Beta2-integrin Mac-1 Research Articles

Leukocyte Beta2-Integrins; Genes and Disease

Integrins are heterodimeric transmembrane receptors that mediate cell-cell and cell-extracellular matrix interactions. Beta2-integrins are expressed exclusively in leukocytes and mediate many important functions in the immune system. Beta2-integrin genes are important in the pathologies of several diseases and genetic syndromes. These include Leukocyte Adhesion Deficiency (LAD) and Systemic Lupus Erythematosus (SLE), disorders which lie at opposite ends of the spectrum of immunological diseases. In LAD-I and LAD-III syndromes, beta2-integrin expression or function is reduced or absent. In SLE, genetic variants of the ITGAM gene, which encodes for the alpha M/CD11b chain of the beta2-integrin Mac-1,are associated with SLE development. In this mini review we summarise current knowledge regarding the involvement of beta2-integrins in LAD and SLE. Interestingly, dysfunctional beta2-integrins have been linked to both disorders, shedding light on the diverse roles of these receptors in the immune system.

Stroke‐associated inflammation: is von Willebrand factor a ‘bad guy’?

Stroke‐associated inflammation: is von Willebrand factor a ‘bad guy’?

Cysteine protease cathepsin X modulates immune response via activation of β2 integrins

Cathepsin X is a lysosomal, cysteine dependent carboxypeptidase. Its expression is restricted to cells of the immune system, suggesting a function related to the processes of inflammatory and immune responses. It has been shown to stimulate macrophage antigen-1 (Mac-1) receptor-dependent adhesion and phagocytosis via interaction with integrin beta2 subunit. Here its potential role in regulating lymphocyte proliferation via Mac-1 and the other beta2 integrin receptor, lymphocyte function-associated antigen-1 (LFA-1) has been investigated. Cathepsin X has been shown to suppress proliferation of human peripheral blood mononuclear cells, by activation of Mac-1, known as a suppressive factor for lymphocyte proliferation. On the other hand, co-localization of cathepsin X and LFA-1 supports the role of cathepsin X in regulating LFA-1 activity, which enhances lymphocyte proliferation. As shown by fluorescence resonance energy transfer, using U-937 and Jurkat cells transfected with alpha(L)-mCFP and beta2-mYFP, recombinant cathepsin X directly activates LFA-1. The activation was confirmed by increased binding of monoclonal antibody 24, recognizing active LFA-1. We demonstrate that cathepsin X is involved in the regulation of two beta2 integrin receptors, LFA-1 and Mac-1, which exhibit opposing roles in lymphocyte activation.

Two-dimensional kinetics of beta 2-integrin and ICAM-1 bindings between neutrophils and melanoma cells in a shear flow.

Cell adhesion, mediated by specific receptor-ligand interactions, plays an important role in biological processes such as tumor metastasis and inflammatory cascade. For example, interactions between beta 2-integrin (lymphocyte function-associated antigen-1 and/or Mac-1) on polymorphonuclear neutrophils (PMNs) and ICAM-1 on melanoma cells initiate the bindings of melanoma cells to PMNs within the tumor microenvironment in blood flow, which in turn activate PMN-melanoma cell aggregation in a near-wall region of the vascular endothelium, therefore enhancing subsequent extravasation of melanoma cells in the microcirculations. Kinetics of integrin-ligand bindings in a shear flow is the determinant of such a process, which has not been well understood. In the present study, interactions of PMNs with WM9 melanoma cells were investigated to quantify the kinetics of beta 2-integrin and ICAM-1 bindings using a cone-plate viscometer that generates a linear shear flow combined with a two-color flow cytometry technique. Aggregation fractions exhibited a transition phase where it first increased before 60 s and then decreased with shear durations. Melanoma-PMN aggregation was also found to be inversely correlated with the shear rate. A previously developed probabilistic model was modified to predict the time dependence of aggregation fractions at different shear rates and medium viscosities. Kinetic parameters of beta 2-integrin and ICAM-1 bindings were obtained by individual or global fittings, which were comparable to respectively published values. These findings provide new quantitative understanding of the biophysical basis of leukocyte-tumor cell interactions mediated by specific receptor-ligand interactions under shear flow conditions.

Proteomic analysis of plasma membrane lipid rafts of HL‐60 cells

Neutrophils acquire phagocytic activity as they differentiate. Recently, plasma membrane lipid rafts have been shown to play important roles in the process of phagocytosis in neutrophils. To characterize the proteins involved in phagocytosis and to elucidate the process by which they acquire phagocytic activity, we investigated by nano-LC-MS/MS analysis the changes in protein composition of plasma membrane lipid rafts during DMSO-induced differentiation of the human leukemia cell line HL-60 cells into neutrophilic lineage. Based on the spectrum counts of 147 proteins identified, 25 proteins were upregulated and 49 were downregulated by DMSO treatment. CD11b/CD18 subunits of beta2-integrin Mac-1, CD35, and GPI-80, which are known to be upregulated during differentiation, were dominantly detected in the lipid rafts of DMSO-treated cells. Many known membrane proteins, G proteins, and cytoskeletal proteins were also detected and they showed characteristic distributions. Absolute quantification of nine proteins in the lipid rafts using internal standard peptides labeled with stable isotopes showed that the amount of protein almost corresponded to the results obtained by spectrum count. Identified proteins, expression of which was altered by DMSO treatment, are expected to be candidate proteins involved in differentiation and functions of neutrophils.

Soluble receptor for advanced glycation end products triggers a proinflammatory cytokine cascade via β2 integrin Mac‐1

Receptor for advanced glycation end products (RAGE) is a cell surface molecule that binds a variety of ligands, including high mobility group box chromosomal protein 1 (HMGB-1), a potent proinflammatory cytokine. RAGE-ligand interaction leads to an inflammatory response. A truncated form of the receptor, soluble RAGE (sRAGE), has been suggested to function as a decoy abrogating cellular activation, but its endogenous activity is not fully understood. We undertook this study to assess the properties of sRAGE in vivo and in vitro and to analyze the role of sRAGE in HMGB-1-induced arthritis. Mice were injected intraarticularly with HMGB-1 and treated systemically with sRAGE prior to histologic joint evaluation. All animals were subjected to peritoneal lavage to assess the local effect of sRAGE treatment. For in vitro studies, mouse splenocytes were incubated with sRAGE followed by assessment of NF-kappaB activation and cytokine production. The chemotactic properties of sRAGE were investigated using in vitro migration assay. Soluble RAGE was determined to have proinflammatory properties since it gave rise to production of interleukin-6, tumor necrosis factor alpha, and macrophage inflammatory protein 2. This effect was triggered by interaction with leukocyte beta2 integrin Mac-1 and was mediated via NF-kappaB. Systemic treatment with sRAGE significantly down-regulated HMGB-1-triggered arthritis, but the observed effect was due to a deviation of the inflammatory response from the joint to the peritoneal cavity rather than a genuine antiinflammatory effect. Apart from its proinflammatory properties, sRAGE was proven to act as a chemotactic stimulus for neutrophils. We conclude that sRAGE interacts with Mac-1, thereby acting as an important proinflammatory and chemotactic molecule.

Expression of MAC‐1 (CD11b) in acute myeloid leukemia (AML) is associated with an unfavorable prognosis

There is evidence to suggest, that cellular adhesion molecules and receptors could play a role in leukemia, e.g., through altered adhesive qualities of leukemic blasts. We have studied the expression of the beta2-integrin Mac-1 (CD11b) on mononuclear cells in 48 patients with AML at first diagnosis by flow cytometry using a direct fluorescein-conjugated antibody. A case was defined as positive if more than 20% of the cells expressed Mac-1. Within the FAB types, we observed a high expression rate in cases with M5 (100% MAC-1+ cases, 73% MAC-1+ cells), M4 (75% MAC-1+ cases, 48% MAC-1+ cells) and in cases with FAB-M1 with 71% MAC-1+ cases and 29% MAC-1+ cells. Separating our patients' cohort in cytogenetic risk groups, we could detect significant higher proportions of MAC-1+, cases (88% vs. 27%, P = 0.005) and cells (51% vs. 16%, P = 0.015) with poor cytogenetic risk compared to the favorable risk group. For clinical evaluations only patients treated according to the protocols of the German AML Cooperative Group (AML-CG) were included (n = 29, cases with AML-M3 were excluded). More MAC-1+ cases and cells were found in the "non-responders" group (n = 8) compared to the "responders" group (n = 24). We can conclude that AML cases with high MAC-1 expression are characterized by a worse prognosis. Evaluation of MAC-1 expression in AML might therefore contribute clinically important data with respect to develop new therapies that influence the interactions between integrins like MAC-1 on leukemic cells and endothelial or immunoreactive cells.

Induction of LFA‐1‐Dependent Neutrophil Rolling on ICAM‐1 by Engagement of E‐Selectin

To study rolling of mouse neutrophils on E-selectin and ICAM-1 in an ex vivo flow chamber system. The authors developed a small autoperfused flow chamber (20 x 200-microm cross section) that allows direct visualization of cells with and without fluorescent labeling and does not require recirculation of blood. Neutrophils rolled on E-selectin alone, but were unable to interact with immobilized ICAM-1. When ICAM-1 was co-immobilized with E-selectin, the number of cells that rolled was doubled, but no significant firm adhesion was observed. This phenomenon was specific for E-selectin, and no enhancement of rolling was observed when P-selectin was immobilized with ICAM-1. The increased neutrophil rolling seen on E-selectin and ICAM-1 substrates required beta2 integrins. Treating mice with antibodies to the beta2 integrins LFA-1 and Mac-1 showed that LFA-1 was primarily responsible for mediating rolling on ICAM-1 in this model. Increased rolling on E-selectin and ICAM-1 was significantly reduced following administration of a specific p38 mitogen-activated protein kinase (MAPK) inhibitor. The data show that neutrophil rolling on E-selectin leads to partial activation of LFA-1, enabling LFA-1-dependent rolling on ICAM-1. This mechanism is likely to amplify and accelerate neutrophil recruitment in inflammation.

Increased Neutrophil Adherence in Psoriasis: Role of the Human Endothelial Cell Receptor Thy-1 (CD90)

The chronic inflammatory skin disease psoriasis is characterized by prominent skin infiltration by neutrophils and microabscess formation. The adhesion of leukocytes and subsequent transmigration through the activated endothelium is one prerequisite for the accumulation of these cells in skin. In recent studies, the human Thy-1 (CD90) was characterized as an adhesion molecule on activated endothelial cells (ECs) mediating the adhesion of neutrophils via the interaction with the beta2-integrin Mac-1. Based on these novel findings, we compared the roles of Thy-1 and ICAM-1 in the adhesion of neutrophils from patients with psoriasis to activated ECs. The adhesion of peripheral blood neutrophils of patients suffering from psoriasis to Thy-1-transfected cells as well as to activated, Thy-1-expressing human dermal microvascular ECs (HDMECs) is distinctly increased in comparison to the adhesion of neutrophils from healthy controls. In contrast, adherence of psoriatic neutrophils to ICAM-1 transfectants is, if at all, only slightly enhanced compared to healthy controls. The interaction of healthy as well as psoriatic polymorphonuclear cells to Thy-1 transfectants and HDMECs was significantly inhibited by blocking Thy-1 on ECs or its receptor Mac-1 on neutrophils, indicating the importance of this interaction for the adhesion of neutrophils to activated endothelium. In conclusion, our data indicate that the adhesion of neutrophils to activated ECs mediated by Thy-1/Mac-1 interaction is an important attachment mechanism facilitating their subsequent migration into lesional psoriatic skin.

Lipoprotein(a) in atherosclerotic plaques recruits inflammatory cells through interaction with Mac‐1 integrin

Lipoprotein(a) [Lp(a)], consisting of LDL and the unique constituent apolipoprotein(a) [apo(a)], which contains multiple repeats resembling plasminogen kringle 4, is considered a risk factor for the development of atherosclerotic disorders. However, the underlying mechanisms for the atherogenicity of Lp(a) are not completely understood. Here, we define a novel function of Lp(a) in promoting inflammatory cell recruitment that may contribute to its atherogenicity. Through its apo(a) moiety Lp(a) specifically interacts with the beta2-integrin Mac-1, thereby promoting the adhesion of monocytes and their transendothelial migration in a Mac-1-dependent manner. Interestingly, the interaction between Mac-1 and Lp(a) was strengthened in the presence of proatherogenic homocysteine and was blocked by plasminogen/angiostatin kringle 4. Through its interaction with Mac-1, Lp(a) induced activation of the proinflammatory transcription factor NFkappaB, as well as the NFkappaB-related expression of prothrombotic tissue factor. In atherosclerotic coronary arteries Lp(a) was found to be localized in close proximity to Mac-1 on infiltrating mononuclear cells. Taken together, our data demonstrate that Lp(a), via its apo(a) moiety, is a ligand for the beta2-integrin Mac-1, thereby facilitating inflammatory cell recruitment to atherosclerotic plaques. These observations suggest a novel mechanism for the atherogenic properties of Lp(a).

The role of junctional adhesion molecule‐C (JAM‐C) in oxidized LDL‐mediated leukocyte recruitment

The junctional adhesion molecule-C (JAM-C) was recently shown to be a counter receptor for the leukocyte beta2-integrin Mac-1 (CD11b/CD18), thereby mediating interactions between vascular cells, particularly in inflammatory cell recruitment. Here, we investigated the role of JAM-C in oxidized low-density lipoprotein (LDL)-mediated leukocyte recruitment. As compared with normal arteries, immunostaining of atherosclerotic vessels revealed a high expression of JAM-C in association with neointimal smooth muscle cells and the endothelium. Moreover, JAM-C was strongly up-regulated in the spontaneous early lesions in ApoE -/- mice. In vitro, cultured human arterial smooth muscle cells (HASMC) were found to express JAM-C, and oxLDL, as well as enzymatically modified LDL (eLDL) significantly up-regulated JAM-C on both HASMC and endothelial cells in a time- and dose-dependent manner. Although under quiescent conditions, JAM-C predominantly localized to interendothelial cell-cell contacts in close proximity to zonula occludens-1 (ZO-1), oxLDL treatment induced a disorganization of JAM-C localization that was no more restricted to the interendothelial junctions. JAM-C thereby mediated both leukocyte adhesion and leukocyte transendothelial migration upon oxLDL treatment of endothelial cells, whereas JAM-C on quiescent endothelial cells only mediates leukocyte transmigration. Thus, upon oxLDL stimulation endothelial JAM-C functions as both an adhesion, as well as a transmigration receptor for leukocytes. Taken together, JAM-C is up-regulated by oxLDL and may thereby contribute to increased inflammatory cell recruitment during atherosclerosis. JAM-C may therefore provide a novel molecular target for antagonizing interactions between vascular cells in atherosclerosis.

The CD11/CD18 (beta2) integrins modulate neutrophil caspase activation and survival following TNF-alpha or endotoxin induced transendothelial migration.

Neutrophils (PMN) are short-lived cells but their survival is often prolonged in inflammation. The beta2 (CD11/CD18) integrins are involved in PMN migration into inflammation but their role in PMN survival is not well understood. We investigated the role of beta2 integrins in PMN caspase activation, a key enzyme cascade in apoptosis. After 20 h, caspase activation (Western blotting) was markedly decreased in PMN cultured on fibrinogen, a ligand for Mac-1 (CD11b/CD18), but not on fibronectin or albumin. In the presence of TNF-alpha or endotoxin (LPS), blockade of CD18 (beta2 chain) with mAb markedly increased caspase activation in PMN on fibrinogen. PMN which migrated through endothelium in vitro in response to TNF-alpha, LPS, IL-1alpha, IL-8 or C5a contained 58% fewer active caspase positive PMN after 20 h than non-migrated PMN remaining on the endothelium. When beta2 (CD18) integrin or lymphocyte function antigen (LFA)-1 (CD11a) plus Mac1 (CD11b) were blocked by mAb (intact or Fab'), the proportion of migrated PMN (but not of non-migrated PMN) with active caspases was significantly increased (2-4-fold) and this was associated with accelerated PMN apoptosis and death. Thus, engagement of ligands on extracellular matrix and endothelium by the beta2 integrins Mac-1 and LFA-1 plays a role in delaying apoptosis in PMN recruited in response to LPS and TNF-alpha. Inhibition of beta2 integrin function may not only inhibit PMN infiltration, but also accelerate PMN clearance from inflamed tissue.

SRC-dependent outside-in signalling is a key step in the process of autoregulation of beta2 integrins in polymorphonuclear cells.

In human PMN (polymorphonuclear cells), challenged by P-selectin, the beta2-integrin Mac-1 (macrophage antigen-1) promoted the activation of the SRC (cellular homologue of Rous sarcoma virus oncogenic protein) family members HCK (haematopoietic cell kinase) and LYN (an SRC family protein tyrosine kinase) and phosphorylation of a P-110 (110 kDa protein). SRC kinase activity in turn was necessary for macrophage antigen-1-mediated adhesion [Piccardoni, Sideri, Manarini, Piccoli, Martelli, de Gaetano, Cerletti and Evangelista (2001) Blood 98, 108-116]. This suggested that an SRC-dependent outside-in signalling strengthens the beta2-integrin interaction with the ligand. To support this hypothesis further, in the present study, we used the monoclonal antibody KIM127 or manganese to lock beta2 integrins in a high-affinity state, and homotypic PMN adhesion was analysed to monitor beta2-integrin adhesive function. KIM127 or manganese induced PMN homotypic adhesion and P-110 phosphorylation. Both these processes were abolished by blocking antibodies against the common beta2 chain, by a combination of antibodies against alphaL and alphaM or by inhibitors of SRC activity. Confocal microscopy showed that activation epitopes were expressed by beta2 integrins co-localized with patches of F-actin at the adhesion sites. Blockade of SRC kinases or of actin polymerization prevented clustering of activated integrins as well as F-actin accumulation. FACS analysis showed that SRC inhibitors modified neither basal nor manganese-induced KIM127 binding. An SRC-dependent outside-in signalling initiated by beta2 integrins was also required for adhesion triggered by interleukin-8. These results confirm the hypothesis that an SRC-dependent outside-in signalling triggered by high affinity and ligand binding is necessary to stabilize beta2-integrin-mediated adhesion. Allowing clustering of activated integrins, SRC might link the high-affinity with the high-avidity state. Proline-rich tyrosine kinase-2 appears to be involved in this process.

Fcγ-receptors Induce Mac-1 (CD11b/CD18) Mobilization and Accumulation in the Phagocytic Cup for Optimal Phagocytosis

Functional interactions between Fcgamma-receptors (FcgammaR) and the beta2 integrin Mac-1 (CD11b/CD18) have been described, but the molecular basis of this relationship remains unclear. Although the glycosylphosphatidylinositol-linked receptor FcgammaRIIIB of human neutrophils is constitutively associated with Mac-1, we found no evidence for direct physical association between Mac-1 and the FcgammaR of mouse macrophages, which are transmembrane proteins. Nevertheless, Mac-1 accumulated in the phagocytic cup following engagement of FcgammaR by IgG-opsonized particles. Blocking the CD18 chains of beta2 integrins by using specific antibodies reduced Mac-1 accumulation in the cup. These antibodies or the addition of the recombinant CD11b I-domain inhibited the ingestion of IgG-opsonized particles. FcgammaR cross-linking stimulated cell adhesion to surfaces coated with Mac-1 ligands and in addition enabled macrophages to bind C3bi-opsonized particles, indicating that FcgammaR-derived signals induce activation of Mac-1. Measurements of fluorescence recovery after photobleaching revealed that whereas most (>80%) of Mac-1 is immobile in resting cells, stimulation of FcgammaR markedly increases the mobile fraction of the integrin. Activation of Mac-1 by FcgammaR required the activity of Src family tyrosine kinases, phosphatidylinositol 3-kinase and phospholipase C, with the release of diacylglycerol and stimulation of protein kinase C. Because elevated cytosolic Ca2+ was not required, we suggest that novel protein kinase C isoforms are involved in Mac-1 activation. These results suggest that FcgammaR stimulation promotes Mac-1 clustering into high avidity complexes in phagocytic cups by releasing the integrin from cytoskeletal constraints and enhancing its lateral diffusion. FcgammaR can enhance host defense by activating Mac-1 (and possibly other integrins), having a synergistic effect on pathogen engulfment and promoting the adherence of phagocytes at sites of infection.

High Molecular Weight Kininogen Regulates Platelet-Leukocyte Interactions by Bridging Mac-1 and Glycoprotein Ib

Leukocyte-platelet interaction is important in mediating leukocyte adhesion to a thrombus and leukocyte recruitment to a site of vascular injury. This interaction is mediated at least in part by the beta2-integrin Mac-1 (CD11b/CD18) and its counter-receptor on platelets, glycoprotein Ibalpha (GPIbalpha). High molecular weight kininogen (HK) was previously shown to interact with both GPIbalpha and Mac-1 through its domains 3 and 5, respectively. In this study we investigated the ability of HK to interfere with the leukocyte-platelet interaction. In a purified system, HK binding to GPIbalpha was inhibited by HK domain 3 and the monoclonal antibody (mAb) SZ2, directed against the epitope 269-282 of GPIbalpha, whereas mAb AP1, directed to the region 201-268 of GPIbalpha had no effect. In contrast, mAb AP1 inhibited the Mac-1-GPIbalpha interaction. Binding of GPIbalpha to Mac-1 was enhanced 2-fold by HK. This effect of HK was abrogated in the presence of HK domains 3 or 5 or peptides from the 475-497 region of the carboxyl terminus of domain 5 as well as in the presence of mAb SZ2 but not mAb AP1. Whereas no difference in the affinity of the Mac-1-GPIbalpha interaction was observed in the absence or presence of HK, maximal binding of GPIbalpha to Mac-1 doubled in the presence of HK. Moreover, HK/HKa increased the Mac-1-dependent adhesion of myelomonocytic U937 cells and K562 cells transfected with Mac-1 to immobilized GPIbalpha or to GPIbalpha-transfected Chinese hamster ovary cells. Finally, Mac-1-dependent adhesion of neutrophils to surface-adherent platelets was enhanced by HK. Thus, HK can bridge leukocytes with platelets by interacting via its domain 3 with GPIbalpha and via its domain 5 with Mac-1 thereby augmenting the Mac-1-GPIbalpha interaction. These distinct molecular interactions of HK with leukocytes and platelets contribute to the regulation of the adhesive behavior of vascular cells and provide novel molecular targets for reducing atherothrombotic pathologies.

Downregulation of mac-1 expression in monocytes by surface-bound IgG.

Physical and functional association between the beta2-integrin Mac-1 (CD11b/CD18) and receptors of immunoglobulin G (IgG) (FcgammaRs) has been previously reported. In this study, we examined the modulation of Mac-1 expression by IgG in different leucocyte populations. Our data show that human monocytes, but not neutrophils, macrophages, dendritic or natural killer cells, downregulate the expression of Mac-1 after overnight exposure to surface-bound IgG. This effect, which requires at least 6 h of incubation, is not associated with a general downmodulation of membrane antigens, and is selectively induced by immobilized IgG (iIgG), as the stimulation of monocytes with N-formyl-methionyl-leucyl-phenylalanine, lipopolysaccharide, tumour necrosis factor-alpha (TNF-alpha) or soluble IgG did not modify the Mac-1 expression after 18 h in culture. The loss of Mac-1 was completely prevented by blocking antibodies (Abs) directed to FcgammaRII (CD32) or CD18. On the other hand, the serine protease inhibitor, phenyl methyl sulphonyl fluoride, but not inhibitors of cysteine proteases or neutral endopeptidases, partially prevented the downregulation of Mac-1 by iIgG. Monocytes cultured overnight on iIgG exhibited a dramatic decrease in their capacity to ingest zymosan particles that could be attributed to the reduced expression of Mac-1. However, there was no inhibition of TNF-alpha production induced by zymosan, suggesting that Mac-1-dependent responses require different levels of its expression to be fully activated.

Regulation of leukocyte recruitment by polypeptides derived from high molecular weight kininogen.

Proteolytic cleavage of single-chain, high molecular weight kininogen (HK) by kallikrein releases the short-lived vasodilator bradykinin and leaves behind a two-chain, high molecular weight kininogen (HKa) reported to bind to the beta2-integrin Mac-1 (CR3, CD11b/CD18, alphaMbeta2) on neutrophils and exert antiadhesive properties by binding to the urokinase receptor (uPAR) and vitronectin. We define the molecular mechanisms for the antiadhesive effects of HK related to disruption of beta2-integrin-mediated cellular interactions in vitro and in vivo. In a purified system, HK and HKa inhibited the binding of soluble fibrinogen and ICAM-1 to immobilized Mac-1, but not the binding of ICAM-1 to immobilized LFA-1 (CD11a/CD18, alphaLbeta2). This inhibitory effect could be attributed to HK domain 5 and to a lesser degree to HK domain 3, consistent with the requirement of both domains for binding to Mac-1. Accordingly, HK, HKa, and domain 5 inhibited the adhesion of Mac-1 but not LFA-1-transfected K562 human erythroleukemic cells to ICAM-1. Moreover, adhesion of human monocytic cells to fibrinogen and to human endothelial cells was blocked by HK, HKa, and domain 5. By using peptides derived from HK domain 5, the sequences including amino acids H475-G497 (and to a lesser extent, G440-H455) were identified as responsible for the antiadhesive effect, which was independent of uPAR. Finally, administration of domain 5 into mice, followed by induction of thioglycollate-provoked peritonitis, decreased the recruitment of neutrophils by approximately 70% in this model of acute inflammation. Taken together, HKa (and particularly domain 5) specifically interacts with Mac-1 but not with LFA-1, thereby blocking Mac-1-dependent leukocyte adhesion to fibrinogen and endothelial cells in vitro and in vivo and serving as a novel endogenous regulator of leukocyte recruitment into the inflamed tissue.

Evidence for the involvement of phosphatidylinositol 3-kinase in fMLP-stimulated neutrophil adhesion to ICAM-1-transfected cells.

Phosphatidylinositol 3-kinase (PI-3K) controls important intracellular steps involved in inflammation, immunity, and cell growth. PI-3K also modulates leukocyte integrin adhesiveness. In this study we evaluated the role of PI-3K on neutrophil adhesion to intercellular adhesion molecule-1 (ICAM-1)-transfected cells. N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated neutrophil adhesion was inhibited by wortmannin and LY294002, two unrelated PI-3K inhibitors, whereas phorbol myristate acetate (PMA)-induced neutrophil adhesion was not inhibited by them. After fMLP stimulation, a rapid activation of AKT and ERK was observed. However, only activation of AKT was reversed by the PI-3K inhibitors. Neutrophil expression of the beta2-integrins Mac-1, lymphocyte function-associated antigen-1(LFA-1), and gp150.95 was not affected by wortmannin, nor was expression of the activation epitope recognized by MAB24. We conclude that (a) PI-3K is involved in fMLP-activated neutrophil adhesion to ICAM-1-transfected cells, (b) the mechanism involved is not mediated by the modulation of beta2-integrin expression or activation, and (c) another mechanism seems to involve the adhesion to ICAM-1 when a cellular system of adhesion is used.

Tumor cell-derived prostaglandin E2 inhibits monocyte function by interfering with CCR5 and Mac-1.

The cyclooxygenases (COX)-1 and COX-2 are key enzymes in the conversion of arachidonic acid to prostaglandins and other eicosanoids. Whereas COX-1 is expressed ubiquitously, COX-2 is an immediate-early gene often associated with malignant transformation, and a role for the COX enzymes in tumor initiation and promotion is discussed. Nonsteroidal anti-inflammatory drugs (NSAIDs) like aspirin and indomethacin that block COX-1 and -2 have been shown to have beneficial effects for tumor patients. Therefore, these compounds have gained interest also among oncologists. However, the molecular mechanism by which NSAIDs inhibit carcinogenesis is not clearly understood. The prostaglandin-dependent and -independent effect may both account for their antineoplastic action. We show here that tumor cells derived from different tumors regularly produce prostaglandin E(2) (PGE(2)) interfering with the function of monocytes. In particular, PGE(2) inhibits the potential of monocytes to migrate in the direction of a chemotactic stimulus and to adhere to endothelial cell. This inhibition is most probably due to a modulation of the chemokine receptor CCR5 and the beta2-integrin Mac-1. Both down-regulation of CCR5 and reduced expression of Mac-1 may diminish the potential of peripheral blood monocytes to leave blood vessels and invade target tissues. Since both dysfunctions can be restored with NSAIDs, our findings help to explain the molecular chemopreventive action of NSAIDs on tumor formation and progression.

Decreased neointimal formation in Mac-1(-/-) mice reveals a role for inflammation in vascular repair after angioplasty.

Inflammation plays an essential role in the initiation and progression of atherosclerosis, but its role in vascular repair after mechanical arterial injury (i.e., percutaneous transluminal coronary angioplasty, PTCA) is unknown. In animal models of vascular injury, leukocytes are recruited as a precursor to intimal thickening. Furthermore, markers of leukocyte activation - in particular, increased expression of the beta2-integrin Mac-1 (alphaMbeta2, or CD11b/CD18), which is responsible for firm leukocyte adhesion to platelets and fibrinogen on denuded vessels - predict restenosis after PTCA. To determine whether Mac-1-mediated leukocyte recruitment is causally related to neointimal formation, we subjected mice lacking Mac-1 to a novel form of mechanical carotid artery dilation and complete endothelial denudation. We now report that the selective absence of Mac-1 impairs transplatelet leukocyte migration into the vessel wall, reducing leukocyte accumulation over time. Diminished medial leukocyte accumulation was accompanied by markedly reduced neointimal thickening after vascular injury. These data establish a role for inflammation in neointimal thickening and suggest that leukocyte recruitment to mechanically injured arteries may prevent restenosis.