Alexander Brill

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)‐α acts as an adhesion‐strengthening and stop signal for T cells migrating toward stromal cell‐derived factor‐1α, while transforming growth factor‐β down‐regulates TNF‐α‐induced matrix metalloproteinase‐9 secretion by monocytes. These findings indicate the importance of how one cytokine, such as TNF‐α, 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.

TGF-β1 enhances SDF-1α-induced chemotaxis and homing of naive T cells by up-regulating CXCR4 expression and downstream cytoskeletal effector molecules

The migration of immunocytes within the extracellular matrix (ECM) is influenced by the activation state of the incoming cell and its responses to the presence of chemokines and cytokines. We studied the regulatory role of TGF‐β1 on T cell homing to secondary lymphatic organs, such as the spleen, and chemotaxis within an ECM‐like environment in using an ECM‐like 3‐dimensional gel system designed to follow the migration of individual leukocytes along chemokine gradients in real time. The numbers of migrating naive, but not memory T cells toward SDF‐1α markedly increased after pre‐incubating the cells with TGF‐β1 (0.25u2004ngu2009/u2009ml) for 24u2004h. The mechanisms underlying TGFβ1‐modulated migration involve the up‐regulation of the expression of the SDF‐1α receptor CXCR4, the enhancement of the SDF‐1α‐induced actin polymerization, and increased phosphorylation of Pyk2, a focal adhesion kinase involved in integrin‐mediated lymphocyte migration, adhesion and interactions with ECM. Interestingly, priming of naive human T cells with TGF‐β1 increased homing of these cells to the spleen of NODu2009/u2009SCID mice in a CXCR4‐dependent manner. We propose that theeffect of TGF‐β1 on the chemotaxis of naive T cells may be important in the locomotion of naive T cells toward SDF‐1α‐rich niches.

Induction of interactions between CD44 and hyaluronic acid by a short exposure of human T cells to diverse pro-inflammatory mediators.

Migration of T cells into extravascular sites of inflammation is mediated by cell–cell and cell–matrix adhesion receptors, including the hyaluronan‐binding glycoprotein, CD44. The biochemical nature of CD44 variants and the ligand specificity, function and the regulation of activation of CD44 expressed on various cell types have been extensively studied. However, little is still known about the short‐term influence of cytokines and chemokines on the activation of CD44 on human T cells. Therefore, we studied the role of inflammatory mediators in regulating the adhesion of T cells from human peripheral blood to immobilized hyaluronan under static or shear stress conditions. We found that the CD44‐dependent adhesion, under static and shear stress (i.e. relative gradual resistance to flow of 150 and 1500u2003s−1) conditions, of T cells to hyaluronan requires a T‐cell activation of 2–3u2003hr and is regulated by the cross‐linking of CD3, cytokines (e.g. interleukin‐2 and tumour necrosis factor‐α), and chemokines (e.g. MIP‐1β, interleukin‐8, and RANTES). This T‐cell adhesion was manifested by polarization, spreading and co‐localization of cell surface CD44 with a rearranged actin cytoskeleton in hyaluronan‐bound T cells. Thus, cytokines and chemokines present in the vicinities of blood vessel walls or present intravascularly in tissues where immune reactions take place, can rapidly activate the CD44 molecules expressed on T cells.

Allicin inhibits SDF‐1α‐induced T cell interactions with fibronectin and endothelial cells by down‐regulating cytoskeleton rearrangement, Pyk‐2 phosphorylation and VLA‐4 expression

Allicin, a major ingredient of fresh garlic extract that is produced during the crushing of garlic cloves, exerts various beneficial biological effects, including a broad spectrum of antimicrobial activity, antihyperlipidaemic and antihypertensive effects. However, how allicin affects the immune system is less well known, and its effect on human T cells has never been studied. Here, we examined the in‐vitro effects of allicin on the functioning of T cells related to their entry to inflamed extravascular sites. We found that allicin (20–100u2003µm) inhibits the SDF‐1α (CXCL12)‐induced T cell migration through fibronectin (FN), and that this inhibition is mediated by the down‐regulation of (i) the reorganization of cortical actin and the subsequent T cell polarization, and (ii) T cell adhesion to FN. Moreover, allicin also inhibited T cell adhesion to endothelial cells and transendothelial migration. The mechanisms underlying these inhibitory effects of allicin are associated with its ability to down‐regulate the phosphorylation of Pyk2, an intracellular member of the focal adhesion kinases, and to reduce the expression of the VCAM‐1‐ and FN‐specific α4β1‐integrin (VLA‐4). The ability of allicin to down‐regulate these chemokine‐induced and VLA‐4‐mediated T cell functions explains its beneficial biological effects in processes where T cells play an important role and suggests that allicin may be used therapeutically with chronic inflammatory diseases.

Increased platelet adhesion under flow conditions is induced by both thalassemic platelets and red blood cells.

Thromboembolic complications are not uncommon in thalassemia. Previous studies suggest increased platelet aggregation and a potential role of pathological changes in the red blood cell (RBC) lipid membrane, induced by oxidative stress. In the present study, platelet adhesion and the effect of thalassemic RBC on platelet adhesion under flow conditions were evaluated, using the Cone and Plate (let) Analyzer(CPA). Twenty-two beta-thalassemia patients and 22 blood type-matched healthy controls were studied. An increased platelet adhesion (% surface coverage, SC), was observed in patients as compared to controls (p < 0.05). When platelet count and haematocrit were normalized by autologous reconstitution, a significant increase in platelet aggregation (average size, AS) was observed (p < 0.05). Increased platelet adhesion (SC and AS), was demonstrated in six patients with a history of thrombosis as compared to 16 patients without any history of thrombosis (p < or = 0.007) and in 17 splenectomized patients as compared to five non-splenectomized patients (p = 0.003). In reconstitution studies, thalassemic RBC mixed with normal platelet-rich plasma significantly increased platelet adhesion compared to normal RBC (SC p < 0.03, AS p < 0.02). Thalassemic platelets reconstituted with normal RBC, had increased aggregation (AS, p < 0.004) in comparison with normal platelets. The results indicate that increased platelet adhesion in beta-thalassemia is induced by both platelets and RBC. Increased platelet adhesion correlated with clinical thrombotic events and thus may suggest a mechanism of thrombosis in thalassemic patients. The potential application of the CPA in identifying thalassemic patients with high risk for thrombosis should be studied prospectively in a larger cohort of patients.

Augmentation of RANTES-Induced Extracellular Signal-Regulated Kinase Mediated Signaling and T Cell Adhesion by Elastase-Treated Fibronectin

T cells migrating across extracellular matrix (ECM) barriers toward their target, the inflammatory site, should respond to chemoattractant cytokines and to the degradation of ECM by specific enzymes. In this study, we examined the effects of RANTES and ECM proteins treated with human leukocyte elastase on T cell activation and adhesion to the ECM. We found that human peripheral blood T cells briefly suspended with RANTES (0.1–100 ng/ml) had increased phosphorylation of their intracellular extracellular signal-regulated kinase (ERK), a mitogen-activated protein kinase involved in the activation of several intracellular downstream effector molecules implicated in cell adhesion and migration. Consequently, a small portion (12–20%) of the responding cells adhered to fibronectin (FN). However, when the T cells were exposed to RANTES in the presence of native immobilized FN, laminin, or collagen type I, ERK phosphorylation was partially inhibited, suggesting that this form of the ECM proteins can down-regulate RANTES-induced intracellular signaling. In contrast, when the T cells were exposed to RANTES in the presence of elastase-treated immobilized FN, but not to elastase-treated laminin, ERK phosphorylation was markedly increased. Furthermore, a large percentage (30%) of RANTES-activated T cells adhered to the enzymatically treated FN in a β1 integrin-dependent fashion. Thus, while migrating along chemotactic gradients within the ECM, T cells can adapt their adhesive performance according to the level of cleavage induced by enzymes to the matrix.

Effects of estradiol and raloxifene on arterial thrombosis in ovariectomized mice.

Objective: The effects of estrogen and selective estrogen receptor modulators (eg, raloxifene) on arterial thrombosis are not well defined. This study assessed the manner and mechanism by which estrogen and raloxifene affect homeostatic pathways in ovariectomized mice after acute arterial injury. Design: Female mice (3 weeks old) underwent ovariectomy or sham operation. Five days after surgery, mice were assigned to treatment with estradiol (5.3 nmol/kg), raloxifene (2.7 &mgr;mol/kg), or placebo (n = 10-12/group). The biological effects of both treatments were assessed by measurements of bone mass and the degree of uterine atrophy. After 4 months of therapy, carotid artery thrombosis was induced by photochemical injury, and the time to vascular occlusion was measured. Results: Both treatments increased bone mineral density (4.1%-7.85%). Reversal of macroscopic uterine atrophy was observed only in estrogen-treated mice. Ovariectomized mice had a shorter time to occlusion compared with sham-operated mice (70.8 ± 7.4 vs 103 ± 11.3 min), suggesting accelerated thrombosis. Both estradiol and raloxifene significantly inhibited intra-arterial thrombosis in ovariectomized mice, prolonging the time to occlusion to 136.33 ± 13.5 and 141.43 ± 9.26 min, respectively. Cyclooxygenase-2 levels in the lung tissue were significantly increased by both raloxifene and estradiol with endothelial nitric oxide synthase expression being unaltered. Platelet adhesion (measured by surface coverage under a shear rate of 1,800 s−1 for 2 min) was significantly reduced in ovariectomized animals, being 4.63% ± 1.47%, 5.78% ± 1.58%, and 10.04% ± 1.33% for raloxifene, estradiol, and placebo, respectively. Conclusions: Ovariectomy amplifies thrombosis. We found that 4 months of treatment with both estradiol and raloxifene attenuates intravascular thrombosis. The antithrombotic effect was accompanied by increased expression of cyclooxygenase-2 and suppression of platelet surface adhesion.

Regulation of T‐cell interaction with fibronectin by transforming growth factor‐β is associated with altered Pyk2 phosphorylation

Although the involvement of transforming growth factor‐β (TGF‐β) in inflammatory reactions has been extensively studied, its mode of action in the context of the extracellular matrix (ECM) is still not fully understood. We undertook this study in an attempt to reveal the putative roles of TGF‐β in T‐cell adhesion and migration. We found that a 60‐min treatment of T cells with TGF‐β regulates T‐cell adhesion to fibronectin (FN), a prototype cell adhesion protein of the ECM, depending on the presence of other activators. At 5u2003pg/ml to 1u2003ng/ml, TGF‐β alone induced T‐cell adhesion to FN in an integrin α4/β1‐ and integrin α5/β1‐dependent manner. TGF‐β also attenuated T‐cell migration on the stromal cell‐derived factor (SDF)‐1α gradients. These effects of TGF‐β were not accompanied by alteration in the expression of very‐late activation antigen type 4 (VLA‐4) and VLA‐5, nor were they mediated by the cyclo‐oxygenase pathway. The cellular mechanism underlying the adhesion‐regulating activities of TGF‐β involves adhesion‐associated cytoskeletal elements. TGF‐β induced the phosphorylation of focal adhesion kinase Pyk2, but not extracellular signal‐regulated kinase (ERK), and this effect was markedly increased in the presence of immobilized FN, suggesting a collaborative role for FN‐specific integrins. Indeed, TGF‐β‐induced Pyk2 phosphorylation was inhibited by monoclonal antibodies against VLA‐4, VLA‐5 and CD29. Thus, TGF‐β, which may appear at extravascular sites during inflammation, affects the adhesion of T cells to ECM glycoproteins and their migration by its ability to differentially induce or inhibit the phosphorylation of Pyk2.

Correction of hemocoagulation changes in pathologic stress by low-power laser irradiation

In experiments on white rats stress was modeled by means of a combined effect of rigid immobilization and sound stimulus (120 dB, 150-500 Hz) for two hours. Just prior to the stress rats of another group were exposed to 15 minute transcutaneous He-Ne laser irradiation (λ - 632.8 nm, power density - 5 mW/cm2). Transcutaneous He-Ne laser irradiation prior to stress prevents its hypercoagulation effect.

Low-power laser irradiation of blood inhibits platelet function: role of cyclic GMP

The aim of the present work was to investigate effect of low power laser irradiation (LPLI) on platelet function in vitro. He-Ne laser (Optronix, USA; (lambda) - 632.8 nm, output power - 7 mW) was employed. Platelet adhesion and aggregation in whole blood (WB) under defined shear conditions were assayed by a Cone and Plate(let) Analyzer. Platelet activation was evaluated by flow cytometry. Level of platelet cGMP was estimated by immunoenzyme assay. Experiments performed showed that LPLI of WB resulted in decrease of platelet deposition on extracellular matrix at high shear rate (1300 s-1). Similar results were obtained using surfaces precoated with either collagen type I or von Willebrand factor. LPLI inhibited fibrinogen binding as well as P-selectin expression on the platelet membrane, induced by thrombin analogue. It was found out that primary acceptor of laser energy responsible for the effect on platelets was located in platelets themselves and not in blood plasma or in other blood cells. LPLI of gel- filtered platelets resulted in increase of intracellular level of cGMP both in the absence and in presence of izobutylmethylxantine (phosphodiesterase inhibitor) suggesting stimulation of synthesis rather than destruction of cGMP under the influence of LPLI. It is suggested that guanylate cyclase and/or NO-synthase might serve as primary acceptors of He-Ne laser light in platelets.