Chemokine-chemokine receptor network in immune cell trafficking

Chemokines play critical roles in leukocyte trafficking in normal and inflammatory conditions. The primary roles of chemokines are to activate integrins for leukocyte adherence on endothelial cells and to induce chemotaxis of leukocytes in tissue microenvironments. Specificity in leukocyte migration is regulated at multiple levels. First, it is achieved through differential tissue expression of chemokines and adhesion molecules. Second, it is achieved by limited and differential expression of chemokine receptors by leukocyte subsets. Third, combinatorial expression of multiple chemokine receptors and adhesion molecules makes leukocyte migration more specific. Homing of leukocytes into various tissue sites (e.g. inflamed skin, small intestine, mucosal tissues, T cell areas vs. B cell follicles) requires different chemokines and chemokine receptors. Furthermore, distinct immune responses and diseases (e.g. Th1 vs. Th2 responses) involve different sets of chemokines and leukocyte subsets. This review examines the recent advances in research on chemokines and chemokine receptors in tissue-specific migration of immune cells, and discusses potential targets of intervention in chemokine-mediated leukocyte migration in normal and diseased conditions.     

Effects of uridine, isomatitol and 4-thiouridine on in vitro cell adhesion and in vivo effects of 4-thiouridine in a lung inflammation model

Since leukocyte adhesion to endothelial cells is crucial for extravasation of leukocytes to sites of inflammation, inhibition of cell-cell adhesion has been suggested as a means to achieve selective modulation of the immune system. We have, using a static in vitro adhesion assay involving adhesion of granulocytes to tumor necrosis factor alpha (TNFalpha)-stimulated human umbilical vein endothelial cells (HUVEC), found three substances--uridine, isomaltitol and 4-thiouridine-that, independently and significantly, reduced leukocyte adhesion by approximately 30-65%. 4-Thiouridine was also tested in an in vivo model of Sephadex (SDX)-induced lung inflammation with Sprague-Dawley rats. Intratracheal instillation of Sephadex (5 mg/kg) alone resulted in a dramatic increase in lung edema and total leukocyte count after 24 h. A differential count of bronchoalveolar lavage (BAL) cells indicated an increased influx of macrophages, eosinophils and neutrophils. Co-administration of 4-thiouridine significantly reduced lung edema by 38%. There was also a significant reduction of the total leukocyte count by 58%. The differential leukocyte count indicated that eosinophil influx alone was reduced by 70%. After Sephadex challenge, we found elevated levels of TNFalpha--an important inflammatory mediator--in the bronchoalveolar lavage fluid (BALF). TNFalpha levels were significantly reduced by more than 80% by co-administration of 4-thoiuridine. These results suggest that uridine, isomaltitol and, especially, 4-thiouridine affect adhesion between leukocytes and activated endothelium, and warrant further in vitro and in vivo studies.     

Integrin/Chemokine Receptor Interactions in the Pathogenesis of Experimental Autoimmune Encephalomyelitis

Excessive infiltration of leukocytes and the elaboration of inflammatory cytokines are believed to be responsible for the observed damage to neurons and oligodendrocytes during multiple sclerosis (MS). Blocking adhesion molecules or preventing the effects of chemotactic mediators such as chemokines can be exploited to prevent immune cell recruitment to inflamed tissues. An anti-α4 integrin antibody (anti-VLA-4mAb/natalizumab (Tysabri®)) has been used as a treatment for MS and reduces leukocyte influx into the brain. In patients, anti-VLA-4 reduces relapses and disability progression. However, its mechanism of action in the brain is not completely understood. The anti-VLA-4mAb was demonstrated to mobilize hematopoietic progenitor cells. Interestingly, the chemokine SDF-1/CXCL12 and its receptor CXCR4 are also key factors regulating the migration of hematopoietic stem cells. Moreover, studies have revealed a crosstalk between SDF-1/CXCR4 and VLA-4 signaling in regulating cell migration. In this study, we address the effects of anti-VLA-4 on chemokine signaling in the brain during MS. We assessed the ability of anti-VLA-4 to regulate Experimental Autoimmune Encephalomyelitis (EAE) and chemokine/receptor signaling. Preclinical administration of anti-VLA-4 delayed clinical signs of EAE. We found that anti-VLA-4 treatment reduced chemokine expression. In order to further explore the interaction of anti-VLA-4 with chemokine/receptor signaling we used dual color transgenic mice. After EAE induction, the expression of both SDF-1/CXCL12 and CXCR4 receptor was upregulated, treatment with anti-VLA-4 inhibited this effect. The effects of anti-VLA-4 on chemokine signaling in the CNS may be of importance when considering its mechanism of action and understanding the pathogenesis of EAE.     

CXC chemokines, MIP-2 and KC, induce P-selectin-dependent neutrophil rolling and extravascular migration in vivo

The purpose of this study was to examine the impact of CXC chemokines, i.e. macrophage inflammatory protein-2 (MIP-2) and KC, on leukocyte-endothelium interactions in detail and to evaluate the role of P-selectin by use of intravital microscopy in the mouse cremaster muscle. Administration of MIP-2 and KC provoked a dose (5 - 500 ng)- and time (0 - 4 h)-dependent increase in leukocyte rolling, adhesion and tissue recruitment. Neutrophils comprised more than 92% of the leukocyte response. Pretreatment with an antibody directed against P-selectin (RB40.34) significantly inhibited MIP-2- and KC-induced leukocyte rolling by more than 96%. This marked decrease in rolling abolished firm adhesion and extravascular accumulation of neutrophils (>89% reduction), suggesting that CXC chemokines induce P-selectin-dependent rolling, which in turn apparently is a precondition for the subsequent stationary adhesion and extravasation of neutrophils. Moreover, the extravascular recruitment of leukocytes was evaluated in whole-mounts of the cremaster muscle without preceding intravital microscopy. Using this approach, it was again observed that MIP-2- and KC-induced neutrophil accumulation was completely dependent onP-selectin function. In contrast to the CXC chemokines, administration of the classical chemoattractant formyl-methionyl leucyl phenylalanine (fMLP) did not provoke extravascular tissue accumulation of neutrophils. We could not detect gene expression of CXCR2 in murine endothelial cells, whereas neutrophils were positive, indicating that the stimulatory effect of CXC chemokines on leukocyte-endothelium interactions is not a direct effect on the endothelium but rather an indirect effect via activation of an intermediary tissue cell. However, challenge with MIP-2 and KC did not increase the number of degranulated mast cells. In conclusion, our data demonstrate that CXC chemokines induce all steps in the extravasation process of leukocytes, including rolling, adhesion and transmigration in vivo. Moreover, these results show that P-selectin plays a critical role in MIP-2 and KC provoked neutrophil recruitment as a critical mediator of initial leukocyte rolling. Additionally, our study suggest that a restricted action of MIP-2 and KC on neutrophils is far too simplistic to explain the complex mechanisms of action of CXC chemokines on neutrophil infiltration in vivo.     

CCR1 antagonists in clinical development

Chemokines (chemotactic cytokines) are a family of low-molecular-weight proteins that direct the cellular migration of leukocytes by binding to and activating the G protein-coupled receptors displayed on the leukocyte cell surface. The inadvertent or excessive generation of chemokines has been associated with the inflammatory component of several disease processes, and consequently, considerable efforts have been made to characterise chemokine/chemokine receptor interactions with the ultimate aim of therapeutic intervention. This review focuses on the biology of CC chemokine receptor 1, which together with its ligands is thought to recruit leukocytes during the progression of rheumatoid arthritis, multiple sclerosis and organ transplant rejection. The developments made in antagonising this receptor and efficacies of these compounds in the clinical setting are also highlighted.     

CCR1 antagonists in clinical development

Chemokines (chemotactic cytokines) are a family of low-molecular-weight proteins that direct the cellular migration of leukocytes by binding to and activating the G protein-coupled receptors displayed on the leukocyte cell surface. The inadvertent or excessive generation of chemokines has been associated with the inflammatory component of several disease processes, and consequently, considerable efforts have been made to characterise chemokine/chemokine receptor interactions with the ultimate aim of therapeutic intervention. This review focuses on the biology of CC chemokine receptor 1, which together with its ligands is thought to recruit leukocytes during the progression of rheumatoid arthritis, multiple sclerosis and organ transplant rejection. The developments made in antagonising this receptor and efficacies of these compounds in the clinical setting are also highlighted.     

CXCR2--the receptor to hit?

Emigration of leukocytes from the microcirculation into inflammatory tissues is one of the hallmarks of our immune system. However, excessive leukocyte recruitment can disturb the integrity of the organism and aggravate acute and chronic inflammatory diseases. Chemokines are chemoattractant peptides that are used as messengers to direct leukocytes to sites of inflammation. They mediate their effects through G-protein-coupled chemokine receptors on the surface of leukocytes and other, nonhematopoietic cells, where they induce a variety of cell responses including cell activation and transmigration. CXC chemokine receptor 2 (CXCR2) has been implicated in numerous inflammatory disorders. In many models of acute and chronic inflammatory diseases, blockade of CXCR2 substantially reduces leukocyte recruitment, tissue damage and mortality. The physiological importance of CXCR2 has led to the development of selective CXCR2 inhibitors that are now being tested in clinical trials. This review will summarize current knowledge about CXCR2 in inflammatory diseases and discuss its potential as a pharmaceutical target.     

Inhibition of Mac-1-dependent leukocyte adhesion by Y-24180, an anti-inflammatory agent, in mice

Y-24180 (4-(2-chlorophenyl)-2-[2-(4-isobutylphenyl)ethyl]-6,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]-diazepine), an antagonist of platelet-activating factor (PAF), has been already clarified to suppress the expression of an adhesion molecule, Mac-1, on human neutrophils in the previous in vitro study. In the present paper, we examined the effect of Y-24180 on in vivo Mac-1 expression on mouse neutrophils using a lipopolysaccharide-induced leukocyte reduction model in which Mac-1-dependent infiltration of neutrophils was involved. Prophylactic oral administration of Y-24180 inhibited the induction of Mac-1-strongly positive neutrophils by intraperitoneal injection of lipopolysaccharide and prevented the reduction of leukocyte number. In contrast, WEB 2086 (3-[4-(2-chlorophenyl)-9-methyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-2-yl]-1-(4-morpholinyl)-1-propanone), another PAF antagonist, showed little effect. PAF injection failed to induce the Mac-1-strongly positive neutrophils in peripheral blood and the reduction of circulating leukocytes, indicating that PAF was not concerned with the lipopolysaccharide-induced up-regulation of Mac-1 expression and leukocyte reduction. Y-24180 inhibited the leukocyte infiltration also in the thioglycollate medium-induced peritonitis, which was mediated by Mac-1-dependent leukocyte adhesion. These results indicate that Y-24180 inhibits the leukocyte infiltration into the inflamed sites by suppressing Mac-1 expression on leukocytes in vivo and can contribute to the improvement of inflammatory diseases in which the Mac-1-dependent leukocyte adhesion is involved.     

Peroxynitrite mediates the failure of neutrophil migration in severe polymicrobial sepsis in mice

BACKGROUND AND PURPOSE: Sepsis is a systemic inflammatory response resulting from the inability of the host to restrict local infection. The failure of neutrophil migration to the infection site is one of the mechanisms involved in this process. Recently, it was demonstrated that this event is mediated by nitric oxide (NO). The present study addresses the possibility that peroxynitrite (ONOO(-)), a NO-derived powerful oxidizing and nitrating compound, could also be involved in neutrophil migration failure. EXPERIMENTAL APPROACH: Male C57Bl/6 mice were subjected to moderate (MSI) or severe (SSI) septic injury, both induced by cecal ligation and puncture (CLP). The leukocyte rolling and adhesion in the mesentery was evaluated by intravital microscopy. Cytokines (TNF-alpha and MIP-1alpha) were measured by ELISA and 3-nitrotyrosine (3-NT) by immunofluorescence. KEY RESULTS: Compared with saline pretreatment of SSI mice, pre-treatment with uric acid, a ONOO(-) scavenger, partially restored the failure of neutrophil rolling, adhesion and migration to the site of infection. These mice also presented low circulating bacterial counts and diminished systemic inflammatory response. Pretreatment with uric acid reduced 3-NT labelling in leukocytes in mesenteric tissues and in neutrophils obtained from peritoneal exudates. Finally, uric acid pretreatment enhanced significantly the survival rate in the SSI mice. Similarly, treatment with FeTPPs, a more specific ONOO(-) scavenger, re-established neutrophil migration and increased mice survival rate. CONCLUSIONS AND IMPLICATIONS: These results indicate that ONOO(-) contributed to the reduction of neutrophil/endothelium interaction and the consequent failure of neutrophil migration into infection foci and hence susceptibility to severe sepsis.     

Regulators of chemokine receptor activity as promising anticancer therapeutics

Chemokines are a family of small proteins inducing directed cell migration via specific chemokine receptors, which play important roles in a variety of biological and pathological processes. Their respective ligands act as proinflammatory mediators that primarily control leukocyte migration into selected tissues and upregulation of adhesion receptors, and also have a role in pathological conditions that require neovascularization. Therapeutic strategies based on modulation of chemokine receptor pathways were reported to be promising clinical strategies in the treatment of inflammatory diseases and viral infections. Recent studies have been also demonstrated that chemokines and chemokine receptors are produced by many different cell types, including tumor cells. Overexpression of many chemokine and chemokine receptors in tumor cells suggests that they are crucial regulators of the levels of tumor infiltrating leukocytes implicated in the tumorigenesis of multiple human cancers. In the tumor microenvironment they control a variety of biological activities, such as production and deposition of collagen, activation of matrix-digesting enzymes, stimulation of cell growth, inhibition of apoptosis and promotion of neo-angiogenesis and metastasis. In this review we elucidate key aspects of chemokine signaling as well as clinically relevant strategies to modulation of chemokine receptor activity in the treatment of cancer with emphasis on small-molecule agents. We also elucidate various research strategies which were found to be useful in the design of chemokine receptor targeted therapeutics.     

Correlation of leukocyte adhesiveness, adhesion molecule expression and leukocyte-induced contraction following balloon angioplasty

1. The aim of this study was to examine the changes in leukocyte adhesion and leukocyte-induced contraction in balloon-injured rabbit subclavian artery and to correlate these changes with vessel morphology and expression of adhesion molecules on the injured arteries. 2. Rabbits were anaesthetized and their left subclavian arteries were injured by balloon inflation and withdrawal followed by sacrifice at 2, 24, 48 h or 8 days after injury. The left and right subclavian arteries were removed and leukocytes were isolated from autologous rabbit blood. Leukocyte-induced contraction was measured in 5-HT precontracted artery rings and leukocyte adhesion was measured using (51)Cr-labelled leukocytes. Immunocytochemistry using paraffin-embedded tissue was employed to detect changes in the expression of adhesion molecules on injured arteries. 3. Autologous leukocytes caused a contraction of rabbit subclavian artery rings, which was prevented by L-NAME (10(-3) M). Balloon-induced injury abolished the contractile response to leukocytes, which correlated with loss of carbachol-induced relaxation 4. Balloon injury markedly enhanced the adhesiveness of the subclavian artery for leukocytes, most notably at 24 and 48 h after injury (1.7 and 1.8 fold respectively). Increased leukocyte adhesion at these two time points correlated with an upregulation of E-selectin, P-selectin and VCAM-1 expression on the remaining endothelium of the injured artery. 5. Vessel morphology revealed that balloon inflation had induced an infiltration of inflammatory cells into the vessel wall, the greatest increase being seen at 24 h after injury. 6. It is concluded that an increase in the expression of E-selectin, P-selectin and VCAM-1 following balloon-induced injury leads to enhanced leukocyte adhesion and migration into the injured vessel.     

Neutrophil function in severe sepsis

Sepsis and septic shock continue to be a major cause of morbidity and mortality in critically ill patients. During the onset of sepsis, several inflammatory mediators, including cytokines, chemokines and nitric oxide are released systemically and mediate most of the pathophysiological events present in sepsis and septic shock, such as cardiovascular dysfunction and target-organ lesions. Polymorphonuclear leukocytes are critical effector cells during the inflammatory process and their migration to the infection focus is extremely important for the local control of bacterial growth and consequently for the prevention of bacterial dissemination. In experimental models and in human sepsis a profound failure of neutrophil migration to the infection focus is observed. It seems that the failure of neutrophil migration is dependent on toll-like receptor 4 (TLR4) and mediated by cytokines and chemokines, which induce the production of nitric oxide that inhibits neutrophil adhesion to venular endothelium and also the neutrophil chemotactic ability.     

Mechanisms in mediating the anti-inflammatory effects of baicalin and baicalein in human leukocytes

To evaluate the possible mechanisms responsible for the anti-inflammatory effects of baicalin or baicalein, phorbol-12-myristate-13-acetate (PMA)- or N-formyl-methionyl-leucyl-phenylalanine (fMLP)-activated inflammatory responses of peripheral human leukocytes were studied. Both baicalin and baicalein diminished fMLP- or PMA-induced reactive oxygen intermediates production in neutrophils or monocytes. Neither baicalin nor baicalein prevented the protein kinase C (PKC)-dependent assembly of the NADPH oxidase. Conversely, myeloperoxidase (MPO) activity was inhibited by baicalin or baicalein. fMLP-induced activation of leukocytes, as reflected by increased surface expression of Mac-1 (CD11b/CD18) and Mac-1-dependent neutrophil adhesion, were also inhibited by baicalin or baicalein. Furthermore, baicalein, but not baicalin, impeded fMLP- or AlF(4)(-)-induced Ca(2+) influx. We conclude that impairment of reactive oxygen intermediates production, through scavenging reactive oxygen intermediates by baicalin, or antagonizing ligand-initiated Ca(2+) influx by baicalein, accounts for the inhibition of Mac-1-dependent leukocyte adhesion that confers the anti-inflammatory activity of baicalin or baicalein.     

Chemokine scavenging by the human cytomegalovirus chemokine decoy receptor US28 does not inhibit monocyte adherence to activated endothelium

The human cytomegalovirus has found smart ways to exploit the chemokine network in order to subvert immune attack. Chemokines trigger the arrest and firm adhesion of inflammatory cells to the vascular wall. Scavenging of chemokines by viral decoy receptors, such as US28, might prevent arrest of leukocytes to the vascular wall and impair an antiviral immune response. We determined the effect of chemokine scavenging by endothelium-expressed signaling mute US28 (US28R129A) on static monocyte adhesion. Despite the chemokine scavenging capacity of US28R129A, expression of this construct by endothelial cells was insufficient to disrupt leukocyte adhesion to cytokine-activated monolayers. Our results suggest that the concentrations of chemokines that trigger firm leukocyte adhesion are too high to be efficiently scavenged by viral chemokine decoy receptors like US28. From the results of this experimental model a role for US28 in viral immune evasion by chemokine scavenging would appear therefore unlikely.     

Local inflammatory events induced by Bothrops atrox snake venom and the release of distinct classes of inflammatory mediators

Bothrops atrox is responsible for most accidents involving snakes in the Brazilian Amazon and its venom induces serious systemic and local effects. The local effects are not neutralized effectively by commercial antivenoms, resulting in serious sequelae in individuals bitten by this species. This study investigates the local inflammatory events induced in mice by B. atrox venom (BaV), such as vascular permeability, leukocyte influx and the release of important inflammatory mediators such as cytokines, eicosanoids and the chemokine CCL-2, at the injection site. The effect of BaV on cyclooxygenase (COX-1 and COX-2) expression was also investigated. The results showed that intraperitoneal (i.p.) injection of BaV promoted a rapid and significant increase in vascular permeability, which reached a peak 1 h after venom administration. Furthermore, BaV caused leukocyte infiltration into the peritoneal cavity between 1 and 8 h after i.p. injection, with mononuclear leukocytes (MNs) predominating in the first 4 h, and polymorphonuclear leukocytes (PMNs) in the last 4 h. Increased protein expression of COX-2, but not of COX-1, was detected in leukocytes recruited in the first and fourth hours after injection of BaV. The venom caused the release of eicosanoids PGD₂, PGE₂, TXA₂ and LTB₄, cytokines TNF-α, IL-6, IL-10 and IL-12p70, but not IFN-γ, and chemokine CCL-2 at different times. The results show that BaV is able to induce an early increase in vascular permeability and a leukocyte influx to the injection site consisting mainly of MNs initially and PMNs during the later stages. These phenomena are associated with the production of cytokines, the chemokine CCL-2 and eicosanoids derived from COX-1 and COX-2.     

Review. Leukocyte and endothelial cell adhesion molecules in inflammation focusing on inflammatory heart disease

In multicellular organisms the development of adhesion bonds, either among cells or among cells and components of the extracellular matrix, is a crucial process. These interactions are mediated by molecules which are named adhesion molecules and play a main role both at the early stages of the development of tissue integrity and later. Cell adhesion molecules (CAMs) have a key role in several pathologies such as cancer and inflammatory diseases. Selectins, integrins and immunoglobulin gene superfamily of adhesion receptors mediate different steps of leukocyte migration from the bloodstream towards the inflammatory foci. Leukocyte interactions with the vascular endothelium are highly orchestrated processes that include the capture of free-flowing leukocytes from the blood with subsequent leukocyte rolling, arrest, firm adhesion and ensuing diapedesis. These interactions occur under high shear stresses within venules and depend on multiple families of adhesion molecules. As a response to infection mediators, leukocyte gathering is considered to be crucial for the adequate defence of the organism to any kind of injury or infection. Endothelial activation contributes significantly to the systemic inflammatory response to bacteraemia and increased expression. Release of soluble endothelial markers into the circulation has been demonstrated together with elevated plasma levels of CAMs and has been reported in bacteraemic patients. It has been proposed that infection of endothelial cells with Staphylococcus aureus, Streptococcus sanguis, or Staphylococcus epidermidis induces surface expression of ICAM-1 and VCAM-1 and monocyte adhesion. In general, leukocyte/endothelial cell interactions such as capture, rolling, and firm adhesion can no longer be viewed as occurring in discrete steps mediated by individual families of adhesion molecules but rather as a series of overlapping synergistic interactions among adhesion molecules resulting in an adhesion cascade. These cascades thereby direct leukocyte migration, which is essential for the generation of effective inflammatory responses and the development of rapid immune responses.     

Critical role of P-selectin-dependent rolling in tumor necrosis factor-α-induced leukocyte adhesion and extravascular recruitment in vivo

Leukocyte-endothelium interactions are dependent on a coordinated expression and function of specific adhesion molecules. The objective of the present study was to examine the role of selectin function and leukocyte rolling in tumor necrosis factor-alpha (TNF-alpha)-induced leukocyte adhesion and extravasation in venules in vivo. For this purpose, we used intravital microscopy in the mouse cremaster muscle stimulated for 2-3 h with TNF-alpha intrascrotally. Pretreatment with fucoidan, which inhibits P- and L-selectin, and a P-selectin monoclonal antibody (RB40.34) abolished TNF-alpha-stimulated leukocyte rolling. This great reduction in rolling caused a marked attenuation of firm adhesion and extravascular accumulation of leukocytes. When fucoidan and RB40.34 were administrated after stimulation with TNF-alpha, it was found that leukocyte rolling was greatly reduced whereas the number of firmly adherent leukocytes was completely unchanged, suggesting that the inhibitory effect of blocking P-selectin function on firm leukocyte adhesion and recruitment was due to the reduction in leukocyte rolling along the endothelium. Moreover, pretreatment with a monoclonal antibody against intercellular adhesion molecule-1 (ICAM-1) and a platelet-activating factor (PAF)-receptor antagonist had no effect of TNF-alpha-induced leukocyte rolling and adhesion, indicating that molecules other than ICAM-1 and PAF mediate firm adhesion and recruitment of leukocytes in TNF-alpha-activated tissues. Taken together, our data demonstrate that P-selectin function plays an important role in TNF-alpha-induced inflammatory cell recruitment by mediating leukocyte rolling as a precondition for cytokine-provoked firm adhesion and transmigration in vivo. These findings, thus, suggest that inhibition of P-selectin may be a central target for pharmacological intervention in inflammatory diseases.     

Delayed polymorphonuclear leukocyte infiltration is an important component of Thalassophryne maculosa venom pathogenesis

Thalassophryne maculosa fish envenomation is characterized by severe pain, dizziness, fever, edema and necrosis. Here, the dynamic of cellular influx, activation status of phagocytic cells, and inflammatory modulator production in the acute inflammatory response to T. maculosa venom was studied using an experimental model. Leukocyte counting was performed (2 h to 21 days) after venom injection in BALB/c mice footpads. Our results showed an uncommon leukocyte migration kinetic after venom injection, with early mononuclear cell recruitment followed by elevated and delayed neutrophil influx. The pattern of chemokine expression is consistent with the delay in neutrophil recruitment to the footpad: T. maculosa venom stimulated an early production of IL-1beta, IL-6, and MCP-1, but was unable to induce an effective early TNF-alpha and KC release. Complementary to these observations, we detected a marked increase in soluble KC and TNF-alpha in footpad at 7 days post-venom injection when a prominent influx of neutrophils was also detected. In addition, we demonstrated that bone marrow-derived macrophages and dendritic cells were strongly stimulated by the venom, showing up-regulated ability to capture FITC-dextran. Thus, the reduced levels of KC and TNF-alpha in footpad of mice concomitant with a defective accumulation of neutrophils at earlier times provide an important clue to uncovering the mechanism by which T. maculosa venom regulates neutrophil movement.     

Leukotrienes are involved in leukocyte recruitment induced by live Histoplasma capsulatum or by the beta-glucan present in their cell wall

1. The inflammatory cell influx towards the peritoneal cavity in mice inoculated i.p. with live or dead Histoplasma capsulatum or with its subcellular preparations was studied. We also evaluated the effects of dexamethasone (Dexa) or MK886, an inhibitor of leukotriene (LT) biosynthesis, on the recruitment of leukocytes. 2. Live yeast form of fungus (LYH) induced an increase in neutrophils (NE) which was highest 4 to 24 h after inoculation. Mononuclear cell (MN) migration beginning at 24 h with a gradual increase over 48 and 168 h, and an eosinophil (EO) recruitment occurs between 24 and 48 h. 3. NE and EO recruitment induced by dead mycelial form of fungus (DMH) was greater than that observed for dead yeast form of fungus (DYH). A similar leukocyte migration pattern was seen after i.p. injection of the alkali-insoluble fraction (F1) from DYH (F1Y) and F1 from DMH (F1M) this being more active than former. The difference in concentration of beta-glucan in DYH and DMH could explain the different inflammatory capacity exhibited by the two forms of H. capsulatum. 4. LT seems to be the principal mediator of leukocyte migration in response to LYH, DYH or DMH or to beta-glucan. However, other mediators appear to contribute to NE and EO migration since the treatment with Dexa was more effective in inhibiting cell migration than MK886. Complement dependent leukocyte migration may participate in this recruitment. Treatment with MK886 completely abolished MN cell migration, indicating its dependence on the presence of LT.