Role In Leukocyte Recruitment Research Articles

A functional heteromeric MIF receptor formed by CD74 and CXCR4

MIF is a chemokine-like inflammatory mediator that triggers leukocyte recruitment by binding to CXCR2 and CXCR4. MIF also interacts with CD74/invariant chain, a single-pass membrane-receptor. We identified complexes between CD74 and CXCR2 with a role in leukocyte recruitment. It is unknown whether CD74 also binds to CXCR4. We demonstrate that CD74/CXCR4 complexes formed when CD74 was expressed with CXCR4 in HEK293 cells. Expression of CD74-variants lacking an ER-retention signal showed CD74/CXCR4 complexes at the cell surface. Importantly, endogenous CD74/CXCR4 complexes were isolated by co-immunoprecipitation from monocytes. Finally, MIF-stimulated CD74-dependent AKT activation was blocked by anti-CXCR4 and anti-CD74 antibodies and AMD3100, whereas CXCL12-stimulated AKT activation was not reduced by anti-CD74. Thus, CD74 forms functional complexes with CXCR4 that mediate MIF-specific signaling. Structured summaryMINT-7234512, MINT-7234528: CD74 (uniprotkb:P04233) physically interacts (MI:0915) with CXCR4 (uniprotkb:P61073) by anti tag coimmunoprecipitation (MI:0007)MINT-7234542: CD74 (uniprotkb:P04233) and CXCR4 (uniprotkb:P61073) physically interact (MI:0915) by anti bait coimmunoprecipitation (MI:0006)MINT-7234499: CXCR4 (uniprotkb:P61073) and CD74 (uniprotkb:P04233) colocalize (MI:0403) by fluorescence microscopy (MI:0416)

Glycosylation in immune cell trafficking

Leukocyte recruitment encompasses cell adhesion and activation steps that enable circulating leukocytes to roll, arrest, and firmly adhere on the endothelial surface before they extravasate into distinct tissue locations. This complex sequence of events relies on adhesive interactions between surface structures on leukocytes and endothelial cells and also on signals generated during the cell-cell contacts. Cell surface glycans play a crucial role in leukocyte recruitment. Several glycosyltransferases such as alpha1,3 fucosyltransferases, alpha2,3 sialyltransferases, core 2 N-acetylglucosaminlytransferases, beta1,4 galactosyltransferases, and polypeptide N-acetylgalactosaminyltransferases have been implicated in the generation of functional selectin ligands that mediate leukocyte rolling via binding to selectins. Recent evidence also suggests a role of alpha2,3 sialylated carbohydrate determinants in triggering chemokine-mediated leukocyte arrest and influencing beta1 integrin function. The recent discovery of galectin- and siglec-dependent processes further emphasizes the significant role of glycans for the successful recruitment of leukocytes into tissues. Advancing the knowledge on glycan function into appropriate pathology models is likely to suggest interesting new therapeutic strategies in the treatment of immune- and inflammation-mediated diseases.

Abstract: 559 CHEMOKINES AS THERAPEUTIC TARGETS IN ATHEROSCLEROSIS

MIF is a chemokine-like inflammatory mediator that triggers leukocyte recruitment by binding to CXCR2 and CXCR4. MIF also interacts with CD74/invariant chain, a single-pass membrane-receptor. We identified complexes between CD74 and CXCR2 with a role in leukocyte recruitment. It is unknown whether CD74 also binds to CXCR4. We demonstrate that CD74/CXCR4 complexes formed when CD74 was expressed with CXCR4 in HEK293 cells. Expression of CD74-variants lacking an ER-retention signal showed CD74/CXCR4 complexes at the cell surface. Importantly, endogenous CD74/CXCR4 complexes were isolated by co-immunoprecipitation from monocytes. Finally, MIF-stimulated CD74-dependent AKT activation was blocked by anti-CXCR4 and anti-CD74 antibodies and AMD3100, whereas CXCL12-stimulated AKT activation was not reduced by anti-CD74. Thus, CD74 forms functional complexes with CXCR4 that mediate MIF-specific signaling.MINT-7234512, MINT-7234528: CD74 (uniprotkb:P04233) physically interacts (MI:0915) with CXCR4 (uniprotkb:P61073) by anti tag coimmunoprecipitation (MI:0007)MINT-7234542: CD74 (uniprotkb:P04233) and CXCR4 (uniprotkb:P61073) physically interact (MI:0915) by anti bait coimmunoprecipitation (MI:0006)MINT-7234499: CXCR4 (uniprotkb:P61073) and CD74 (uniprotkb:P04233) colocalize (MI:0403) by fluorescence microscopy (MI:0416)

Serum amyloid A protein stimulates CCL20 production in rheumatoid synoviocytes

Although serum amyloid A (SAA) has been used as a marker of inflammation, its role in leucocyte recruitment and angiogenesis has not been well established in RA. CCL20 is a chemokine involved in the migration of CCR6-expressing Th17 cells. To study the contribution of SAA to the recruitment of Th17 cells, we investigated the effects of SAA on CCL20 production by RA synoviotytes. Synoviocytes isolated from RA patients were stimulated with recombinant SAA and cellular supernatants were analysed by CCL20-specific ELISA. CCL-20 mRNA expression was analysed by RT-PCR. SAA is a most potent inducer of CCL20 secretion in RA synoviocytes compared with other inflammatory cytokines (IL-1beta, TNF-alpha and IL-17A). SAA stimulation induced CCL20 mRNA expression in RA synoviocytes, which was not affected by polymyxin B pre-treatment. SAA-induced CCL20 production was down-regulated by NF-kappaB inhibition and partially by c-jun N-terminal kinase (JNK) inhibition. SAA-induced CCL20 production was also suppressed by dexamethasone or FK506. These findings suggest that SAA may be implicated in the recruitment of lymphocytes, including CCR6-expressing Th17 cells, in RA synovium by up-regulating CCL20 production in synoviocytes.

Severe impairment of leukocyte recruitment into inflamed tissue of ppGalNAcT1-deficient mice (94.1)

Abstract P-selectin glycoprotein ligand-1 (PSGL-1) plays an important role in leukocyte recruitment. Its binding affinity to selectins is modulated by posttranslational modifications. The polypeptide N-acetylgalactosamine transferase-1 (ppGalNAcT1) initiates core-type protein O-glycosylation. To address whether the glycosylation of PSGL-1 by ppGalNAcT1 is important for leukocyte recruitment, we investigated leukocyte rolling and velocity in untreated and TNF-α treated cremaster muscles and autoperfused flow chambers comparing ppGalNAcT1-/-(Galnt1-/-) with WT mice. In untreated and TNF-α treated Galnt1-/- mice, leukocyte rolling was significantly reduced with markedly increased rolling velocity compared to control mice. Flow chamber experiments showed that Galnt1-/-- and WT-neutrophils had the same rolling velocity on E-selectin, but the rolling velocity of Galnt1-/--neutrophils on E-selectin/ICAM-1 was significantly elevated, suggesting that E-selectin-dependent neutrophil activation may be defective. Thioglycollate-induced peritonitis experiments with chimeric mice revealed that hematopoietic ppGalNAcT1 is important for leukocyte recruitment. These data show that the loss of ppGalNAcT1 led to reduced leukocyte rolling and recruitment and increased rolling velocity, suggesting a predominant role of ppGalNAcT1 in attaching functionally relevant O-linked glycans to PSGL-1.

ICAM-1 targeting of doxorubicin-loaded PLGA nanoparticles to lung epithelial cells

Interaction of leukocyte function associated antigen-1 (LFA-1) on T-lymphoctytes and intercellular adhesion molecule-1 (ICAM-1) on epithelial cells controls leukocyte adhesion, spreading, and extravasation. This process plays an important role in leukocyte recruitment to a specific site of inflammation and has been identified as a biomarker for certain types of carcinomas. Cyclo-(1,12)-PenITDGEATDSGC (cLABL) has been shown to inhibit LFA-1 and ICAM-1 interaction via binding to ICAM-1. In addition, cLABL has been shown to internalize after binding ICAM-1. The possibility of using cLABL conjugated nanoparticles (cLABL-NP) as a targeted and controlled release drug delivery system has been investigated in this study. The cLABL peptide was conjugated to a modified Pluronic ® surfactant on poly ( dl-lactic-co-glycolic acid) (PLGA) nanoparticles. The cLABL-NP showed more rapid cellular uptake by A549 lung epithelial cells compared to nanoparticles without peptide. The specificity of ICAM-1-mediated internalization was confirmed by blocking the uptake of cLABL-NP to ICAM-1 using free cLABL peptide to block the binding of cLABL-NP to ICAM-1 on the cell surface. Cell studies suggested that cLABL-NPs targeted encapsulated doxorubicin to ICAM-1 expressing cells. Cytotoxicity assay confirmed the activity of the drug incorporated in nanoparticles. Sustained release of doxorubicin afforded by PLGA nanoparticles may enable cLABL-NP as a targeted, controlled release drug delivery system.

Chemoattractant Receptors and Leukocyte Recruitment: More Than Cell Migration

Chemoattractants induce cell migration through the activation of a distinct family of structurally related heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors. Over the past few years, several receptors in this family have been identified that recognize different classes of chemoattractants but do not induce cell migration. These atypical "chemoattractant receptors" are unable to activate transduction events that lead directly to cell migration, but appear nonetheless to play a nonredundant role in leukocyte recruitment by shaping the chemoattractant gradient, either by removing, transporting, or concentrating their cognate ligands.

Involvement of TRPC Channels in CCL2-Mediated Neuroprotection against Tat Toxicity

Chemokine (C-C motif) ligand 2 (CCL2), also known as monocyte chemoattractant protein-1, plays a critical role in leukocyte recruitment and activation. In the present study, we identify an additional role for CCL2 that of neuroprotection against HIV-1 transactivator protein (Tat) toxicity in rat primary midbrain neurons. Furthermore, we report the involvement of transient receptor potential canonical (TRPC) channels in CCL2-mediated neuroprotection. TRPC are Ca(2+)-permeable, nonselective cation channels with a variety of physiological functions. Blockage of TRPC channels resulted in suppression of both CCL2-mediated neuroprotection and intracellular Ca(2+) elevations. Parallel but distinct extracellular signal-regulated kinase (ERK)/cAMP response element-binding protein (CREB) and Akt/nuclear factor kappaB (NF-kappaB) pathways were involved in the CCL2-mediated neuroprotection. Blocking TRPC channels and specific downregulation of TRPC channels 1 and 5 resulted in suppression of CCL2-induced ERK/CREB activation but not Akt/NF-kappaB activation. In vivo relevance of these findings was further corroborated in wild-type and CCR2 knock-out mice. In the wild-type but not CCR2 knock-out mice, exogenous CCL2 exerted neuroprotection against intrastriatal injection of HIV-1 Tat. These findings clearly demonstrate a novel role of TRPC channels in the protection of neurons against Tat through the CCL2/CCR2 axis.

Adhesion molecule levels in serum and cerebrospinal fluid in children with bacterial meningitis and sepsis.

Background:Adhesion molecules play a role in leukocyte recruitment during central nervous system (CNS) inflammation.Aim:This study was designed to compare serum, cerebrospinal fluid (CSF) concentrations of adhesion molecules in children with meningitis and sepsis, and to evaluate their sources.Setting:This study was carried out at Pediatric Department, King Abdulaziz University Hospital from January 2007 to June 2008.Design:Serum and CSF samples were collected on admission from meningitis (n = 40), sepsis (n = 20) patients, and sera from controls (n = 20).Materials and Methods:Endothelial (E), leukocyte (L), platelet (P) selectins intercellular cell adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecules-1 (VCAM-1) were measured using ELISA.Statistics:ANOVA and Spearman's correlations were used. Adhesion molecules with albumin concentration were estimated in CSF/serum to calculate concentration quotients.Results:In meningitis, serum sE-, sL-, sP-selectins sICAM-1, sVCAM-1 levels were higher than controls. Compared to sepsis, serum sE-selectin, sL-selectin, sVCAM-1, CSF-sL-selectin, CSF-sVCAM-1, VCAM-1 ratio and index were higher, while serum sP-selectin was lower than meningitis. sE-selectin ratio, CSF sICAM-1 were higher in meningitis with positive than negative culture. The sE-selectin index was higher in meningitis with neurological complication than those without it. In meningitis, correlation was found between CSF protein and CSF white blood cell counts (WBCs), CSF sICAM-1, CSF sVCAM-1 and between CSF sE-selectin and CSF sICAM-1.Conclusions:This study supports the role of adhesion molecules especially sL-selectin, sVCAM-1 in meningitis and suggests further research to determine their use as biomarkers for meningitis and use of their antagonists as therapeutic for CNS inflammation. The presence of discrepancy of CSF/serum ratios for molecules of same molecular weight suggest intrathecal shedding in addition to diffusion through the blood-CSF barrier.

Antagonists of CD117 (cKit) signaling inhibit mast cell accumulation in healing skin wounds

Mast cells (MCs) have important functional roles in leukocyte recruitment, pain, and wound healing, and increased tissue resident MC function has been associated with several fibrotic diseases. Consequently, the study of MCs in situ can be a direct approach to studying the pharmacodynamic impact of MC-directed therapeutics in tissues. Here we describe an automated laser scanning cytometry assay that was used to characterize the kinetics of MC accumulation in healing skin wounds and to study the effect of inhibiting CD117 (cKit) signaling. The number of tryptase-positive MCs approximately doubled 14 days after cutaneous injury in nonhuman primates. Treatment of animals with anti-CD117 or imatinib mesylate (Gleevec) reduced MC accumulation at the edge of healing wounds in mice and nonhuman primates, respectively. In translating this MC assay to become a biomarker for human studies, no differences in dermal MC numbers were evident between genders, ages or body mass index from 20 healthy donors. These data suggest that skin is a practical and useful tissue for tracking pharmacodynamic effects of MC-directed therapies.

Cytokines That Promote Periodontal Tissue Destruction

Although periodontal diseases are initiated by bacteria that colonize the tooth surface and gingival sulcus, the host response is believed to play an essential role in the breakdown of connective tissue and bone, key features of the disease process. An intermediate mechanism that lies between bacterial stimulation and tissue destruction is the production of cytokines, which stimulates inflammatory events that activate effector mechanisms. These cytokines can be organized as chemokines, innate immune cytokines, and acquired immune cytokines. Although they were historically identified as leukocyte products, many are also produced by a number of cell types, including keratinocytes, resident mesenchymal cells (such as fibroblasts and osteoblasts) or their precursors, dendritic cells, and endothelial cells. Chemokines are chemotactic cytokines that play an important role in leukocyte recruitment and may directly or indirectly modulate osteoclast formation. This article focuses on aspects of osteoimmunology that affect periodontal diseases by examining the role of cytokines, chemokines, and immune cell mediators. It summarizes some of the key findings that attempt to delineate the mechanisms by which immune factors can lead to the loss of connective tissue attachment and alveolar bone. In addition, a discussion is presented on the importance of clarifying the process of uncoupling, a process whereby insufficient bone formation occurs following resorption, which is likely to contribute to net bone loss in periodontal disease.

LEUKOTRIENE B4/LEUKOTRIENE B4 RECEPTOR PATHWAY IS INVOLVED IN HEPATIC MICROCIRCULATORY DYSFUNCTION ELICITED BY ENDOTOXIN

Leukotrienes (LTs), metabolites of arachidonic acid through 5-lipoxygenase (5-LOX), have been known to play a role in leukocyte recruitment. However, the contribution of LTB4 to liver microcirculatory dysfunction during endotoxemia remains unknown. LTB4 receptor (BLT1) has been identified as a high-affinity receptor specific for LTB4. The present study was conducted to examine the roles of LTB4 and BLT1 in hepatic microcirculatory dysfunction elicited by LPS in mice. The number of leukocytes adhering to the endothelial cells of the hepatic microvessels and perfused sinusoids was determined 4 h after the administration of LPS (0.3 mg/kg, i.v.) to male C57Bl6 mice by in vivo microscopy. A 5-LOX synthase inhibitor, AA-861 (10 or 100 mg/kg, s.c.), was administered 30 min before LPS injection. BLT1 knockout mice were used to investigate whether LPS-induced hepatic microcirculatory dysfunction is mediated by BLT1 signaling. The expression of 5-LOX, intercellular adhesion molecule (ICAM) 1, and TNF-[alpha] in the liver was measured by real-time reverse-transcriptase-polymerase chain reaction. The administration of LPS caused significant accumulation of leukocyte adhesion to the hepatic microvessels and reduced sinusoidal perfusion when compared with saline-treated mice. The hepatic microcirculatory dysfunction elicited by LPS was minimized in mice pretreated with AA-861 or in BLT1 knockout mice. This was associated with the suppression of hepatic expression of 5-LOX, ICAM-1, and TNF-[alpha]. These findings suggest that the LTB4/BLT1 pathway mediates hepatic microcirculatory dysfunction by enhanced expression of ICAM-1 and TNF-[alpha] in a murine model of endotoxemia.

The role of tumour necrosis factor-alpha and IL-1 in polymorphonuclear leucocyte and T lymphocyte recruitment to joint inflammation in adjuvant arthritis.

The mediators involved in leucocyte recruitment to joints during arthritis are not fully defined, but two important proinflammatory cytokines, IL-1 and tumour necrosis factor-alpha (TNF-alpha), are produced in joints in rheumatoid arthritis (RA). We investigated in the rat adjuvant arthritis model whether endogenous IL-1 and TNF-alpha contribute to joint inflammation and polymorphonuclear leucocyte (PMNL) and T lymphocyte infiltration. The migration of 51Cr-labelled rat blood PMNL and 111In-labelled T lymphocytes to the joints of rats with adjuvant arthritis was measured along with plasma protein extravasation, which was quantified using 125I-labelled human albumin. Rats with active arthritis of 5 days' duration received i.p. non-immune serum, polyclonal neutralizing anti-serum to rat TNF-alpha, antiserum to IL-1 alpha and IL-1 beta, or both anti-TNF plus anti-IL-1 for 5 days. Treatment with anti-IL-1 alpha and IL-1 beta did not affect plasma protein extravasation, or PMNL or T lymphocyte accumulation in the joints (i.e. talar joint, hind paws, and tail) despite the fact that this treatment inhibited 80-90% of the PMNL migration into dermal sites injected with IL-1 alpha or IL-1 beta. In contrast, anti-TNF-alpha treatment significantly improved clinical scores, decreased plasma protein extravasation by 60-80%, inhibited PMNL accumulation by 40-50% and decreased T lymphocyte accumulation by 30-50%. Treatment with anti-IL-1, together with anti-TNF-alpha, significantly potentiated the inhibition of T lymphocyte accumulation observed with anti-TNF-alpha alone. These results indicate that endogenous TNF-alpha production may play an important role in the inflammatory changes and leucocyte recruitment in this experimental model of human arthritis, while IL-1 may have a less important role in leucocyte recruitment to these joints.

Intercellular adhesion molecule-1 genetic markers (+241G/A and +469A/G) in Iranian women with breast cancer

Breast cancer is the most common cancer among women, the second-most leading cause of women's death after lung cancer. ICAM-1 is a cell adhesion molecule that belongs to the Ig-superfamily, with a glycoprotein structure playing a key role in leukocyte recruitment into inflammatory sites, as well as in leukocyte activation and effector function. Proteolytic cleavage of ICAM-1 results in the formation of a soluble form, sICAM-1, which is present in low-serum levels in healthy individuals but becomes elevated in inflammatory and malignant conditions. The ICAM1 gene is located on chromosome 19 and contains two well-known single-nucleotide polymorphisms (SNP) of +241G/A (G241R) and +469A/G (K469E). In this study, the frequencies of the two polymorphisms were investigated in breast cancer patients and healthy individuals. For G241R, we selected 276 breast cancer patients and 235 healthy sex-matched controls, and for K469E, 264 patients and 200 healthy sex-matched controls were chosen. The results of this study show that the frequency of the GA genotype was significantly higher in breast cancer patients in comparison to the control group ( P = 0.007). In addition, the frequency of the R allele was significantly higher in breast cancer patients compared to controls ( P = 0.008). However, both the genotype and allele frequency of K469E did not differ significantly between patients and controls. A significant difference was observed in the frequency of genotype combination A/G (+241 G/A and +469 A/G, respectively) between patients and controls (6.2 vs. 2.2%; ∗ P = 0.007). These findings indicate that individuals carrying the A allele of the ICAM1 gene as well as the A/G haplotype may have a higher risk of developing breast cancer.

Expression of cytokines and cytokine receptors in human Schwann cells

The expression of cytokines and cytokine receptors was investigated in enriched populations of human fetal Schwann cells by reverse transcribed-PCR and enzyme-linked immunosorbent assay. Human fetal Schwann cells constitutively expressed mRNA of IL-1beta, IL-6, IL-8, IL-11, IL-12, IL-15 and TGF-beta, and also cytokine receptors for IL-1, IL-4, IL-6, IL-8, IL-13, tissue necrosis factor and gp130. The expression of IL-1beta, IL-6 and IL-15 was upregulated following treatment with IL-1beta or TGF-beta. The protein levels of IL-6 were increased with IL-1beta treatment, but were decreased with IFN-gamma treatment. Human Schwann cells may respond to cytokine signals in the nerve injury sites and modify the pathological conditions by secreting cytokines. The secreted cytokines may play a role in leukocyte recruitment and exacerbation of axonal injury process.

Possible Mechanism of the Anti-inflammatory Activity of Ruscogenin: Role of Intercellular Adhesion Molecule-1 and Nuclear Factor-κB

Ruscogenin (RUS), first isolated from Ruscus aculeatus, also a major steroidal sapogenin of traditional Chinese herb Radix Ophiopogon japonicus, has been found to exert significant anti-inflammatory and anti-thrombotic activities. Our previous studies suggested that ruscogenin remarkably inhibited adhesion of leukocytes to a human umbilical vein endothelial cell line (ECV304) injured by tumor necrosis factor-Α (TNF-Α) in a concentration-dependent manner. Yet the underlying mechanisms remain unclear. In this study, the in vivo effects of ruscogenin on leukocyte migration and celiac prostaglandin E2 (PGE2) level induced by zymosan A were studied in mice. Furthermore, the effects of ruscogenin on TNF-Α-induced intercellular adhesion molecule-1 (ICAM-1) expression and nuclear factor-κB (NF-κB) activation were also investigated under consideration of their key roles in leukocyte recruitment. The results showed that ruscogenin significantly suppressed zymosan A-evoked peritoneal total leukocyte migration in mice in a dose-dependent manner, while it had no obvious effect on PGE2 content in peritoneal exudant. Ruscogenin also inhibited TNF-Α-induced over expression of ICAM-1 both at the mRNA and protein levels and suppressed NF-κB activation considerably by decreasing NF-κB p65 translocation and DNA binding activity. These findings provide some new insights that may explain the possible molecular mechanism of ruscogenin and Radix Ophiopogon japonicus for the inhibition of endothelial responses to cytokines during inflammatory and vascular disorders.

Abstract 1193: Mast Cell Activation Promotes Leukocyte Recruitment And Adhesion To The Atherosclerotic Plaque

Activated mast cells have been identified in the perivascular tissue of human coronary artery plaques. As mast cells have been described to release a whole array of chemokines including interleukin 8 (IL-8) and MIP1 α, we propose that activated mast cells play a pivotal role in leukocyte recruitment at advanced stages of atherosclerotic plaque development. Peritoneal mast cells of either C57Bl/6 or mast cell deficient Kit(W −sh /W −sh ) mice were activated by injection of compound 48/80 (1.2 mg/kg). Interestingly, mast cell activation led to a massive neutrophil influx into the peritoneal cavity at 3 hours after activation (controls: 1 ± 0.7*10 4 Gr1 + -neutrophils/ml up to 8 ± 0.2*10 4 Gr1 + neutrophils/ml at 3 hours after activation, *P<0.05), while neutrophil numbers in Kit(W −sh /W −sh ) mice were not affected by compound 48/80 administration. Moreover, increased levels of CXCR2 + Gr1 + neutrophils (t=0: 0.55 ± 0.07% versus t=3 hours: 1.00 ± 0.12%, *P<0.05) were observed after mast cell activation. Next, we investigated whether mast cell activation also translated in induced leukocyte adhesion to advanced atherosclerotic plaques. Adventitial mast cells of advanced collar aided carotid artery plaques were activated by local application of a dinitrophenyl-BSA (DNP) challenge in ApoE −/− mice. Three days later, the carotid artery segments carrying the plaques were isolated and perfused ex vivo with rhodamine labeled leukocytes, showing a dramatically increased number of adherent leukocytes after mast cell activation (49 ± 6 versus 19 ± 4 leukocytes/microscopic field for DNP versus control plaques, respectively, **P<0.001). Strikingly, antibody blockade of either the CXCR2 or VCAM-1 receptor VLA-4 on labeled leukocytes completely inhibited leukocyte adhesion to the atherosclerotic plaque (*P<0.05), while blockade of CCR1, -3 and -5 with Met-RANTES had no effect. In conclusion, our data suggest that chemokines such as IL-8 released from activated perivascular mast cells induce leukocyte recruitment and adhesion to the atherosclerotic plaque, aggravating the ongoing inflammatory response and thus effecting plaque destabilization. We propose that mast cell stabilization could be a new therapeutic approach in the prevention of acute coronary syndromes.

Monocyte chemoattractant protein 1 contributes to an adequate immune response in influenza pneumonia

Monocyte chemoattractant protein 1 (MCP-1) and its receptor CCR2 have been shown to play an import role in leukocyte recruitment to sites of infection and inflammation. To investigate the role of MCP-1 during infection with influenza we inoculated wild-type (WT) and MCP-1 knockout (KO) mice with a non-lethal dose of a mouse adapted strain of influenza A. Influenza infection of WT mice resulted in a profound increase in pulmonary MCP-1 levels. MCP-1 KO mice had enhanced weight loss and did not fully regain their body weight during the 14-day observation period. In addition, MCP-1 KO mice demonstrated elevated viral loads 8 days after infection, which was accompanied by reduced leukocyte recruitment into the infected lungs, primarily caused by a diminished influx of macrophages and granulocytes. Moreover, pulmonary levels of IgA were reduced in MCP-1 KO mice. The pulmonary concentrations of tumor necrosis factor-α, interleukin-6, macrophage inflammatory protein 2 and interferon-γ were higher in MCP-1 KO mice. This study shows that MCP-1 contributes to an adequate protective immune response against influenza infection in mice.

Chronic Interleukin-1β Expression in Mouse Brain Leads to Leukocyte Infiltration and Neutrophil-Independent Blood–Brain Barrier Permeability without Overt Neurodegeneration

The proinflammatory cytokine interleukin-1beta (IL-1beta) plays a significant role in leukocyte recruitment to the CNS. Although acute effects of IL-1beta signaling in the mouse brain have been well described, studies elucidating the downstream effects of sustained upregulation have been lacking. Using the recently described IL-1beta(XAT) transgenic mouse model, we triggered sustained unilateral hippocampal overexpression of IL-1beta. Transgene induction led to blood-brain barrier leakage, induction of MCP-1 (monocyte chemoattractant protein 1) (CCL2), ICAM-1 (intercellular adhesion molecule 1), and dramatic infiltration of CD45-positive leukocytes comprised of neutrophils, T-cells, macrophages, and dendritic cells. Despite prolonged cellular infiltration of the hippocampus, there was no evidence of neuronal degeneration. Surprisingly, neutrophils were observed in the hippocampal parenchyma as late as 1 year after transgene induction. Their presence was coincident with upregulation of the potent neutrophil chemotactic chemokines KC (keratinocyte-derived chemokine) (CXCL1) and MIP-2 (macrophage inflammatory protein 2) (CXCL2). Knock-out of their sole receptor CXCR2 abrogated neutrophil infiltration but failed to reduce leakage of the blood-brain barrier.

Leukocyte PI3Kγ and PI3Kδ have temporally distinct roles for leukocyte recruitment in vivo

AbstractPhosphoinositide 3-kinases (PI3Ks) have been considered important in leukocyte motility. PI3Kγ, the class IB PI3K, expressed prominently in leukocytes and also in endothelial cells, mediates leukocyte functional responses induced by chemoattractants. To reveal its role in leukocyte recruitment, we used intravital microscopy to directly visualize leukocyte rolling, adhesion, and emigration in postcapillary venules in PI3Kγ-deficient (PI3Kγ-/-) mice. We report here that PI3Kγ deficiency had no significant effects on leukocyte rolling flux or rolling velocity and minor effects on adhesion (30% to 35%) in response to CXC chemokine MIP-2 (CXCL2) or KC (CXCL1). However, leukocyte emigration was severely impaired in PI3Kγ-/- mice in an early (first 90 minutes) response to MIP-2 or KC. Chimeric mice receiving bone marrow transplants revealed that this early response was entirely dependent upon PI3Kγ in neutrophils but not parenchymal cells (endothelium and others). Identical responses were observed when endogenous chemokine production was induced by TNFα; leukocyte emigration was reduced in PI3Kγ-/- mice. More prolonged responses to MIP-2 (for 4 to 5 hours) or TNFα (6 to 8 hours) were almost entirely PI3Kγ independent and largely dependent on PI3Kδ. Our results reveal that leukocyte emigration response to CXC chemokines is entirely dependent upon PI3Kγ or PI3Kδ, but these are nonoverlapping, temporally distinct events in inflamed tissues in vivo.