Antigen-driven bystander suppression after oral administration of antigens.

Suppression of experimental autoimmune encephalomyelitis (EAE) in Lewis rats by the oral administration of myelin basic protein (MBP) is mediated by CD8+ T cells that can be isolated from the spleens of MBP-fed animals. These cells adoptively transfer protection to naive animals subsequently immunized with MBP and complete Freund's adjuvant (CFA) and suppress in vitro MBP proliferative responses. Using a transwell system in which the modulator spleen cells from MBP-fed animals are separated by a semipermeable membrane from responder cells, MBP, or OVA-specific T cell lines, we have found that cell contact is not required for in vitro suppression to occur. In vitro suppression is dependent, however, upon antigen-specific triggering of modulator T cells. Once antigen-specific triggering occurs, suppression across the transwell is mediated by an antigen-nonspecific soluble factor that equally suppresses an MBP line or an ovalbumin (OVA) line. This phenomenon of antigen-driven bystander suppression was also demonstrated in vivo. Specifically, Lewis rats fed OVA which were then immunized with MBP/CFA plus OVA given separately subcutaneously were protected from EAE. Animals fed OVA and then immunized with MBP/CFA without OVA given subcutaneously were not protected. The protective effect of feeding OVA could be adoptively transferred by CD8+ T cells from OVA-fed animals into MBP/CFA plus OVA-injected animals. Feeding bovine serum albumin (BSA) or keyhole limpet hemocyanin did not suppress EAE in animals immunized with MBP/CFA plus OVA. EAE was suppressed, however, if BSA was fed and animals then immunized with MBP/CFA plus BSA given subcutaneously. Antigen-driven bystander suppression appears to be an important mechanism by which antigen-driven peripheral tolerance after oral administration of antigen is mediated, and presumably occurs in the microenvironment accounting for the antigen specificity of suppression generated by oral tolerization to antigens.

Molecular behavior adapts to context: heparanase functions as an extracellular matrix-degrading enzyme or as a T cell adhesion molecule, depending on the local pH.

Migration of lymphocytes into inflammatory sites requires their adhesion to the vascular endothelium and subendothelial extracellular matrix (ECM). The ensuing penetration of the ECM is associated with the expression of ECM-degrading enzymes, such as endo-beta-D glucuronidase (heparanase), which cleaves heparan sulfate (HS) proteoglycans. We now report that, depending on the local pH, a mammalian heparanase can function either as an enzyme or as an adhesion molecule. At relatively acidified pH conditions, heparanase performs as an enzyme, degrading HS. In contrast, at the hydrogen ion concentration of a quiescent tissue, heparanase binds specifically to HS molecules without degrading them, and thereby anchors CD4+ human T lymphocytes. Thus, the local state of a tissue can regulate the activities of heparanase and can determine whether the molecule will function as an enzyme or as a proadhesive molecule.

Extracellular K(+) and opening of voltage-gated potassium channels activate T cell integrin function: physical and functional association between Kv1.3 channels and beta1 integrins.

Elevated extracellular K(+) ([K(+)](o)), in the absence of "classical" immunological stimulatory signals, was found to itself be a sufficient stimulus to activate T cell beta1 integrin moieties, and to induce integrin-mediated adhesion and migration. Gating of T cell voltage-gated K(+) channels (Kv1.3) appears to be the crucial "decision-making" step, through which various physiological factors, including elevated [K(+)](o) levels, affect the T cell beta1 integrin function: opening of the channel leads to function, whereas its blockage prevents it. In support of this notion, we found that the proadhesive effects of the chemokine macrophage-inflammatory protein 1beta, the neuropeptide calcitonin gene-related peptide (CGRP), as well as elevated [K(+)](o) levels, are blocked by specific Kv1.3 channel blockers, and that the unique physiological ability of substance P to inhibit T cell adhesion correlates with Kv1.3 inhibition. Interestingly, the Kv1.3 channels and the beta1 integrins coimmunoprecipitate, suggesting that their physical association underlies their functional cooperation on the T cell surface. This study shows that T cells can be activated and driven to integrin function by a pathway that does not involve any of its specific receptors (i.e., by elevated [K(+)](o)). In addition, our results suggest that undesired T cell integrin function in a series of pathological conditions can be arrested by molecules that block the Kv1.3 channels.

Autocrine secretion of interferon gamma negatively regulates homing of immature B cells.

The mechanism by which immature B cells are sequestered from encountering foreign antigens present in lymph nodes or sites of inflammation, before their final maturation in the spleen, has not been elucidated. We show here that immature B cells fail to home to the lymph nodes. These cells can actively exclude themselves from antigen-enriched sites by downregulating their integrin-mediated adhesion to the extracellular matrix protein, fibronectin. This inhibition is mediated by interferon gamma secretion. Perturbation of interferon gamma activity in vivo leads to the homing of immature B cells to the lymph nodes. This is the first example of autocrine regulation of immune cell migration to sites of foreign antigen presentation.

Anti-idiotypic network induced by T cell vaccination against experimental autoimmune encephalomyelitis.

In a study of the mechanism of resistance to autoimmune disease induced by T cell vaccination, rats were vaccinated against experimental autoimmune encephalomyelitis (EAE) by injecting them once in the hind footpads with a subencephalitogenic dose (10(4)) of a clone of T lymphocytes specific for myelin basic protein (BP). The response to vaccination was assayed by challenging the rats with an encephalitogenic dose (3 X 10(6)) of T lymphocytes of this BP-specific clone. Five to six days after vaccination, the cells responsible for mediating resistance to adoptively transferred EAE were concentrated in the popliteal lymph nodes draining the vaccination site. Transfer of the draining lymph node cells to unvaccinated rats led to loss of resistance in the donor rats and acquisition of resistance by the recipient rats. Limiting-dilution cultures of the draining lymph node cells were established with irradiated cells of the BP-specific clone as stimulators. Two sets of T lymphocytes specifically responsive to the BP-specific T cells from the clone were isolated: CD4+CD8- helper and CD4-CD8+ suppressor cells. The helper T cells, like the BP antigen, specifically stimulated the BP-specific vaccinating clone. In contrast, the suppressor T cells specifically suppressed the response of the BP-specific vaccinating clone to its BP antigen. These results suggest that T cell vaccination induces resistance to autoimmune disease by activating an antiidiotypic network.

Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4.

Stem cell homing and repopulation are not well understood. The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 were found to be critical for murine bone marrow engraftment by human severe combined immunodeficient (SCID) repopulating stem cells. Treatment of human cells with antibodies to CXCR4 prevented engraftment. In vitro CXCR4-dependent migration to SDF-1 of CD34+CD38-/low cells correlated with in vivo engraftment and stem cell function. Stem cell factor and interleukin-6 induced CXCR4 expression on CD34+ cells, which potentiated migration to SDF-1 and engraftment in primary and secondary transplanted mice. Thus, up-regulation of CXCR4 expression may be useful for improving engraftment of repopulating stem cells in clinical transplantation.

Suppression of experimental autoimmune diseases and prolongation of allograft survival by treatment of animals with low doses of heparins.

The ability of activated T lymphocytes to penetrate the extracellular matrix and migrate to target tissues was found to be related to expression of a heparanase enzyme (Naparstek, Y., I. R. Cohen, Z. Fuks, and I. Vlodavsky. 1984. Nature (Lond.). 310:241-243; Savion, N., Z. Fuks, and I. Vlodavsky. 1984. J. Cell. Physiol. 118:169-176; Fridman, R., O. Lider, Y. Naparstek, Z. Fuks, I. Vlodavsky, and I. R. Cohen. 1987. J. Cell. Physiol. 130:85-92; Lider, O., J. Mekori, I. Vlodavsky, E. Baharav, Y. Naparstek, and I. R. Cohen, manuscript submitted for publication). We found previously that heparin molecules inhibited expression of T lymphocyte heparanase activity in vitro and in vivo, and administration of a low dose of heparin in mice inhibited lymphocyte traffic and delayed-type hypersensitivity reactions (Lider, O., J. Mekori, I. Vlodavsky, E. Baharav, Y. Naparstek, and I. R. Cohen, manuscript submitted for publication). We now report that treatment with commercial or chemically modified heparins at relatively low doses once daily (5 micrograms for mice and 20 micrograms for rats) led to inhibition of allograft rejection and the experimental autoimmune diseases adjuvant arthritis and experimental autoimmune encephalomyelitis. Higher doses of the heparins were less effective. The ability of chemically modified heparins to inhibit these immune reactions was associated with their ability to inhibit expression of T lymphocyte heparanase. There was no relationship to anticoagulant activity. Thus heparins devoid of anticoagulant activity can be effective in regulating immune reactions when used at appropriate doses.

Intravenous immunoglobulin treatment of experimental T cell-mediated autoimmune disease. Upregulation of T cell proliferation and downregulation of tumor necrosis factor alpha secretion.

It has been reported previously that intravenous administration of normal human immunoglobulins (IVIg) to human patients can suppress the clinical signs of certain autoimmune diseases. However, the mechanism(s) by which normal Ig interferes with the various disorders and the scheduling of treatment have been poorly delineated. To study these questions, we examined IVIg treatment of two experimentally induced T cell autoimmune diseases in rats: experimental autoimmune encephalomyelitis (EAE) and adjuvant arthritis (AA). We now report that IVIg treatment (0.4 g/kg) inhibited the active induction of both EAE and AA, and that this treatment did not affect the acquisition of resistance to reinduction of EAE. The importance of the site of administration and schedule of treatment were studied in the AA model. Ig was effective when given intravenously, but not when administrated subcutaneously or intraperitoneally. IVIg treatment was effective when given daily from immunization to outbreak of disease; but it was also effective when given once at the time of immunization or once 2 wk after induction of AA, just at the clinical outbreak of disease. Administration of IVIg between immunization and outbreak of AA was less effective. Prevention of disease by IVIg occurred despite the presence of T cell reactivity to the specific antigens in the disease. In fact, IVIg administrated to naive rats activated T cell reactivity to some self-antigens. Nevertheless, IVIg treatment led to decreased production of the inflammatory cytokine TNF alpha. Thus, IVIg treatment may exert its therapeutic power not by inhibiting T cell recognition of self-antigens, but by inhibiting the biological consequences of T cell recognition.

Halofuginone inhibits NF-kappaB and p38 MAPK in activated T cells.

Halofuginone, a low molecular weight plant alkaloid, inhibits collagen alpha1 (I) gene expression in several animal models and in patients with fibrotic disease, including scleroderma and graft-versus-host disease. In addition, halofuginone has been shown to inhibit angiogenesis and tumor progression. It was demonstrated recently that halofuginone inhibits transforming growth factor-beta (TGF-beta), an important immunomodulator. The present study was undertaken to explore the effects of halofuginone on activated T cells. Peripheral blood T cells were activated by anti-CD3 monoclonal antibodies in the absence and presence of halofuginone and assessed for nuclear factor (NF)-kappaB activity, production of tumor necrosis factor alpha (TNF-alpha) and interferon-gamma (IFN-gamma), T cell apoptosis, chemotaxis, and phosphorylation of p38 mitogen-activated protein kinase (MAPK). A delayed-type hypersensitivity (DTH) model was applied to investigate the effect of halofuginone on T cells in vivo. Preincubation of activated peripheral blood T cells with 10-40 ng/ml halofuginone resulted in a significant dose-dependent decrease in NF-kappaB activity (80% inhibition following incubation with 40 ng halofuginone, P = 0.002). In addition, 40 ng/ml halofuginone inhibited secretion of TNF-alpha, IFN-gamma, interleukin (IL)-4, IL-13, and TGF-beta (P < 0.005). Similarly, halofuginone inhibited the phosphorylation of p38 MAPK and apoptosis in activated T cells (P = 0.0001 and 0.005, respectively). In contrast, T cell chemotaxis was not affected. Halofuginone inhibited DTH response in mice, indicating suppression of T cell-mediated inflammation in vivo. Halofuginone inhibits activated peripheral blood T cell functions and proinflammatory cytokine production through inhibition of NF-kappaB activation and p38 MAPK phosphorylation. It also inhibited DTH response in vivo, making it an attractive immunomodulator and anti-inflammatory agent.

Interactions of migrating T lymphocytes, inflammatory mediators, and the extracellular matrix.

Leukocytes are mobile units of the immune system. The process of leukocytes migration from blood vessels to inflamed tissues involves two major steps: (1) extravasation through the vessel wall and (2) movement through the underlying basement membrane and extracellular matrix (ECM). The ECM is a complex macromolecular mesh composed of proteoglycans and adhesive glycoproteins, such as collagen, laminin, and fibronectin, and serves as a supportive structure surrounding cells and can also provide co-stimulatory signals to immune cells. Hence, the basement membrane and the ECM play important roles as contexts in which biological processes take place, and therefore these moieties should be considered as microenvironment milieu in which extravasating cells function, communicate, and signal their messages; the outcome of which can result in the immunological eradication of hazardous elements. During migration, leukocytes continuously exchange information with the surrounding microenvironment. This cross-talk, which is also influenced by cytokines and chemokines, determines the type and the strength of the resulting immune response to foreign determinants. As suggested in the present article, these signals determine the response to a specific antigen and enable the migrating leukocytes to recognize any insult in their vicinity and to rapidly modify their activities.

Effect of Linomide on adhesion molecules, TNF-alpha, nitrogen oxide, and cell adhesion.

Linomide (quinoline-3-carboxamide) is an immunomodulator with anti-inflammatory effects in rodents with autoimmune diseases. Its mode of action still remains to be elucidated. We hypothesized that an investigation of T cell interactions with the extracellular matrix (ECM), composed of glycoproteins such as fibronectin (FN) and laminin (LN), might provide better understanding of their in vivo mode of action in extravascular inflammatory sites. We examined the effect of Linomide on T cell adhesion to intact ECM, and separately to LN, and FN, and on the release and production of tumor necrosis factor (TNFalpha) and nitrogen oxide (NO) in relation to adhesive molecules in non-obese diabetic (NOD) female spleen cells, focusing on intracellular adhesion molecule-1 (ICAM-1) and CD44. NOD female mice that developed spontaneous autoimmune insulitis, which destroys pancreatic islets and subsequently leads to insulin-deficient diabetes mellitus, were studied. Linomide, given in the drinking water or added to tissue cultures in vitro, inhibited the beta1 integrin-mediated adhesion of T cells to ECM, FN and LN, as well as the production and release of TNFalpha and NO, which play a major role in the induction and propagation of T cell-mediated insulitis. In addition, exposure of T cells to Linomide resulted in increased expression of CD44 and ICAM-1 molecules on spleen cells of Linomide-treated mice; such an increase in adhesion molecule expression may lead to more effective arrest of T cell migration in vivo. The regulation of T-cell adhesion, adhesion receptor expression, and inhibition of TNFalpha and NO secretion by Linomide may explain its beneficial role and provide a new tool for suppressing self-reactive T cell-dependent autoimmune diseases.

Extracellular matrix moieties, cytokines, and enzymes: dynamic effects on immune cell behavior and inflammation.

Tissue injury caused by infection or physical damage evokes inflammatory reactions and events that are necessary for regaining homeostasis. Central to these events is the translocation of leukocytes, including monocytes, neutrophils, and T lymphocytes, from the vascular system, through endothelium, and into the extracellular matrix (ECM) surrounding the injured tissue. This transition from the vasculature into the site of inflammation elicits remarkable changes in leukocyte behavior as cells adhere to and migrate across ECM before carrying out their effector functions. Growing evidence suggests that, through its interactions with cytokines and degradative enzymes, the ECM microenvironment has a specialized role in providing intrinsic signals for coordinating leukocyte actions. Recent advances also reveal that enzymatic modifications to ECM moieties and cytokines induce distinctive cellular responses, and are likely part of the mechanism regulating the perpetuation or arrest of inflammation. This article reviews the findings that have elucidated the dynamic relationships among these factors and how they communicate with immune cells during inflammation.

Pervanadate-induced adhesion of CD4+ T cell to fibronectin is associated with tyrosine phosphorylation of paxillin.

The initial stages of T cell activation involve tyrosine protein kinase-mediated intracellular signaling events. Integrin-mediated adhesion of CD4+ T lymphocytes to extracellular matrix glycoproteins, such as fibronectin, is an activation-dependent process. The involvement of tyrosine protein kinases in the adhesion of CD4+ T cells to fibronectin was examined using pervanadate, a protein-tyrosine phosphatase inhibitor. Pervanadate induced the adhesion of human CD4+ T cells to immobilized fibronectin in a beta1 integrin-mediated fashion, and adhesion was associated with an increase of protein tyrosine phosphorylation. Tyrosine protein kinase inhibitors abrogated both T cell adhesion and intracellular protein tyrosine phosphorylation. Participation of cytoskeletal proteins in the pervanadate-induced T cell adhesion was indicated because cytoskeleton disruption by cytochalasin B inhibited cell adhesion to fibronectin. We demonstrate that the cytoskeletal protein paxillin underwent time-dependent tyrosine phosphorylation simultaneously with pervanadate-induced T cell adhesion to fibronectin. Tyrosine phosphorylation of paxillin was related to cell adhesion, since pretreatment of T cells with cytochalasin B abrogated both adhesion and phosphorylation. This study demonstrates a correlation between activation of protein tyrosine kinases, tyrosine phosphorylation of paxillin, and integrin-mediated T cell adhesion to extracellular matrix glycoproteins.

Inhibition of T cell adhesion to extracellular matrix glycoproteins by histamine: a role for mast cell degranulation products.

Mast cells, which are capable of releasing a multitude of preformed and newly generated biological mediators and cytokines, are involved in various inflammatory processes. We studied whether histamine, a mast cell degranulation product, influences the adhesive interactions of T cells with extracellular matrix (ECM) glycoproteins, an event that occurs at sites of inflammation and is mediated primarily by virtue of cell-surface receptors of the beta 1-integrin subfamily. A prerequisite of lymphocyte-ECM interactions is activation of the cells, which modulates the affinity of the otherwise inactive integrins. Isolated rat CD4+ T cells were preincubated with histamine and activated with phorbol myristate acetate (PMA), and their ability to adhere to immobilized ECM components (fibronectin and laminin) was determined. Preincubation with histamine resulted in a 40-50% decrease in the adhesion of the CD4+ cells to both fibronectin or laminin. The notion that inhibition of T cell adhesion to ECM proteins by histamine-induced increase of the cells' intracellular levels of cAMP, thus interfering with calcium influx-associated events that occur during T cell activation, is supported by the finding that T cell adhesion was also abrogated by pharmacological inducers of cAMP. When the T cells were preincubated with supernatants of immunologically activated mast cells and then activated with PMA, a 40-50% inhibition of their adhesion to fibronectin or laminin was also observed. The inhibitory moiety present in the mast cell degranulation supernatants was resistant to heat (80 degrees C). Histamine exerted its suppressive effect on adhesion of T cells via their H2 receptors, as pretreatment with H2 antagonists abrogated the inhibitory effect. Thus, both purified histamine and mast cell-secreted histamine appear to be capable of affecting T cell interactions with ECM.

Transforming growth factor-beta suppresses tumor necrosis factor alpha-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)-alpha and transforming growth factor (TGF)-beta on MMP-9 synthesis. We found that TGF-beta suppressed TNF-alpha-induced MMP-9 secretion by MonoMac-6 monocytic cells in a dose-dependent manner, with a maximal effect of TGF-beta observed at 1 ng/ml. Such suppression was likely regulated at the pretranslational level, because steady-state mRNA levels of TNF-alpha-induced MMP-9 were reduced by TGF-beta, and pulse-chase radiolabeling also showed a decrease in new MMP-9 protein synthesis. The suppressive effects of TGF-beta were time dependent, because short exposures to TNF-alpha before TGF-beta or simultaneous exposure to both cytokines efficiently reduced MMP-9 secretion. Expression of the tissue inhibitor of metalloproteinases (TIMP)-1 and TNF-alpha receptors was unaffected by either cytokine individually or in combination. Affinity binding with radiolabeled TGF-beta demonstrated that levels of TGF-beta receptors were not increased after preincubation with TGF-beta. Suppression of TNFalpha-induced MMP-9 secretion by TGF-beta correlated with a reduction in prostaglandin E2 (PGE2) secretion. Furthermore, the effect of TGF-beta or indomethacin on blockage of TNF-alpha-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-beta acts as a potent suppressor of TNF-alpha-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.

Fibronectin-bound TNF-alpha stimulates monocyte matrix metalloproteinase-9 expression and regulates chemotaxis.

Tumor necrosis factor alpha (TNF-alpha) is a proinflammatory cytokine implicated in the stimulation of matrix metalloproteinase (MMP) production by several cell types. Our previous studies demonstrated that TNF-alpha avidly binds fibronectin (FN) and laminin, major adhesive glycoproteins of extracellular matrix (ECM) and basement membranes. These findings suggested that TNF-alpha 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-alpha by examining its effects on MMP-9 secretion by monocytes. Immunofluorescent staining indicated that LPS-activated monocytes deposited newly synthesized TNF-alpha into ECM-FN. FN-bound TNF-alpha (FN/TNF-alpha) 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-alpha activity and binding to its receptors TNF RI (p55) and TNF RII (p75). Cheniotaxis through ECM gels in the presence of soluble or bound TNF-alpha was inhibited by a hydroxamic acid inhibitor of MMPs (GM6001). It is interesting that, although the adhesion of MonoMac-6 cells to FN/TNF-alpha required functional activated beta1 integrins, FN/TNF-alpha-induced MMP-9 secretion was independent of binding to beta1 integrins, since MMP-9 secretion was unaffected by: (1) neutralizing nAb to alpha4, alpha5, and beta1 subunits, which blocked cell adhesion; (2) a mAb that stimulated beta1 integrin-mediated adhesion; and (3) binding TNF-alpha 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-alpha-ECM interactions may serve to limit the cytokine's availability and bioactivity to target areas of inflammation.

Combinatorial signals by inflammatory cytokines and chemokines mediate leukocyte interactions with extracellular matrix.

On their extravasation from the vascular system into inflamed tissues, leukocytes must maneuver through a complex insoluble network of molecules termed the extracellular matrix (ECM). Leukocytes navigate toward their target sites by adhering to ECM glycoproteins and secreting degradative enzymes, while constantly orienting themselves in response to specific signals in their surroundings. Cytokines and chemokines are key biological mediators that provide such signals for cell navigation. Although the individual effects of various cytokines have been well characterized, it is becoming increasingly evident that the mixture of cytokines encountered in the ECM provides important combinatorial signals that influence cell behavior. Herein, we present an overview of previous and ongoing studies that have examined how leukocytes integrate signals from different combinations of cytokines that they encounter either simultaneously or sequentially within the ECM, to dynamically alter their navigational activities. For example, we describe our findings that tumor necrosis factor (TNF)-alpha acts as an adhesion-strengthening and stop signal for T cells migrating toward stromal cell-derived factor-1alpha, while transforming growth factor-beta down-regulates TNF-alpha-induced matrix metalloproteinase-9 secretion by monocytes. These findings indicate the importance of how one cytokine, such as TNF-alpha, can transmit diverse signals to different subsets of leukocytes, depending on its combination with other cytokines, its concentration, and its time and sequence of exposure. The combinatorial effects of multiple cytokines thus affect leukocytes in a step-by-step manner, whereby cells react to cytokine signals in their immediate vicinity by altering their adhesiveness, directional movement, and remodeling of the ECM.

Keratinocytes-associated chemokines and enzymatically quiescent heparanase induce the binding of resting CD4+ T cells.

Whether the chemokines macrophage inflammatory protein-1 beta (MIP-1 beta) and regulated on activation normal T expressed and secreted (RANTES), which interact specifically with glycosaminoglycans and thus mediate the recruitment, attachment, and migration of leukocytes to vascular endothelia and extracellular matrix, are also involved in interactions between CD4+ murine T lymphocytes and keratinocytes was examined. We have previously observed that depending on the local pH, a mammalian extracellular matrix-degrading enzyme, endo-beta-D glucuronidase (heparanase), which cleaves heparin sulfate proteoglycans, can function wither as an enzyme or as an adhesion molecule for CD4+ T lymphocytes. Herein, the involvement of heparanase in T cell-keratinocyte interactions was also probed. At 37 degree C and pH 7.2, radioactively labeled MIP-1 beta, RANTES, and heparanase bound to confluent layers of resting keratinocytes in a saturable and an heparan sulfate- or heparin-dependent manner, and thereby induced the adhesion of resting CD4+ T cells to keratinocytes. At a relatively acidic pH characteristic of inflammatory milieu, enzymatically active heparanase did not bind to the keratinocytes but, rather, inhibited the binding of MIP-1beta, RANTES, and the enzymatically quiescent heparanase to keratinocytes. These results suggest that certain chemokines and heparanase may function to restrict passing leukocytes, notable T lymphocytes, in the cutaneous micro-environment, a site which is continuously challenged with antigens. These keratinocyte-bound lymphocytes can serve as a reservoir of immediate responders to immunological stimuli.

Serum amyloid A-derived peptides, present in human rheumatic synovial fluids, induce the secretion of interferon-gamma by human CD(4)(+) T-lymphocytes.

Serum amyloid A (SAA) is a major acute-phase protein whose biochemical functions remain largely obscure. Human rheumatic synovial fluids were screened by high performance liquid chromatography mass spectrometry for SAA-derived peptides, specifically the sequence AGLPEKY (SAA(98-104)) which was previously shown to modulate various leukocyte functions. Two such fluids were found to contain a truncated version of SAA(98-104). Synthetic SAA(98-104) and several of its analogs were shown capable of binding isolated human CD(4)(+) T-lymphocytes and stimulating them to produce interferon-gamma. Given the high acute-phase serum level of SAA and its massive proteolysis by inflammatory related enzymes, SAA-derived peptides may be involved in host defense mechanisms.

Differential response of platelets to chemokines: RANTES non-competitively inhibits stimulatory effect of SDF-1 alpha.

BACKGROUND: Among the chemokines related to CXC and CC receptor groups and released from platelets, leukocytes and endothelial cells, SDF-1, TARC and MDC have been found to be platelet agonists. Platelets do not contain SDF-1 alpha. In contrast, RANTES is constitutively present in platelet alpha-granules and released upon platelet activation. OBJECTIVES: To study a possible role of RANTES as a modulator of SDF-1 alpha effect on platelets, in relation to CXCR4 and various CC receptors. METHODS: CXCR-4 (CXCL12) receptor expression and platelet activation were evaluated by flow cytometry, platelet deposition was studied by cone and plate(let) analyzer, and platelet aggregation by turbidometric aggregometry. RESULTS: Flow cytometry studies revealed similar expression of CXCR-4, the specific receptor of SDF-1 alpha on intact, inactivated, and activated platelets. Preincubation of platelets with RANTES affected neither CXCR-4 expression, nor SDF-1 alpha binding to the platelet membrane. In the presence of fibrinogen, SDF-1 alpha activated gel-filtered platelets. RANTES did not activate platelets, but substantially (by 70%) inhibited SDF-1 alpha-induced fibrinogen binding. Similarly, RANTES abrogated the promoting effect of SDF-1 alpha on whole blood platelet adhesion to endothelial cell monolayer under venous flow conditions. In platelet-rich plasma, RANTES moderately inhibited SDF-1 alpha-induced platelet aggregation, while it did not affect aggregation induced by thrombin-receptor activation peptide, adenosine diphosphate, or phorbol 12-myristate 13-acetate. A synergistic inhibitory effect of RANTES and prostaglandin E1 used at subthreshold concentrations, on SDF-1 alpha-induced aggregation and SDF-1 alpha-induced fibrinogen binding to platelets was observed, which may suggest involvement of RANTES in a cAMP-dependent signal transduction pathway. CONCLUSIONS: RANTES non-competitively inhibits activation of platelets by SDF-1 alpha, and thus may play a regulatory role in platelet response to inflammation.