MIP-3alpha, MIP-3beta and fractalkine induce the locomotion and the mobilization of intracellular calcium, and activate the heterotrimeric G proteins in human natural killer cells

We demonstrate here that the CC chemokines macrophage inflammatory protein-3alpha (MIP-3alpha), macrophage inflammatory protein-3beta (MIP-3beta) and the CX3C chemokine fractalkine induce the chemotaxis of interleukin-2 (IL-2)-activated natural killer (IANK) cells. In addition, these chemokines enhance the binding of [gamma-35S]guanine triphosphate ([gamma-35S]GTP) to IANK cell membranes, suggesting that receptors for these chemokines are G protein-coupled. Our results show that MIP-3alpha receptors are coupled to Go, Gq and Gz, MIP-3beta receptors are coupled to Gi, Gq and Gs, whereas fractalkine receptors are coupled to Gi, and Gz. All three chemokines induced a robust calcium response flux in IANK cells. Cross-desensitization experiments show that MIP-3alpha, MIP-3beta or fractalkine use receptors not shared by each other or by the CC chemokine regulated on activation, normal, T-cell expressed, and secreted (RANTES), the CXC chemokines stromal-derived factor-1alpha (SDF-1alpha) and interferon-inducible protein-10 (IP-10), or the C chemokine lymphotactin.     

Chemokine production by a human alveolar epithelial cell line in response to Mycobacterium tuberculosis

To investigate the role of chemokines during the initial local response to Mycobacterium tuberculosis in the human lung, we studied chemokine production by the human alveolar epithelial cell line A549 after infection with M. tuberculosis. M. tuberculosis-infected A549 cells produced mRNAs and protein for monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8) but not mRNAs for macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, and RANTES. Chemokine production in response to M. tuberculosis was not dependent on production of tumor necrosis factor alpha, IL-1beta, or IL-6. Two virulent clinical M. tuberculosis isolates, the virulent laboratory strain H37Rv, and the avirulent strain H37Ra elicited production of comparable concentrations of MCP-1 and IL-8, whereas killed M. tuberculosis and three Mycobacterium avium strains did not. The three virulent M. tuberculosis strains grew more rapidly than the avirulent M. tuberculosis strain in the alveolar epithelial cell line, and the three M. avium strains did not grow intracellularly. These findings suggest that intracellular growth is necessary for mycobacteria to elicit production of MCP-1 and IL-8 by alveolar epithelial cells but that virulence and the rate of intracellular growth do not correlate with chemokine production. Alveolar epithelial cells may contribute to the local inflammatory response in human tuberculosis by producing chemokines which attract monocytes, lymphocytes, and polymorphonuclear cells.     

High expression of the chemokine receptor CCR3 in human blood basophils. Role in activation by eotaxin, MCP-4, and other chemokines

Eosinophil leukocytes express high numbers of the chemokine receptor CCR3 which binds eotaxin, monocyte chemotactic protein (MCP)-4, and some other CC chemokines. In this paper we show that CCR3 is also highly expressed on human blood basophils, as indicated by Northern blotting and flow cytometry, and mediates mainly chemotaxis. Eotaxin and MCP-4 elicited basophil migration in vitro with similar efficacy as regulated upon activation normal T cells expressed and secreted (RANTES) and MCP-3. They also induced the release of histamine and leukotrienes in IL-3-primed basophils, but their efficacy was lower than that of MCP-1 and MCP-3, which were the most potent stimuli of exocytosis. Pretreatment of the basophils with a CCR3-blocking antibody abrogated the migration induced by eotaxin, RANTES, and by low to optimal concentrations of MCP-4, but decreased only minimally the response to MCP-3. The CCR3-blocking antibody also affected exocytosis: it abrogated histamine and leukotriene release induced by eotaxin, and partially inhibited the response to RANTES and MCP-4. In contrast, the antibody did not affect the responses induced by MCP-1, MCP-3, and macrophage inflammatory protein-1alpha, which may depend on CCR1 and CCR2, two additional receptors detected by Northern blotting with basophil RNA. This study demonstrates that CCR3 is the major receptor for eotaxin, RANTES, and MCP-4 in human basophils, and suggests that basophils and eosinophils, which are the characteristic effector cells of allergic inflammation, depend largely on CCR3 for migration towards different chemokines into inflamed tissues.     

Migration of eosinophils through basement membrane components in vitro: role of matrix metalloproteinase-9

In general, inflammatory cells cross basement membranes by producing proteinases. To investigate the role of proteinases in eosinophil basement membrane migration, we studied peripheral blood eosinophils in Matrigel-coated chemotaxis chambers. Electron microscopy showed degradation of the Matrigel layer when eosinophils, added to the upper chamber, transmigrated the membrane in the presence of both platelet-activating factor (PAF) in the lower chamber and interleukin (IL)-5 in both chambers. In the absence of either or both PAF and IL-5, no changes occurred in the Matrigel layer. Matrigel transmigration of eosinophils induced by PAF and IL-5 was inhibited by 1,10-phenanthroline, batimastat, 3,4-dichloroisocoumarin, chymostatin, and a neutralizing antibody for the matrix metalloproteinase (MMP)-9, indicating that serine proteinase(s) and MMP, specifically MMP-9, were involved in the transmigration of eosinophils through Matrigel. In contrast, eosinophil migration through a bare membrane was not affected by batimastat. Using gelatin zymography and immunoblotting, MMP-9 was detected in the migration upper chamber supernatant of the eosinophil transmigration assay and in the conditioned medium of eosinophils. Release of MMP-9 by eosinophils was increased by IL-5, PAF, or both, but the substrate-degrading activity of MMP-9 was increased only in the presence of both IL-5 and PAF, indicating that the releasing and activating mechanisms of MMP-9 are involved in eosinophil basement membrane migration. This study implicates MMP-9 in basement membrane migration of eosinophils and suggests its involvement in inflammatory diseases where tissue eosinophilia plays a role.     

Glomerular endothelial cells synthesize collagens but little gelatinase A and B

Mesangial sclerosis is a major feature of progressive renal disease. The mesangium contains mesangial cells and is bounded by the peripheral glomerular basement membrane and endothelial cells. Mesangial cells synthesize and degrade extracellular matrix. Whereas both mesangial and endothelial cells synthesize extracellular matrix components, the degradative pathway, well studied in the former, has not been investigated in endothelial cells. This study examines lines of all three glomerular cell types derived from female B6SJLF1/J mice, as well as mRNA levels for collagens alpha1(I), alpha1(IV), alpha3 (IV), alpha5 (IV), and alpha1 (VI), laminin, tenascin, matrix metalloproteinase-2 (MMP-2), and MMP-9. Type I and IV collagen synthesis was confirmed by enzyme-linked immunosorbent assay. MMP-2 and MMP-9 enzyme activity was measured by zymography. It was found that glomerular endothelial cells are a significant source of collagens, laminin, and tenascin. However, they express only low levels of MMP-2 and no detectable MMP-9. Stimulation with exogenous transforming growth factor-beta1 leads to a significant increase in collagen I, tissue inhibitors of metalloproteinase-1, and MMP-9 in conditioned media. These data suggest that glomerular endothelial cells may play an active role in extracellular matrix remodeling in glomerular disease.     

CD40 ligand (CD154) stimulation of macrophages to produce HIV-1-suppressive beta-chemokines

beta-chemokines play an important role in the development of immunologic reactions. Macrophages are major beta-chemokine-producing cells during T-cell directed, delayed-type hypersensitivity reactions in tissues, and have been reported to be important producers of beta-chemokines in the lymph nodes of HIV-1-infected individuals. However, the physiological signals responsible for inducing macrophages to produce beta-chemokines have not been established. Two soluble T cell products, interferon-gamma and granulocyte-macrophage colony stimulating factor, were added to cultured macrophages, but failed to stimulate the production of macrophage inflammatory protein-1alpha and -1beta; regulated upon activation, normal T cell expressed and secreted (RANTES); or monocyte chemoattractant protein-1. Instead, direct cell-cell contact between macrophages and cells engineered to express CD40L (also known as CD154) resulted in the production of large amounts of macrophage inflammatory protein-1alpha and -1beta, and RANTES (all ligands for CCR5), and monocyte chemoattractant protein-1 (a ligand for CCR2). Supernatants from CD40L-stimulated macrophages protected CD4(+) T cells from infection by a nonsyncytium-inducing strain of HIV-1 (which uses CCR5 as a coreceptor). These results have implications for granulomatous diseases, and conditions such as atherosclerosis and multiple sclerosis, where CD40L-bearing cells have been found in the macrophage-rich lesions where beta-chemokines are being produced. Overall, these findings define a pathway linking the specific recognition of antigen by T cells to the production of beta-chemokines by macrophages. This pathway may play a role in anti-HIV-1 immunity and the development of immunologic reactions or lesions.     

In vivo production of cytokines and beta (C-C) chemokines in human recurrent herpes simplex lesions--do herpes simplex virus-infected keratinocytes contribute to their production?

Recurrent human herpes simplex lesions are infiltrated by macrophages and CD4 and CD8 lymphocytes, which secrete cytokines and chemokines. Vesicle fluid was examined by ELISA for the presence of cytokines and beta (C-C) chemokines. On the first day of the lesion, high concentrations of interleukin (IL)-1beta, and IL-6, moderate concentrations of IL-1alpha and IL-10, and low concentrations of IL-12 and beta chemokines were found; levels of macrophage inflammatory protein (MIP)-1beta were significantly higher than levels of MIP-1alpha and RANTES. At day 3, the concentrations of IL-1beta, IL-6, and MIP-1beta were lower, whereas the levels of IL-10, IL-12, and MIP-1alpha remained similar, and the level of tumor necrosis factor-alpha was now detectable. Herpes simplex virus infection of keratinocytes in vitro stimulated production of beta chemokines followed by IL-12 and then IL-10, IL-1alpha, IL-1beta, and IL-6, indicating a potential role for these events in early recruitment, activation, and interferon-gamma production of CD4 cells in herpetic lesions.     

Extension of recombinant human RANTES by the retention of the initiating methionine produces a potent antagonist

Extension of recombinant human RANTES by a single residue at the amino terminus is sufficient to produce a potent and selective antagonist. RANTES is a proinflammatory cytokine that promotes cell accumulation and activation in chronic inflammatory diseases. When mature RANTES was expressed heterologously in Escherichia coli, the amino-terminal initiating methionine was not removed by the endogenous amino peptidases. This methionylated protein was fully folded but completely inactive in RANTES bioassays of calcium mobilization and chemotaxis of the promonocytic cell line THP-1. However, when assayed as an antagonist of both RANTES and macrophage inflammatory polypeptide-1 alpha (MIP-1 alpha) in these assays, the methionylated RANTES (Met-RANTES) inhibited the actions of both chemokines. T cell chemotaxis was similarly inhibited. The antagonistic effect was selective since Met-RANTES had no effect on interleukin-8- or monocyte chemotractant protein-1-induced responses in these cells. Met-RANTES can compete with both [125I]RANTES and [125I]IMP-1 alpha binding to THP-1 cells or to stably transfected HEK cells recombinantly expressing their common receptor, CC-CKR-1. These data show that the integrity of the amino terminus of RANTES is crucial to receptor binding and cellular activation.     

Regulation of matrix-degrading enzymes in gynecologic cancer tissues and cells

1. INTRODUCTION: Studies of tumor invasion and metastases have focused on the degradation of the basement membrane, which is predominantly made up of type IV collagen, laminin, and heparan sulfate proteoglycans. Matrix metalloproteinase-2 (MMP-2) and MMP-9, which can degrade type IV collagen, are implicated in cancer invasion and metastasis. Released and activated MMPs are controlled by specific tissue inhibitors of metalloproteinase (TIMP). In the present study, we have examined gelatinolytic and TIMP activity in the conditioned medium of human normal and cancer tissues by zymography and reverse zymography. 2. MATERIALS AND METHODS: 1) Tissues. Tissues were obtained at operation after informed consent was got from each patient. Sliced tissues were incubated in serum-free medium for 4 or 24 h at 37 degrees C. Human ovarian cancer cells (SAOV) were cultured for 24 h in serum-free medium containing conditioned medium of stromal tissues. After washing by PBS 3 times, SAOV cells were cultured for a further 24 h. 2) Zymography. Conditioned medium was subjected to SDS polyacrylamide gel containing 0.3 mg/ml of gelatin in zymography, and purified MMPs were added further in reverse zymography. After electrophoresis the gel was washed with Triton X-100, and incubated for 20 h at 37 degrees C in the reaction buffer. The gel was then stained with Coomassie brilliant blue. The gelatinase and TIMP activities were detected as unstained and stained bands, respectively. The photographs of the gels were scanned with a densitometer. 3) Other method. TIMP-1 levels of conditioned medium were assayed by ELISA kit. 4) Statistics. Statistical comparisons were made by Mann-Whiteny U test. 3. RESULTS AND DISCUSSION: We have examined the gelatinolytic activity in gynecologic normal and cancer tissues by zymography and reverse zymography. Ovarian, cervical, and endometrial cancer tissues demonstrated higher gelatinolytic activity than normal tissues. The major gelatinases were those with molecular weight of 92 and 72kD, which corresponded to MMP-9 and MMP-2, respectively. The ratio of MMP 9 to MMP-2 was significantly higher in 3 types of cancer tissues than in normal tissues. Reverse zymography demonstrated that TIMP-1 and TIMP-2 were present in all tissues, and the ratio of TIMP-1 to TIMP-2 was significantly higher in 3 types of cancer tissues than in normal tissues. These findings suggested that MMP-9 and TIMP-1 were more associated with cancer phenotype than other types of MMP and TIMP. The influence of human stromal tissues (peritoneum, myometrium, ovary) on the secretion of MMPs and TIMPs was examined by addition of these stromal tissues culture medium to human ovarian cancer cells (SAOV). All conditioned medium of stromal tissues could increase in both MMP-2, MMP-9, TIMP-1, and TIMP-2 activity in SAOV cells. Fraction (> 100kD) of conditioned medium of peritoneum could increase remarkably in MMP-9, and this increase could be inhibited by anti alpha 5 antibody, which is the most popular receptor of fibronectin. Furthermore, the addition of fibronectin to SAOV cells induced increase in the secretion of MMP-9. These results demonstrated that one of the factors included in conditioned medium of peritoneum was fibronectin. We found that interferon beta could suppressed the secretion of MMP-2 and invasion in choriocarcinoma cells. However, no effect of interferon beta was observed in SAOV cells. Several flavonoids were screened to have ability to suppress the secretion of MMPs. All trans retinoic acid (RA) could suppress the secretion of MMPs in SAOV cells in time and concentration dependent manners. Further, RA could inhibited the invasion of SAOV cells by invasion assay using boyden chamber coated with matrigel.     

Leishmania donovani infection initiates T cell-independent chemokine responses, which are subsequently amplified in a T cell-dependent manner

Control of Leishmania donovani infection in immunocompetent mice is associated with hepatic inflammation and granuloma formation, both of which are absent in severe combined immunodeficient (scid) mice. In both BALB/c and scid mice, L. donovani infection induced a rapid hepatic accumulation of mRNA encoding macrophage inflammatory protein-1alpha (MIP-(1alpha), monocyte chemoattractant protein-1 (MCP-1) and interferon-gamma inducible protein-10 (gammaIP-10). This response was not preceded by increased IL-4 production in either strain, unlike that reported in other infectious disease models. Interestingly, only gammaIP-10 mRNA was maintained at elevated levels throughout the first 7 days of infection, by mechanisms involving CD4+ and CD8+ T cells, and CD4+CD8+ cells not activated in scid mice. By in vivo depletion and reconstitution of scid mice it was demonstrated that T cells regulate the expression of all three chemokines studied, while they themselves only produce gammaIP-10 in appreciable quantities.     

Inhibition of head and neck squamous cell carcinoma growth and invasion by the calcium influx inhibitor carboxyamido-triazole

Local invasion and lymph node metastasis are correlated with a decreased overall survival in head and neck cancer patients and warrant new strategies to intervene in the metastatic cascade. One approach is to focus on the intracellular signaling pathways underlying the metastatic process. A common regulatory point in several signal transduction pathways is intracellular calcium homeostasis. We assessed the effect of a novel calcium influx inhibitor, carboxyamido-triazole (CAI), on the growth and invasive phenotype of cell lines derived from head and neck squamous cell carcinoma (HNSCC). CAI inhibited the growth of FaDu and EVSCC17M cells in a dose-dependent (IC50, 13-15 microM) and reversible manner. CAI also caused a generalized attenuation of receptor-mediated calcium elevation to several calcium mobilization agonists, including epidermal growth factor and bradykinin. The effects of CAI on the invasive phenotype of HNSCC cell lines were assessed by a chemo-invasion assay. HNSCC cell lines exhibited a range of invasive potential as measured by the capacity of tumor cells to penetrate a reconstituted basement membrane of Matrigel. HNSCCs were classified as highly invasive (EVSCC14M and EVSCC17M) or weakly invasive (EVSCC18, EVSCC19M, UMSCC10A, and FaDu). Treatment of HNSCC cell lines with 10 microM CAI for 24 h reduced invasion 2-14-fold in a dose-dependent manner. HNSCCs also exhibited different motilities as measured by a chemotaxis assay. EVSCC14M and EVSCC17M were highly motile, whereas EVSCC18, EVSCC19M, UMSCC10A, and FaDu were less motile. CAI reduced the migration of all cell lines. Conditioned medium from HNSCC cell lines was analyzed by zymography for production of Mr 72,000 type IV collagenase [matrix metalloproteinase (MMP)-2)] and Mr 92,000 type IV collagenase (MMP-9). All HNSCC cell lines secreted MMP-2 and/or MMP-9 into conditioned medium. Treatment of cells with 10 microM CAI for 24 h resulted in a reduction of both MMP-2 and MMP-9 production. The results demonstrate that CAI blocks cellular proliferation, migration, chemoinvasion, and MMP production by HNSCC in vitro and identify calcium-dependent signaling as a new target for inhibition of the malignant phenotype of HNSCC.     

Matrix-mesangial cell interaction modulates migration of macrophages

BACKGROUND: Macrophages (M?s) have been demonstrated to play an important role in immune-mediated renal injury. Accumulation of macrophages in the mesangium has been reported to be a key event in the development of focal glomerulosclerosis. We hypothesized that mesangial cells (MCs) and matrix interaction may be a determinant for the migration of M?s into the mesangium. Therefore, we studied the effect of the interaction between matrix and MCs on the migration of M?s. METHODS: Mouse MCs were plated on Petri dishes coated either with buffer, collagen type I, III, IV, or Matrigel in media containing 1% fetal calf serum for 48 hours. Subsequently, supernatants were collected and stored. The effect of these supernatants (conditioned media) was evaluated on the migration of M?s across a filter in a modified Boyden chamber. RESULTS: Conditioned media from MCs grown on Matrigel (MC-Matrigel interaction products, MC-MGP) enhanced the migration of macrophages across a filter in a modified Boyden chamber when compared with conditioned media from MCs grown on plastic, collagen type I, type III, or type IV (MC-PP, MC-CI, MC-CIII, and MC-CIV). MC-MGP enhanced the migration of M?s in a dose dependent manner. Anti-MCP-1 antibodies attenuated (P < 0.05) the MC-MGP-induced M? migration (MC-MGP, 16.8 +/- 2.5 vs MC-MGP + anti-MCP-1 antibody, 6.5 +/- 1.2 migrated macrophages/field, n = 12). Anti-TGF-beta antibodies did not attenuate MC-MGP-induced M? migration. MCs grown on Matrigel showed a 5-fold increase of MCP-1 mRNA when compared with cells grown on plastic or collagen type IV. CONCLUSIONS: The present study suggests that matrix components may modulate the migration of M?s. This effect of MC-matrix interaction on macrophage migration may be mediated through the generation of MCP-1.     

Cytogenetic analysis of sister chromosome sets in the second polar body and in pronuclei of unicellular mouse embryos. I. Frequency and origin of aneuploidy in embryos heterozygous for the reciprocal chromosome translocation T[14;15]6Ca]

Matrix metalloproteinases have been implicated to play a vital role in glioma invasion as they degrade extracellular matrix to facilitate the subsequent migration of tumor cells into the surrounding brain tissue. The cytokine Interleukin-10 (IL-10) was detected recently in glial tumors in vivo. Expression of specific IL-10 mRNA as well as blood serum levels of IL-10 in glioma patients increased with malignancy suggesting a functional role of IL-10 in glioma progression. Moreover, glioma cell migration in vitro was enhanced in the presence of IL-10. We therefore investigated the expression of the matrix metalloproteinases (MMPs) stromelysin-1 (MMP-3), 72-kDa collagenase (MMP-2), 92-kDa collagenase (MMP-9), matrilysin (MMP-7) and the human macrophage metalloelastase (MMP-12). In addition, a possible relation between exposure of glioma cells to IL-10 and invasiveness of these cells due to MMP expression was analyzed. Experiments with Matrigel coated Boyden chambers revealed a pronounced dose dependent effect of IL-10 on glioma invasiveness. The synthetic MMP-inhibitor Marimastat markedly reduced cell invasion in the Boyden chambers confirming the significance of MMPs in the process of invasion. Subsequently, the expression level of MMPs and the serine protease uPA was investigated in 7 glioma cell lines (U373, GaMG, U251, GHE, SNB19, U138 and D54) by RT-PCR. In all but one cell line no enhancement of MMP expression by IL-10 was detected. Matrilysin in U373 cells was the only protease found to be upregulated in the presence of IL-10 dependent on cell density. The present data suggest that IL-10 related effects on the invasive properties of the cell lines are not directly mediated by an upregulation of matrix metalloproteinase expression.     

Retinoic acid-induced type II collagen degradation does not correlate with matrix metalloproteinase activity in cartilage explant cultures

OBJECTIVE: To determine the role of matrix metalloproteinases (MMPs) in retinoic acid (RetA)-induced degradation of type II collagen in cartilage. METHODS: Bovine nasal cartilage explants were cultured with 1 microM RetA or in 3 nM interleukin-1alpha (IL-1alpha). Release of proteoglycan and type II collagen into the medium was measured by colorimetric assay and immunoassay, respectively. MMP activity in the medium was determined using a quenched fluorescent substrate assay, while specific collagenases were identified by Western immunoblotting. In some cases the effects of low molecular mass synthetic MMP inhibitors and serum on collagen degradation were studied. RESULTS: RetA promoted maximal breakdown of type II collagen after 4 or 5 weeks in culture, compared with 3 weeks in culture with IL-1alpha. In IL-1alpha-stimulated cultures, collagen degradation was coincident with a large increase in MMP activity in the culture medium, whereas in RetA-stimulated cultures, there was only a small increase. In Western immunoblots of culture media containing RetA, prointerstitial collagenase and active collagenase 3 were sometimes detected, but not in all experiments. In IL-1alpha cultures, active interstitial collagenase was always detected, and active collagenase 3 was detectable in some experiments. Neutrophil collagenase was not detected in any cultures. IL-1alpha-stimulated collagen degradation was effectively inhibited by a potent, broad-spectrum inhibitor of MMPs, whereas it was poorly inhibited by a weak MMP inhibitor. The same 2 compounds were both only weak inhibitors of RetA-induced collagen degradation. When fetal calf serum was included in cartilage cultures, MMP activity in the culture medium was reduced to low levels. This resulted in a marked inhibition of IL-1alpha-induced type II collagen degradation, whereas there was no inhibition of RetA-induced collagen degradation. CONCLUSION: Unlike IL-1alpha, RetA induces degradation of type II collagen in cartilage explants by a mechanism that is mainly independent of those MMPs that can be detected in the culture medium.     

High affinity binding of latent matrix metalloproteinase-9 to the alpha2(IV) chain of collagen IV

Association of matrix metalloproteinases (MMPs) with the cell surface and with areas of cell-matrix contacts is critical for extracellular matrix degradation. Previously, we showed the surface association of pro-MMP-9 in human breast epithelial MCF10A cells. Here, we have characterized the binding parameters of pro-MMP-9 and show that the enzyme binds with high affinity (Kd approximately 22 nM) to MCF10A cells and other cell lines. Binding of pro-MMP-9 to MCF10A cells does not result in zymogen activation and is not followed by ligand internalization, even after complex formation with tissue inhibitor of metalloproteinase-1 (TIMP-1). A 190-kDa cell surface protein was identified by ligand blot analysis and affinity purification with immobilized pro-MMP-9. Microsequencing and immunoblot analysis revealed that the 190-kDa protein is the alpha2(IV) chain of collagen IV. Specific pro-MMP-9 surface binding was competed with purified alpha2(IV) and was significantly reduced after treatment of the cells with active MMP-9 before the binding assay since alpha2(IV) is hydrolyzed by MMP-9. A pro-MMP-9.TIMP-1 complex and MMP-9 bind to alpha2(IV), suggesting that neither the C-terminal nor the N-terminal domain of the enzyme is directly involved in alpha2(IV) binding. The closely related pro-MMP-2 exhibits a weaker affinity for alpha2(IV) compared with that of pro-MMP-9, suggesting that sites other than the gelatin-binding domain may be involved in the binding of alpha2(IV) to pro-MMP-9. Although pro-MMP-9 forms a complex with alpha2(IV), the proenzyme does not bind to triple-helical collagen IV. These studies suggest a unique interaction between pro-MMP-9 and alpha2(IV) that may play a role in targeting the zymogen to cell-matrix contacts and in the degradation of the collagen IV network.     

Chemokine synthesis in the HSV-1-infected cornea and its suppression by interleukin-10

Herpes simplex virus type 1 (HSV-1) infection of the murine cornea results in a tissue-destructive inflammatory response. In this study we show that virus infection induces the synthesis of macrophage inflammatory protein-2 (MIP-2), MIP-1alpha, and monocyte chemoattractant protein-1 (MCP-1). However, only the production of MIP-2 and MIP-1alpha coincided with the influx of leukocytes into the cornea. IL-10 treatment markedly suppressed chemokine message and protein synthesis in vivo. Local administration of IL-10 also dramatically reduced the number of T cells and neutrophils migrating into the cornea and suppressed the severity of corneal disease. The inflammatory response could also be suppressed by the passive transfer of neutralizing antibody to MIP-1alpha but not MCP-1. We conclude that local IL-10 administration can suppress chemokine synthesis, thereby ameliorating corneal disease. Furthermore, our results indicate that MIP-1alpha plays a major role in herpes stromal keratitis development, whereas MCP-1 does not.     

The T cell-directed CC chemokine TARC is a highly specific biological ligand for CC chemokine receptor 4

Thymus and activation-regulated chemokine (TARC) is a recently identified CC chemokine that is expressed constitutively in thymus and transiently in stimulated peripheral blood mononuclear cells. TARC functions as a selective chemoattractant for T cells that express a class of receptors binding TARC with high affinity and specificity. To identify the receptor for TARC, we produced TARC as a fusion protein with secreted alkaline phosphatase (SEAP) and used it for specific binding. By stably transfecting five orphan receptors and five known CC chemokine receptors (CCR1 to -5) into K562 cells, we found that TARC-SEAP bound selectively to cells expressing CCR4. TARC-SEAP also bound to K562 cells stably expressing CCR4 with a high affinity (Kd = 0.5 nM). Only TARC and not five other CC chemokines (MCP-1 (monocyte chemoattractant protein-1), RANTES (regulated upon activation, normal T cells expressed and secreted), MIP-1alpha (macrophage inflammatory protein-1alpha), MIP-1beta, and LARC (liver and activation-regulated chemokine)) competed with TARC-SEAP for binding to CCR4. TARC but not RANTES or MIP-1alpha induced migration and calcium mobilization in 293/EBNA-1 cells stably expressing CCR4. K562 cells stably expressing CCR4 also responded to TARC in a calcium mobilization assay. Northern blot analysis revealed that CCR4 mRNA was expressed strongly in human T cell lines and peripheral blood T cells but not in B cells, natural killer cells, monocytes, or granulocytes. Taken together, TARC is a specific functional ligand for CCR4, and CCR4 is the specific receptor for TARC selectively expressed on T cells.