recruited into the lung whereas the gelatinase B-deficient mice had the same numbers of lung neutrophils as did the injured WT controls. These studies indicate, first, that both gelatinase B and stromelysin 1 are involved in the development of experimental acute lung injury, and second, that the mechanisms by which these individual MMPs function appear to differ.     

Early cytokine production is associated with protection from murine cerebral malaria

Cerebral malaria (CM) is an infrequent but serious complication of Plasmodium falciparum infection in humans. Animal and human studies suggest that the pathogenesis of CM is immune mediated, but the precise mechanisms leading to cerebral pathology are unclear. In mice, infection with Plasmodium berghei ANKA results in CM on day 6 postinoculation (p.i.), while infection with the closely related strain P. berghei K173 does not result in CM. Infection with P. berghei K173 was associated with increased plasma gamma interferon (IFN-gamma) at 24 h p.i. and with increased splenic and hepatic mRNAs for a range of cytokines (IFN-gamma, interleukin-10 [IL-10], and IL-12) as well as the immunoregulatory enzyme indoleamine 2,3-dioxygenase. In contrast, P. berghei ANKA infection was associated with an absence of cytokine production at 24 h p.i. but a surge of IFN-gamma production at 3 to 4 days p.i. When mice were coinfected with both ANKA and K173, they produced an early cytokine response, including a burst of IFN-gamma at 24 h p.i., in a manner similar to animals infected with P. berghei K173 alone. These coinfected mice failed to develop CM. In addition, in a low-dose P. berghei K173 infection model, protection from CM was associated with early production of IFN-gamma. Early IFN-gamma production was present in NK-cell-depleted, gammadelta-cell-depleted, and Jalpha281(-/-) (NKT-cell-deficient) mice but absent from beta2-microglobulin mice that had been infected with P. berghei K173. Taken together, the results suggest that the absence of a regulatory pathway involving IFN-gamma and CD8(+) T cells in P. berghei ANKA infection allows the development of cerebral immunopathology.     

High parasite burdens cause liver damage in mice following Plasmodium berghei ANKA infection independently of CD8(+) T cell-mediated immune pathology

Infection of C57BL/6 mice with Plasmodium berghei ANKA induces a fatal neurological disease commonly referred to as experimental cerebral malaria. The onset of neurological symptoms and mortality depend on pathogenic CD8(+) T cells and elevated parasite burdens in the brain. Here we provide clear evidence of liver damage in this model, which precedes and is independent of the onset of neurological symptoms. Large numbers of parasite-specific CD8(+) T cells accumulated in the liver following P. berghei ANKA infection. However, systemic depletion of these cells at various times during infection, while preventing neurological symptoms, failed to protect against liver damage or ameliorate it once established. In contrast, rapid, drug-mediated removal of parasites prevented hepatic injury if administered early and quickly resolved liver damage if administered after the onset of clinical symptoms. These data indicate that CD8(+) T cell-mediated immune pathology occurs in the brain but not the liver, while parasite-dependent pathology occurs in both organs during P. berghei ANKA infection. Therefore, we show that P. berghei ANKA infection of C57BL/6 mice is a multiorgan disease driven by the accumulation of parasites, which is also characterized by organ-specific CD8(+) T cell-mediated pathology.     

Is Ischemia Involved in the Pathogenesis of Murine Cerebral Malaria?

Sequestration of parasitized erythrocytes in the central nervous system microcirculation and increased cerebrospinal fluid lactate are prominent features of cerebral malaria (CM), suggesting that sequestration causes mechanical obstruction and ischemia. To examine the potential role of ischemia in the pathogenesis of CM, Plasmodium berghei ANKA (PbA) infection in CBA mice was compared to infection with P. berghei K173 (PbK) which does not cause CM (the non-CM model, NCM). Cerebral metabolite pools were measured by (1)H nuclear magnetic resonance spectroscopy during PbA and PbK infections. Lactate and alanine concentrations increased significantly at the terminal stage of CM, but not in NCM mice at any stage. These changes did not correlate with parasitemia. Brain NAD/NADH ratio was unchanged in CM and NCM mice at any time studied, but the total NAD pool size decreased significantly in the CM mice on day 7 after inoculation. Brain levels of glutamine and several essential amino acids were increased significantly in CM mice. There was a significant linear correlation between the time elapsed after infection and small, progressive decreases in the cell density/cell viability markers glycerophosphocholine and N-acetylaspartate in CM, indicative of gradual loss of cell viability. The metabolite changes followed a different pattern, with a sudden significant alteration in the levels of lactate, alanine, and glutamine at the time of terminal CM. In NCM, there were significant decreases with time of glutamate, the osmolyte myo-inositol, and glycerophosphocholine. These results are consistent with an ischemic change in the metabolic pattern of the brain in CM mice, whereas in NCM mice the changes were more consistent with hypoxia without vascular obstruction. Mild obstructive ischemia is a likely cause of the metabolic changes during CM, but a role for immune cell effector molecules cannot be ruled out.     

Down-regulation of tissue inhibitor of matrix metalloprotease-1 (TIMP-1) in aged human skin contributes to matrix degradation and impaired cell growth and survival

Up regulation of matrix metalloproteinases (MMPs), particularly collagenase-1 (MMP-1), stromelysin-1 (MMP-3) and gelatinase A (MMP-2) is responsible for the lysis of dermal collagen and elastin fibers during chronological skin aging. Tissue inhibitor of metalloproteinase-1 (TIMP-1) is one representative of the natural MMP inhibitor family, encompassing four members. Its expression is decreased with fibroblast senescence, both ex vivo and in vivo, thus contributing to increased catabolic activity within dermis. TIMP-1 displays multiple biological functions. It inhibits most MMPs, except membrane-type MMP subfamily, with Ki in the subnanomolar range, but also interacts with the hemopexin-like (PEX) domain of pro MMP-9. Besides, it exhibits keratinocyte and fibroblast growth factor-like activity and has been described as a cell survival factor.