Nitza Lahat

Regulation of Endothelial Matrix Metalloproteinase-2 by Hypoxia/Reoxygenation

Among the consequences resulting from the exposure of endothelial cells (ECs) to ischemia/reperfusion is angiogenesis, involving degradation of vascular basement membrane and extracellular matrix. Matrix metalloproteinase (MMP)-2, a member of the MMP family, partakes in this process. MMP-2, secreted as a proenzyme, undergoes activation through interaction with membrane type (MT)1-MMP and the endogenous tissue inhibitor of MMPs (TIMP)-2. Although hypoxia and reoxygenation (H/R) are major constituents of ischemia/reperfusion processes, their direct effects on endothelial MMP-2 have been scarcely investigated. This study examined the in vitro effects of H/R on human macrovascular ECs (EAhy 926). The level of MMP-2 mRNA (Northern blot) and protein (zymography, ELISA) and the mRNA of its activator (MT1-MMP) and inhibitor (TIMP-2) were analyzed. Short (6-hour) hypoxia inhibited the mRNA expression of MMP-2, MT1-MMP, and TIMP-2, culminating in reduced latent and active MMP-2 protein. Prolonged (24-hour) hypoxia further suppressed MT1-MMP and TIMP-2 mRNA, whereas it enhanced MMP-2 mRNA and enzyme secretion (after 48-hour hypoxia). Reoxygenation did not influence the inhibited TIMP-2 but upregulated MMP-2 and MT1-MMP mRNA expression, leading to enhanced secretion of active MMP-2 protein. These results demonstrate H/R-mediated modulation of EC MMP-2 at both transcriptional and posttranscriptional levels. Prolonged hypoxia of ECs appears to enhance MMP-2 production and secretion, whereas reoxygenation further increases its level. These H/R-mediated effects on MMPs have the potential of enabling EC migration and possible angiogenesis.

The Role of Macrophage-Derived IL-1 in Induction and Maintenance of Angiogenesis

Inflammation and angiogenesis are pivotal processes in the progression of many diseases, including malignancies. A hypoxic microenvironment often results in a milieu of proinflammatory and proangiogenic cytokines produced by infiltrating cells. We assessed the role of macrophage-derived hypoxia-associated cytokines in promoting inflammation and angiogenesis. Supernatants of macrophages, stimulated under hypoxia with or without an inflammatory stimulus (LPS), promoted angiogenesis when incorporated into Matrigel plugs. However, neutralization of IL-1 in the supernatants, particularly IL-1β, completely abrogated cell infiltration and angiogenesis in Matrigel plugs and reduced vascular endothelial growth factor (VEGF) levels by 85%. Similarly, supernatants from macrophages of IL-1β knockout mice did not induce inflammatory or angiogenic responses. The importance of IL-1 signaling in the host was demonstrated by the dramatic reduction of inflammatory and angiogenic responses in Matrigel plugs that contained macrophage supernatants from control mice which had been implanted in IL-1 receptor type I knockout mice. Myeloid cells infiltrating into Matrigel plugs were of bone marrow origin and represented the major source of IL-1 and other cytokines/chemokines in the plugs. Cells of endothelial lineage were the main source of VEGF and were recruited mainly from neighboring tissues, rather than from the bone marrow. Using the aortic ring sprouting assay, it was shown that in this experimental system, IL-1 does not directly activate endothelial cell migration, proliferation and organization into blood vessel-like structures, but rather activates infiltrating cells to produce endothelial cell activating factors, such as VEGF. Thus, targeting IL-1β has the potential to inhibit angiogenesis in pathological situations and may be of considerable clinical value.

Matrix metalloproteinases and their tissue inhibitors as markers of disease subtype and response to interferon-β therapy in relapsing and secondary-progressive multiple sclerosis patients

Matrix metalloproteinases (MMPs) have recently been implicated in the pathogenesis of multiple sclerosis. Their suggested role includes the disruption of the blood–brain barrier, immune cell transmigration into the central nervous system, and myelin degradation. The present study characterized the mRNA level of a wide spectrum of MMPs and tissue inhibitors of metalloproteinases (TIMPs) expressed by peripheral blood leukocytes from relapsing‐remitting (n = 16) and secondary‐progressive (n = 12) multiple sclerosis patients. The expression of the same MMPs and TIMPs was evaluated also in a prospective 12‐month follow‐up of 6 patients randomly chosen from each of the 2 groups during interferon‐β‐1a treatment. Reverse transcription‐polymerase chain reaction assessment demonstrated elevated levels of MT1‐MMP and MMP‐7 mRNA levels in both groups of patients, and no significant differences in MMP‐9 levels, compared with healthy controls. Divergent expression of MMP‐2 between relapsing‐remitting and secondary‐progressive patients compared with controls was observed. IFN‐β treatment was associated with significant suppression of MMP‐9 and MMP‐7 mRNA in relapsing‐remitting patients, though no significant changes were observed in the secondary‐progressive group. These results contribute to the understanding of the interferon‐β‐mediated immunomodulatory and therapeutic effects in multiple sclerosis patients and also support evidence for distinct immune mechanism(s) underlying relapsing‐remitting‐ versus secondary‐progressive multiple sclerosis. The study also suggests that MMPs may be considered as potential biomarkers for response to treatment as well as targets for immunotherapy in multiple sclerosis.

The use of a single serological marker underestimates the prevalence of celiac disease in Israel: a study of blood donors.

OBJECTIVES:Recent studies suggest that celiac disease was previously underdiagnosed. To find out whether antiendomysial antibodies underestimate the prevalence of celiac disease, we elected to use a strategy combining multiple serological markers to explore the prevalence of celiac disease in Israel and the usefulness of the various antibodies in screening for celiac disease.METHODS:Serum samples from 1571 healthy blood donors were tested. A small intestinal biopsy was offered to all patients who tested positive for either human tissue transglutaminase antibodies, an ELISA kit based on antiendomysium (EMA-ELISA), immunoglobulin A antigliadin verified by antiendomysial immunofluorescence antibodies, and to patients who were IgA deficient with elevated antigliadin IgG.RESULTS:A total of 59 subjects (3.8% of study population) were offered an intestinal biopsy based on serological findings, and 30 of 59 patients agreed to undergo intestinal biopsy (1.9% of study population). Celiac disease was diagnosed in 10 patients, establishing a prevalence of at least 1:157 in the general population (0.6%, CI = 0.3–1.1%). Using any serological marker alone would have underestimated the prevalence of celiac disease, as it was diagnosed in only two patients who tested positive for endomysial immunofluorescence antibodies (prevalence of 1:786, 0.1%, CI = 0.02–0.5%), six patients positive for tissue transglutaminase (prevalence of 1:262, 0.4%, CI = 0.1–0.9%), and seven patients positive for ELISA-EMA (prevalence of 1:224, 0.45%, CI = 0.2–0.9%).CONCLUSIONS:The prevalence of celiac disease in Israel is at least 1:157 in the general population, confirming its underdiagnosis in previous studies. The disparity between the various serological markers suggest that the use of one serological marker is insufficient for establishing the true prevalence of celiac disease.

Transforming growth factor-β suppresses tumor necrosis factor α-induced matrix metalloproteinase-9 expression in monocytes

The inflammatory response is marked by the release of several cytokines with multiple roles in regulating leukocyte activities, including the secretion of matrix metalloproteinases (MMPs). Although the effects of individual cytokines on monocyte MMP expression have been studied extensively, few studies have examined the influence of combinations of cytokines, which are likely present at inflammatory sites. Herein, we report our investigation of the combinatorial effects of tumor necrosis factor (TNF)‐α and transforming growth factor (TGF)‐β on MMP‐9 synthesis. We found that TGF‐β suppressed TNF‐α‐induced MMP‐9 secretion by MonoMac‐6 monocytic cells in a dose‐dependent manner, with a maximal effect of TGF‐β observed at 1 ng/ml. Such suppression was likely regulated at the pretranslational level, because steady‐state mRNA levels of TNF‐α‐induced MMP‐9 were reduced by TGF‐β, and pulse‐chase radiolabeling also showed a decrease in new MMP‐9 protein synthesis. The suppressive effects of TGF‐β were time dependent, because short exposures to TNF‐α before TGF‐β or simultaneous exposure to both cytokines efficiently reduced MMP‐9 secretion. Expression of the tissue inhibitor of metalloproteinases (TIMP)‐1 and TNF‐α receptors was unaffected by either cytokine individually or in combination. Affinity binding with radiolabeled TGF‐β demonstrated that levels of TGF‐β receptors were not increased after preincubation with TGF‐β. Suppression of TNFα‐induced MMP‐9 secretion by TGF‐β correlated with a reduction in prostaglandin E2 (PGE2) secretion. Furthermore, the effect of TGF‐β or indomethacin on blockage of TNF‐α‐stimulated MMP‐9 production was reversed by the addition of either exogenous PGE2 or the cyclic AMP (cAMP) analogue Bt2cAMP. Thus, we concluded that TGF‐β acts as a potent suppressor of TNF‐α‐induced monocyte MMP‐9 synthesis via a PGE2‐ and cAMP‐dependent mechanism. These results suggest that various combinations of cytokines that are present at inflammatory sites, as well as their balance during different stages of inflammation, may provide the signals necessary for directing MMP‐mediated leukocyte activities.

Three different GB virus C/hepatitis G virus genotypes: Phylogenetic analysis and a genotyping assay based on restriction fragment length polymorphism

The 5′‐untranslated region (5′‐UTR) sequences of 33 GB virus C/hepatitis G virus (GBV‐C/HGV) obtained from different geographic areas were determined through reverse‐transcription polymerase chain reaction and dideoxy chain termination sequencing, the alignment of sequences, the estimation of the number of nucleotide substitution per site, and construction of phylogenetic trees. The 5′‐UTR of GBV‐HGV was found to be heterogeneous, with 70.9–99.5% homology. Three distinct phylogenetic branches were observed consistently in all phylogenetic trees. GBV‐C is the prototype for one, HGV for another, and there is a new branch which consisted of GBV‐C/HGV isolates from Asia. Genotype‐specific restriction sites for the restriction enzymes, ScrFI and BsmFI, were identified, and a simple restriction fragment polymorphism analysis was developed for genotyping. These data provide evidence that GBV‐C/HGV consists of three different genotypes. Our simple genotyping assay will also provide a tool for epidemiological studies of GBV‐C/HGV infection.

Differential effects of proclatic upon activation and differentiation of human B lymphocytes

Abstract The effects of human prolactin on enriched peripheral B lymphocytes obtained from healthy males were examined. Immunoregulation by prolactin was studied in B cells activated with either anti-IgM alone, or anti-IgM antibodies and recombinant interleukin-2 (r-IL-2), as well as control resting B cells. Expression of IL-2 receptors (IL-2R), and IgM and IgG in the culture supernatants were used as a measure of B cell activation and differentiation. Prolactin significantly synergized with IL-2 in the enhancement of surface expression of IL-2R on anti-IgM treated B cells. Although no differentiating effect was observed on resting B cells, prolactin (0.2–100 ng/ml) exhibited a dose-dependent enhancement upon both IgM and IgG secretion from B cells treated with anti-IgM and IL-2. In the absence of exogenously added IL-2 similar differentiating effect were observed in B cells treated with anti-IgM at prolactin concentrations of 0.2–10 ng/ml, but not 100 ng/ml. Thus, the present results demonstrate the modulatory effect of prolactin on activation and differentiation of anti-IgM triggered human B cells, and emphasize the importance of co-stimulatory signal mediated by IL-2 in B cell responses to high prolactin levels. These findings extend the immunoregulatory effects of prolactin, previously confirmed for T cells, to the B cell arm of the immune response, and suggest an important role of prolactin in mediating adaptation and communication between the nerve and immune systems.

Serum levels of IL-1, IL-6 and tumour necrosis factors in patients undergoing coronary artery bypass grafts or cholecystectomy.

Plasma levels of biologically active IL‐1, tumour necrosis factor (TNF) and IL‐6 were measured before, during and after coronary artery bypass graftings (CABG) (n= 9) and cholecystectomy (CHO. n= 9), and in normal controls (nine healthy volunteers). Mean pre‐operative IL‐1 concentration in four of the nine CABG patients was 0.452 ± 0.03 ng/ml, significantly (P < 0.001) higher than that of the other five (0.045 ± 0.009 ng/ml), CHO patients (0.035 ± 0.005 ng/ml) and controls (0.029 ± 0.008 ng/ml). Three of the four patients with high pre‐operative IL‐1 had functional capacity IV, while the other five had functional capacity IIa or IIb. Slight IL‐1 elevation after anaesthesia, followed by reduction after initiation of bypass, elevation on completion of surgery and reduction to basal levels after 7 days was found in patients undergoing CABG. Mean basal TNF levels of CABG and CHO patients did not differ, but were higher than those of controls (2.85 ± 0.5 ng/ml for CABG, 2.05 ± 0.06 ng/ml for CHO, 0.72 ± 007 ng/ml for normals, P < 0.001). A unique kinetics of release during CABG was observed also for TNF. Mean pre‐operative IL‐6 levels were normal (50 ± 3 ng/ml for CABG. 50 ± 0.5 ng/ml for CHO and 65 ± 10 ng/ml for controls). Gradual elevation to a mean peak of725 ± 100 ng/ml on completion of CABG was observed as compared with 275 ± 50 ng/ml in CHO (P < 0.01). On the seventh post‐operative day mean IL‐6 levels returned to normal. Two patients with post‐operative low‐grade fever (38°C) had high, late eytokine levels. One of these two patients had leucocytosis, sterile discharge from the operative wound and was diagnosed as suffering from the Dressler syndrome. In this study elevated cytokine values and unique kinetics of release into the serum were found in patients undergoing CABG.

Hypoxia reduces the output of matrix metalloproteinase-9 (MMP-9) in monocytes by inhibiting its secretion and elevating membranal association

Cellular hypoxia, characterizing tumors, ischemia, and inflammation induce recruitment of monocytes/macrophages, immobilize them at the hypoxic site, and alter their function. To migrate across the extracellular matrix and as part of their inflammatory functions, monocytes and macrophages secrete proteases, including matrix metalloproteinase‐9 (MMP‐9), whose expression is induced by proinflammatory cytokines [e.g., tumor necrosis factor α (TNF‐α)]. We show that hypoxia (<0.3% O2 for 48 h) reduced the output of TNF‐α‐induced proMMP‐9 by threefold (P<0.01) in the U937 monocytic cell line and in primary human monocytes. TNF‐α induced MMP‐9 transcription by threefold, but no significant difference was observed in MMP‐9 mRNA steady‐state between normoxia and hypoxia, which inhibited the trafficking of proMMP‐9 via secretory vesicles and increased the intracellular accumulation of proMMP‐9 in the cells by 47% and 62% compared with normoxia (P<0.05), as evaluated by zymography of cellular extracts and confocal microscopy, respectively. Secretion of proMMP‐9 was reduced by the addition of cytochalazin B or nocodazole, which inhibits the polymerization of actin and tubulin fibers, or by the addition of the Rho kinase inhibitor Y27632, suggesting the involvement of the cytoskeleton and the Rho GTPases in the process of enzyme secretion. Furthermore, attachment of proMMP‐9 to the cell membrane increased after hypoxia via its interactions with surface molecules such as CD44. In addition, the reduced migration of monocytes in hypoxia was shown to be mediated, at least partially, by secreted MMP‐9. Thus, hypoxia post‐translationally reduced the secreted amounts of proMMP‐9 by using two mutually nonexclusive mechanisms: mostly, inhibition of cellular trafficking and to a lesser extent, attachment to the membrane.

Fibronectin‐bound TNF‐α stimulates monocyte matrix metalloproteinase‐9 expression and regulates chemotaxis

Tumor necrosis factor α (TNF‐α) is a proinflammatory cytokine implicated in the stimulation of matrix metalloproteinase (MMP) production by several cell types. Our previous studies demonstrated that TNF‐α avidly binds fibronectin (FN) and laminin, major adhesive glycoproteins of extracellular matrix (ECM) and basement membranes. These findings suggested that TNF‐α complexing to insoluble ECM components may serve to concentrate its activities to distinct inflamed sites. Herein, we explored the bioactivity and possible function of ECM‐bound TNF‐α by examining its effects on MMP‐9 secretion by monocytes. Immunofluorescent staining indicated that LPS‐activated monocytes deposited newly synthesized TNF‐α into ECM‐FN. FN‐bound TNF‐α (FN/TNF‐α) significantly up‐regulated MMP‐9 expression and secretion by the human monocytic cell line MonoMac‐6 and peripheral blood monocytes. Such secretion could be inhibited by antibodies that block TNF‐α activity and binding to its receptors TNF RI (p55) and TNF RII (p75). Chemotaxis through ECM gels in the presence of soluble or bound TNF‐α was inhibited by a hydroxamic acid inhibitor of MMPs (GM6001). It is interesting that, although the adhesion of MonoMac‐6 cells to FN/TNF‐α required functional activated β1 integrins, FN/TNF‐α‐induced MMP‐9 secretion was independent of binding to β1 integrins, since MMP‐9 secretion was unaffected by: (1) neutralizing mAb to α4, α5, and β1 subunits, which blocked cell adhesion; (2) a mAb that stimulated β1 integrin‐mediated adhesion; and (3) binding TNF‐α to the 30‐kDa amino‐terminal fragment of FN, which lacks the major cell adhesive binding sites. Thus, in addition to their cell‐adhesive roles, ECM glycoproteins, such as FN, may play a pivotal role in presenting proinflammatory cytokines to leukocytes within the actual inflamed tissue, thereby affecting their capacities to secrete ECM‐degrading enzymes. These TNF‐α‐ECM interactions may serve to limit the cytokine’s availability and bioactivity to target areas of inflammation.