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Complement has recently been implicated in developmental pathways and noninflammatory processes. The expression of various complement components and receptors has been shown in a wide range of circulating myeloid and lymphoid cells, but their role in normal hematopoiesis and stem cell homing has not yet been investigated. We report that normal human CD34(+) cells and lineage-differentiated hematopoietic progenitors express the complement anaphylatoxin C3a receptor (C3aR) and respond to C3a. Moreover, C3a, but not the biologically inactive desArg-C3a, induces calcium flux in these cells. Furthermore, we found that C3 is secreted by bone marrow stroma and that, although C3a does not influence directly the proliferation/survival of hematopoietic progenitors, it (1) potentiates the stromal cell-derived factor 1 (SDF-1)-dependent chemotaxis of human CD34(+) cells and lineage-committed myeloid, erythroid, and megakaryocytic progenitors; (2) primes SDF-1-dependent trans-Matrigel migration; and (3) stimulates matrix metalloproteinase-9 secretion and very late antigen 4 (VLA-4)-mediated adhesion to vascular cell adhesion molecule 1 (VCAM-1). Furthermore, we found that murine Sca-1(+) cells primed by C3a engrafted faster in lethally irradiated animals. These results indicate that normal human hematopoietic stem and progenitor cells express functional C3aR and that the C3aR-C3a axis sensitizes the responses of these cells to SDF-1 and thus may be involved in promoting their homing into the bone marrow via cross talk with the SDF-CXC chemokine receptor-4 (CXCR4) signaling axis. C3a is the first positive regulator of this axis to be identified.

The alpha-I domain, found in the alpha-subunit of the leucocyte integrins such as alphaMbeta2 and alphaLbeta2, switches between the open and closed tertiary conformations, reflecting the high- and low-affinity ligand-binding states of the integrin that are required for regulated cell adhesion and migration. In the present study we show, by using point mutations and engineered disulphide bonds, that ligand affinity can be reduced or increased allosterically by altering the equilibrium between the closed and open states. We determined equilibrium constants for the binding of two ligands, fibrinogen and intercellular cell-adhesion molecule 1, to the alphaM-I domain by surface plasmon resonance, and determined crystal structures of a low-affinity mutant. Locking the domain in the open conformation increases affinity by a factor of no greater than 10, consistent with a closely balanced equilibrium between the two conformations in the absence of ligand. This behaviour contrasts with that of the unliganded alphaL-I domain, for which the equilibrium lies strongly in favour of the closed conformation. These results suggest significant differences in the way the parent integrins regulate I domain conformation and hence ligand affinity.

In a transgenic model of multi-stage squamous carcinogenesis induced by human papillomavirus (HPV) oncogenes, infiltrating CD4+ T cells can be detected in both premalignant and malignant lesions. The lymph nodes that drain sites of epidermal neoplasia contain activated CD4+ T cells predominantly reactive toward Staphylococcal bacterial antigens. HPV16 mice deficient in CD4+ T cells were found to have delayed neoplastic progression and a lower incidence of tumors. This delay in carcinogenesis is marked by decreased infiltration of neutrophils, and reduced activity of matrix metalloproteinase-9, an important cofactor for tumor progression in this model. The data reveal an unexpected capability of CD4 T cells, whereby, proinflammatory CD4+ T cells, apparently responding to bacterial infection of dysplastic skin lesions, can inadvertently enhance neoplastic progression to invasive cancer.

While the specificity and development of natural killer (NK) cells have been intensely studied, little is known about homeostasis of the mature NK population. Here we show that mouse NK cells undergo homeostatic proliferation when transferred into NK-deficient Rag-/- gammaC-/- hosts. Normal NK functional activity is maintained during this process, although there are some changes in NK phenotype. Using cell sorting, we demonstrate that mature (Mac-1hi) NK cells undergo homeostatic proliferation in an NK-deficient environment, yet immature (Mac-1lo) NK cells also proliferate in such hosts. We find that mature NK cells survive but do not proliferate in hosts which possess an endogenous NK pool. However, we go on to show that mature NK survival is critically dependent on interleukin (IL)-15. Surprisingly, NK survival is also compromised after transfer of cells into IL-15Ralpha-/- mice, implying that IL-15 responsiveness by bystander cells is critical for NK maintenance. These data imply that, similar to T cells, homeostasis of the NK pool is much more dynamic than previously appreciated and this may be relevant to manipulation of NK cells for therapeutic purposes.

The trichothecenes are a group of mycotoxins that target leukocytes and have a wide range of immunomodulatory effects. Differential display analysis was applied to assess the effects of the trichothecenes deoxynivalenol (vomitoxin, DON) and satratoxin G (SG), on mRNA in the RAW 264.7 macrophage cell line. Cells were incubated with DON (1 microg/ml) or SG (5 ng/ml) for 2 h and total RNA then subjected to RT-PCR with a set of oligo(dT) primers. Resultant cDNA was amplified using an oligo (dT) downstream primer and an arbitrary decanucleotide upstream primer to make 35S-labeled PCR products. After separation of the products in denaturing polyacrylamide gel, 23 differentially expressed cDNA fragments were isolated and sequenced. Two of these were identified as known genes, namely, macrophage inflammatory protein-2 (MIP-2), a potent neutrophil chemoattractant involved in tissue injury and inflammation, and complement 3a receptor (C3aR), a proinflammatory mediator. Both MIP-2 and C3aR mRNAs were up-regulated by DON while only MIP-2 mRNA was induced by SG. Using commercially available antibodies, MIP-2 protein was also found to be induced by both DON and SG in RAW 264.7 cell cultures. When mice were treated with DON (12.5 mg/kg), splenic MIP-2 mRNA and serum MIP-2 levels were increased. MIP-2 mRNA and serum MIP-2 levels were synergistically increased when mice were co-treated with DON and LPS. Up-regulation of MIP-2 and C3aR are consistent with previous reports of trichothecene-induced inflammatory gene up-regulation and suggest that the specific genes affected may depend on trichothecene structures.

Previous studies demonstrated that integrin alpha(M)beta(2) (CD11b/18, Mac-1) forms a physical complex with the urokinase-type plasminogen activator receptor (uPAR/CD87) on leukocytes. In this study, we used human peripheral blood neutrophils and transfected cells expressing alpha(M)beta(2), uPAR, or both receptors to show that the integrin can directly interact with urokinase (uPA). We demonstrate that alpha(M)beta(2) supported adhesion and migration of these cells to uPA, and, in each case, blockade of alpha(M)beta(2) suppressed the response. Within uPA, both the kringle and proteolytic domains are recognized by alpha(M)beta(2), which are distinct from the growth factor domain that binds to uPAR. Within the alpha(M) subunit of the integrin, the I domain interacts with uPA, which is distinct from the region that interacts with uPAR. On cells expressing uPAR and alpha(M)beta(2), both receptors mediated adhesion and migration. This cooperation was particularly apparent in the responses of neutrophils to uPA, where blockade of alpha(M)beta(2) reduced uPAR-mediated responses and engagement of uPAR enhanced recognition of uPA by alpha(M)beta(2). Thus, recognition of uPA by alpha(M)beta(2) allows for formation of a multicontact trimolecular complex, in which a single uPA ligand may bind simultaneously to both uPAR and alpha(M)beta(2). This complex may play an important role in the control of inflammatory cell migration and vascular homeostasis.

A directed migration of leukocytes through the extracellular matrix requires the regulated engagement of integrin cell adhesion receptors. The integrin alpha(M)beta(2) (CD11b/CD18, Mac-1) is progressively upregulated to high levels on migrating phagocytic leukocytes in response to inflammatory stimuli and is able to bind numerous ligands in the interstitial matrix. The role of alpha(M)beta(2) in migration of leukocytes through the extracellular matrix and its cooperation with other leukocyte integrins during migration are not understood. Using a model system consisting of cells that express different levels of alpha(M)beta(2) and an invariable level of endogenous integrin alpha(5)beta(1), we have explored a situation relevant to migrating neutrophils when alpha(M)beta(2) and alpha(5)beta(1) engage the same ligand, fibronectin. We show that fibronectin is a ligand for alpha(M)beta(2) and that both alpha(M)beta(2) and alpha(5)beta(1) on the alpha(M)beta(2)-expressing cells contribute to adhesion to fibronectin. However, migration of these cells to fibronectin is mediated by alpha(5)beta(1), whereas alpha(M)beta(2) retards migration. The decrease in migration correlates directly with the increased alpha(M)beta(2) density. Ligation of alpha(M)beta(2) with function-blocking antibodies can reverse this effect. The restorative effects of antibodies are caused by the removal of restraint imposed by the excess of alpha(M)beta(2)-fibronectin adhesive bonds. These findings indicate that alpha(M)beta(2) can increase general cell adhesiveness which results in braking of cell migration mediated by integrin alpha(5)beta(1). Because alpha(M)beta(2) binds numerous proteins in the extracellular matrix with a specificity overlapping that of the beta(1) integrins, the results suggest that alpha(M)beta(2) can affect the beta(1) integrin-mediated cell migration., (Copyright 2003 Elsevier Science (USA))

Antibody-reliant destruction of tumor cells by immune effector cells is mediated by antibody-dependent cellular cytotoxicity, in which Fc receptor (FcR) engagement is crucial. This study documents an important role for the beta(2) integrin Mac-1 (CD11b/CD18) in FcR-mediated protection against melanoma. CD11b-deficient mice, those that lack Mac-1, were less protected by melanoma-specific monoclonal antibody TA99 than wild-type (WT) mice. Significantly more lung metastases and higher tumor loads were observed in Mac-1(-/-) mice. Histologic analyses revealed no differences in neutrophil infiltration of lung tumors between Mac-1(-/-) and WT mice. Importantly, Mac-1(-/-) phagocytes retained the capacity to bind tumor cells, implying that Mac-1 is essential during actual FcR-mediated cytotoxicity. In summary, this study documents Mac-1 to be required for FcR-mediated antimelanoma immunity in vivo and, furthermore, supports a role for neutrophils in melanoma rejection.

The majority of B lymphocytes, virgin T lymphocytes and a subpopulation of memory T cells express the addressin, L-selectin. Natural killer (NK) cells in rodents and humans also express L-selectin. We have shown that a similar proportion (40%) of NK cells in mouse spleen also express the integrin, CD18Mac-1, and moreover, that NK cells express both the addressin and the integrin constitutively. It was the aim of the present study to quantify, in knock-out mice deficient for either the L-selectin addressin, or the CD18:Mac-1/LFA-1 integrins, NK cells and B cells in both the spleen and their bone marrow birth site. These cells, in both organs, were immunophenotypically stained with FITC-conjugated anti-NK1.1 (to identify NK cells), and FITC-conjugated anti-mouse B220 (to identify B lymphocytes) and subjected to flow-cytometric analysis using a FACScan equipped with a doublet discrimination module. From the known total organ (spleen, femurs) cellularity, obtained by means of an electronic cell counter, at the time of extraction of each organ, the absolute numbers of NK cells and B lymphocytes from each mouse were obtained. The results revealed that there are significantly more NK cells and B lymphocytes in the spleens of CD18:Mac-1/LFA-1 knockout mice than in control (same strain) mice. Moreover, in L-selectin knockout mice spleens, NK cells and B lymphocytes were elevated by 26.2% and 17.8% respectively. NK cells and B lymphocytes in the bone marrow of the integrin knockout showed no difference from control, however, both cell types in the bone marrow of the L-selectin knockout mice fell to only 3/4 their control levels. Collectively, the results demonstrated that there are organ-specific, but not cell lineage-specific differences in the absolute numbers of NK cells and B lymphocytes, in integrin-deficient (CD18:Mac-1/LFA-1 knockout) mice and addressin-deficient (L-selectin knockout) mice.

The leukocyte integrin alpha(M)beta(2) is a highly promiscuous leukocyte receptor capable of binding a multitude of unrelated ligands. To understand the molecular basis for the broad ligand recognition of alpha(M)beta(2), the inter-integrin chimera was created. In the chimeric integrin, the betad-alpha5 loop-alpha5 helix segment comprised of residues Lys(245)-Arg(261) from the alpha(M)I domain of alpha(M)beta(2) was inserted into the framework of alpha(L)beta(2). The construct was expressed in HEK 293 cells, and the ability of generated cells to adhere to fibrinogen and its derivatives was characterized first. Grafting the alpha(M)(Lys(245)-Arg(261)) sequence converted alpha(L)beta(2) into a fibrinogen-binding protein capable of mediating efficient and specific adhesion similar to that of wild-type alpha(M)beta(2). Verifying a switch in the binding specificity of alpha(L)beta(2), the chimeric receptor became competent to support cell migration to fibrinogen. Mutations at positions Phe(246), Asp(254), and Pro(257) within Lys(245)-Arg(261) of alpha(M)beta(2) produced significant decreases in cell adhesion, illustrating the critical role of these residues in ligand binding. The insertion of alpha(M)(Lys(245)-Arg(261)) imparted to the chimeric integrin the ability to recognize many typical alpha(M)beta(2) protein ligands. Furthermore, cells expressing the chimeric receptor, but not alpha(L)beta(2), were able to stick to uncoated plastic, which represents the hallmark of wild-type alpha(M)beta(2). These results suggest that alpha(M)(Lys(245)-Arg(261)) serves as a consensus binding site for interaction with a variety of distinct molecules and, thus, may define the degenerate recognition properties inherent to alpha(M)beta(2).

Pathogenic leptospires adhere to phagocytes even in the absence of specific antibodies; we demonstrate that the CR3 integrin (known as Mac-1 or CD11b/CD18 receptor), expressed on neutrophils and CHO Mac-1 transfected cells, recognizes and binds leptospires. The I-domain of the molecule seems to be involved in the recognition and data suggest that this occurs via fibronectin absorbed at the Leptospira surface. On the other hand, since N-acetyl-D-glucosamine does not behave as antagonist of the binding, the lectin-like domain of the receptor is not involved in the recognition.

The alpha(M)beta(2) integrin plays an important role in leukocyte biology through its interactions with a diverse set of ligands. Efficient ligand binding requires the involvement of both the alpha(M) and beta(2) subunits. Past ligand binding studies have focused mainly on the alpha(M) subunit, with the beta(2) subunit being largely unexplored. Therefore, in this study we conducted homolog-scanning mutagenesis on the I-domain (residues 125-385) within the beta(2) subunit. We identified four noncontiguous sequences (Arg(144)-Lys(148), Gln(199)-Ala(203), Leu(225)-Leu(230), and Gly(305)-His(309)) that are critical for fibrinogen and C3bi binding to alpha(M)beta(2). Molecular modeling revealed that these four sequences reside within a narrow region on the surface of the beta(2)I-domain, in close proximity to three potential cation-binding sites. Among these sequences, Gln(199)-Ala(203), Leu(225)-Leu(230), and Gly(305)-His(309) are important for the binding of both ligands, whereas Arg(144)-Lys(148) is more critical for fibrinogen than for C3bi binding. These sequences within the beta(2)I-domain are directly involved in ligand binding, since 1) switching these segments to their corresponding beta(1) sequences destroyed ligand binding; 2) loss of function was not due to a nonspecific gross conformational change, since the defective alpha(M)beta(2) mutants reacted well with a panel of conformation-dependent mAbs; 3) mutation of these functional sequences did not effect Ca(2+) binding; and 4) synthetic peptides corresponding to sequences Gln(199)-Ala(203) and Gly(305)-His(309) blocked ligand binding to alpha(M)beta(2), and the peptides interacted directly with fibrinogen and C3bi. Given the similarity among all integrin beta subunits, our results may help us to understand the underlying mechanism of integrin-ligand interactions in general.

IgA, the principal ligand for FcalphaRI, exists in serum as monomeric IgA and at mucosal sites as secretory IgA (SIgA). SIgA consists of dimeric IgA linked by joining chain and secretory components. Human polymorphonuclear leukocytes (PMN) and mouse PMN transgenic for human FcalphaRI exhibited spreading and elicited respiratory burst activity upon interaction with either serum or SIgA. However, PMN devoid of the beta(2) integrin Mac-1 (Mac-1(-/-)) were unable to bind SIgA, despite expression of FcalphaRI. Consistent with this, serum IgA stimulated Mac-1(-/-) PMN oxygen radical production, in contrast to SIgA. Binding studies showed the secretory component, by itself, to interact with Mac-1-expressing PMN, but not with Mac-1(-/-) PMN. These data demonstrate an essential role for Mac-1 in establishing SIgA-FcalphaRI interactions.

Histoplasma capsulatum is a fungus found intracellularly in neutrophils and peripheral blood mononuclear cells (PBMCs), suggesting that it is capable of evading damage and survives inside these cells. In this study, we report that neutrophils from H. capsulatum-infected mice, and human neutrophils and mononuclear cells exposed to H. capsulatum presented less apoptosis than those from noninfected animals or cells exposed to medium only. Moreover, cells harvested from infected animals are resistant to apoptosis induced by dexamethasone - a proapoptotic stimulant. We also show that neutrophils harvested from infected mice and PBMCs from humans exposed to the fungus had a greatly decreased Mac-1 expression. We conclude that H. capsulatum induces an antiapoptotic state on leucocytes, which correlates with decreased cell-surface Mac-1 expression. These facts may represent an escape mechanism for the fungus by delaying cell death and allowing the fungus to survive inside leucocytes.

In leukocytes, C3a and C5a cause chemotaxis in a G(i)-dependent, pertussis toxin (PT)-sensitive fashion. Because we found that HUVECs and immortalized human dermal microvascular endothelial cells express small numbers of C3aRs and C5aRs, we asked what the function of these receptors was on these cells. Activation of the C3aR caused transient formation of actin stress fibers, which was not PT-sensitive, but depended on rho activation implying coupling to G(alpha12) or G(alpha13). Activation of the C5aR caused a delayed and sustained cytoskeletal response, which was blocked by PT, and resulted in cell retraction, increased paracellular permeability, and facilitated eosinophil transmigration. C5a, but not C3a, was chemotactic for human immortalized dermal microvascular endothelial cells. The response to C5a was blocked by inhibitors of phosphatidylinositol-3-kinase, src kinase, and of the epidermal growth factor (EGF) receptor (EGFR) as well as by neutralizing Abs against the EGFR and heparin-binding EGF-like factor. Furthermore, immune precipitations showed that the EGFR was phosphorylated following stimulation with C5a. The C5aR in endothelial cells thus uses a signaling cascade-transactivation of the EGFR-that does not exist in leukocytes, while the C3aR couples to a different G protein, presumably G(alpha12/13).

Patients with sepsis and acute lung injury have increased interleukin (IL)-18 levels systemically. We hypothesize that IL-18 stimulates neutrophils (PMNs) at physiologic concentrations. IL-18 primed the oxidase at 15 min (10-100 ng/ml), 30 min (0.1-100 ng/ml), and 60 min (100 ng/ml; P<0.05) and caused translocation of p47(phox) to the membrane similar to lipopolysaccharides. CD11b surface expression was increased by IL-18 in a time- and concentration-dependent manner. IL-18 caused up-regulation of the formyl-Met-Leu-Phe receptor, changes in PMN size, and elastase release. Investigation of signaling demonstrated IL-18-mediated activation of p38 mitogen-activated protein (MAP) kinase in a concentration (0.1-100 ng/ml)-, time (5-15 min)-, and Ca2+-dependent manner. IL-18 directly increased cytosolic Ca2+ concentration. IL-18 activation of PMNs was blocked by inhibition of p38 MAP kinase activity (SB203580) or by inhibition of p38 MAP kinase activation by chelation of cytosolic Ca2+. We conclude that IL-18, at physiologic concentrations, is an effective PMN priming agent that requires p38 MAP kinase activity.

To determine the biological functions of membrane expressed CD45 isoforms on polymorphonuclear neutrophils (PMN), the monoclonal IgG F(ab')2 antibody against CD45, CD45RA or CD45RO was used as surrogate ligand for binding with these molecules on PMN. We found 99.5 +/- 3.2%, 42.3 +/- 5.8% and 96.7 +/- 2.6% PMN expressed CD45, CD45RA and CD45RO molecules on the cell surface, respectively. The interaction of CD45, CD45RA or CD45RO with its specific antibody on PMN enhanced phagocytosis markedly (34-83% increase), mainly via increased expression of complement receptor type 3 (CR3, CD11b) on the cells. The production of IL-8 by PMN was also increased significantly after binding with antibodies (anti-CD45 > anti-CD45RO > anti-CD45RA). Anti-CD45RA and anti-CD45RO, but not anti-CD45, enhanced TNF-alpha mRNA expression and decreased protein tyrosine phosphorylation of PMN. However, only anti-CD45RO suppressed Src family protein tyrosine kinase p56lck expression in the cells. These results suggest that the cross-linking of CD45 isoforms by their specific antibodies stimulated different PMN activities by differential suppression on protein tyrosine phosphorylation and Src family tyrosine kinase p56lck.

Activation of the complement system generates the anaphylatoxic peptide C5a, which elicits a broad range of inflammatory activities. The biological activities of C5a are mediated through its binding to the widely expressed C5a receptor (C5aR), a G-protein-coupled seven transmembrane domain receptor. In experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, the C5aR is expressed on monocytes/macrophages, reactive astrocytes and T cells infiltrating the central nervous system (CNS). To investigate the role of the C5aR in this T cell-driven autoimmune model, we induced EAE in C5aR-deficient mice (C5aR(-/-)) and wild-type mice using a myelin oligodendrocyte glycoprotein (MOG) peptide as the immunogen. We found that C5aR(-/-) mice were fully susceptible to MOG-induced EAE with no difference in disease onset or severity in C5aR(-/-) mice compared to control mice. Cellular infiltrates (macrophages and T cells) were similar in the spinal cords of both animal groups and splenic T cells from C5aR(-/-) mice and control mice responded identically to MOG in T cell proliferation assays. Ribonuclease protection assays demonstrated no significant differences in pro-inflammatory gene expression between receptor-deficient and sufficient mice. These results indicate that the C5aR is not an essential mediator in the induction and progression of EAE.

The femtomolar-acting eight-amino-acid peptide (NAP), derived from activity-dependent neuroprotective protein (ADNP), provides long-term protection against the deleterious effects of closed head injury (CHI) in mice. Fifteen minutes after injury, mice were divided into two groups, control and NAP-treated and a single subcutaneous injection of NAP or vehicle was administered. A third group served as sham-treated (not subjected to head trauma). Each mouse was assessed for its clinical function, using neurological severity score, at various time intervals following CHI, up to 30-45 d. Total cerebral cortex RNA was prepared from the site of injury of CHI mice, and from parallel regions in peptide-treated and sham brains. RNA was then reversed transcribed to yield radioactive cDNA preparations that were hybridized to Atlas array membranes containing 1200 cDNAs spots. Comparison of sham-treated individual mice showed differential expression levels of at least 15 mRNA species. Furthermore, results indicated that one of the genes that did not change among individuals but specifically increased after CHI and decreased after NAP treatment was the cell surface glycoprotein Mac-1 (CD11B antigen). Thus, Mac-1 is suggested as a marker for the long-term outcome of head injury and as a potential target for NAP protective actions.

Adherence of lymphocytes to the fungus is the first step in the direct lymphocyte-mediated antifungal effect against Candida albicans. In this study we identified macrophage-1 antigen (Mac-1) (CD11b/CD18, alpha(M)/beta(2)) as the lymphocyte surface structure responsible for the adhesion of activated lymphocytes to the hyphal form of the fungus. Antibodies specific for epitopes of the alpha-subunit (CD11b) and the beta(2)-subunit (CD18) of Mac-1 were shown to completely eliminate lymphocyte adhesion to C. albicans hyphae. Lymphocyte adhesion to C. albicans was also inhibited significantly by known ligands of Mac-1, including the extracellular matrix proteins laminin and fibrinogen, as well as engineered peptides containing arginine-glycine-aspartic acid sequences and the disintegrin echistatin. N-Acetyl-D-glucosamine and beta-glucan, which inhibit Mac-1-mediated adhesion to the yeast, blocked lymphocyte adhesion to hyphae. NIH 3T3 fibroblast transfectants expressing human CD11b/CD18 bound to C. albicans, and their binding was inhibited by antibodies specific for CD11b/CD18. Finally, antibodies specific for CD11b/CD18 effectively inhibited the capacity of activated lymphocytes to have an antifungal effect against hyphae. Our results clearly identify Mac-1 (CD11b/CD18) as the lymphocyte surface structure that mediates activated lymphocyte adhesion to C. albicans and the resultant antifungal effect of the lymphocytes.

Previously it was shown that beta(2)-integrins are necessary for slow leukocyte rolling in inflamed venules. In this study, mice that are deficient for either one of the beta(2)-integrins, alpha(L)beta(2) (LFA-1) or alpha(M)beta(2) (Mac-1), were used to determine which of the beta(2)-integrins are responsible for slowing rolling leukocytes. The cremaster muscles of these mice were treated with tumor necrosis factor-alpha and prepared for intravital microscopy. The average rolling velocities in venules were elevated in LFA-1(-/-) mice (11.0 +/- 0.7 microm/s) and Mac-1(-/-) mice (10.1 +/- 1.1 microm/s) compared to wild-type mice (4.8 +/- 0.3 microm/s; P <.05), but were lower than in CD18(-/-) mice (28.5 +/- 2.1 microm/s). When both LFA-1 and Mac-1 were absent or blocked, rolling velocity became dependent on shear rate and approached that of CD18(-/-) mice. In addition, leukocyte adhesion efficiency was decreased in LFA-1(-/-) mice to near CD18(-/-) levels, but decreased only slightly in Mac-1(-/-) mice. Thus, both LFA-1 and Mac-1 contribute to slowing down rolling leukocytes, although LFA-1 is more important than Mac-1 in efficiently inducing firm adhesion.

Macrophage-like development of myeloid leukemia cells which can be induced by agents such as phorbol esters (TPA) is accompanied by integrin expression and cell adhesion. Thus, in differentiating myeloid leukemia cells CD11b is predominantly expressed which can associate with CD18 to form the functional heterodimeric integrin Mac-1. To elucidate the role of cell adhesion during macrophage-like differentiation, we transfected human U937 myeloid leukemia cells with a vector containing the CD11b gene in antisense orientation. Expression of the CD11b antisense gene in stably transfected U937 cells (as-CD11b cells) resulted in an attenuated response to TPA. As-CD11b cells demonstrated poor adhesion to solid substrate upon TPA treatment in contrast to U937 control cells. Constitutive expression of c-myc in as-CD11b transfectants was higher than in control cells and failed to be repressed by TPA treatment. Moreover, unlike control cells, antisense transfectants failed to induce expression of early response genes such as c-jun and the redox factor ref-1 upon TPA stimulation. Consequently, the induction of monocytic differentiation markers such as the activity of alpha-naphthyl acetate esterase, the capacity to reduce nitroblue tetrazolium and the expression of the vimentin gene was much lower in antisense transfectants than in control U937 cells. According to the failure to undergo a monocytic differentiation program, TPA treatment of as-CD11b cells resulted in a progressively increasing amount of apoptotic cells whereas the differentiated population of U937 control cells remained alive. Taken together, these data suggest that the integrin-mediated (particularly CD11b-mediated) adhesion of myeloid leukemia cells in the course of induced monocytic differentiation is crucial for cell attachment, development of a monocytic phenotype and subsequent survival.

The present review describes a hypothesis for a critical role of cell adhesive interactions in tumorigenesis. Dysregulation of tissue cell-cell interactions initiates first of all local (in the tissue) and then general (in whole body) conditions for tumor growth. Otherwise imbalance of tissue-specific adhesion factor at the very beginning of carcinogenesis is considered to trigger a cascade of pathological reactions responsible for more severe adhesive disorders that are in turn critical for the "totalitarian" behavior of a tumor and its "colonization" of other tissues and organs. Impaired disturbance is likely to be the key mechanism of carcinogenesis since it is significantly associated with the main features of a tumor: tissue proliferation control loss, anaplasia, invasion, metastasis, and immune surveillance deficit. The hypothesis is supported by evolutionary, biological, histological, immunological, and clinical arguments whose combination does not characterize any other known mechanisms of oncogenesis. The concept of adhesiveness opens new possibilities for the diagnosis, prevention, and treatment of tumors and also improves a strategy for designing new drugs.

Functional studies of granulocytes in polycythemia vera (PV) and essential thrombocythemia (ET) are contradictory. In order to study the possible link between adhesion molecules and functional activity of polymorphonuclear leukocytes (PMNs) in these patients we measured the expression of CD11/CD18 glycoproteins and the oxidative burst of PMNs in patients with PV (13) and ET (18) and in 15 healthy voluntary donors. Experiments were performed by flow cytometry in total blood. The metabolism was stimulated by incubation with Escherichia coli and fMLP. Increased expression of CD11b/CD18 adhesion was obtained in PV and ET patients (p < 0.05 and p < 0.001). Studies of oxidative metabolism, stimulated by E. coli, showed a significantly reduced production of oxygen radicals in both groups of patients (p < 0.001). These data confirm the presence of a complex neutrophil dysfunction in PV and ET that probably reflects intrinsic abnormalities induced by changes in the cells' surface of the neoplastic clone and impaired signal transduction., (Copyright 2002 S. Karger AG, Basel)

Recently, it has been clarified that interaction between hematopoietic cells and endothelial cells is important in normal hematopoiesis and leukemogenesis. In this study, we examined the relationship between AML cells and endothelial cells by analyzing the expression profile of angiogenic factors, angiopoietin-1 (Ang-1), Ang-2, Tie-2 (a receptor for angiopoietins) and vascular endothelial growth factor (VEGF). Our results demonstrated that CD7(+)AML expressed Ang-2 mRNA frequently and integrin-family adhesion molecules (CD11c and CD18) intensively, suggesting the close correlation with endothelial cells. On the other hand, in t(8;21) AML cells, expression of Ang-2 was infrequent and expression of integrin-family adhesion molecules (CD11b, CD11c and CD18) was weak, suggesting the sparse association with endothelial cells. As for CD7(+)AML cells, despite the frequent and intense expression of endothelial cell-associated molecules (such as Ang-2, CD11c and CD18), intensity of Tie-2 expression was quite low (P < 0.05). Ang-2 expressed in CD7(+)AML cells is not considered to act in an autocrine fashion, but to work on endothelial cells to "feed" leukemic cells. Although Ang-2 is recognized as a natural antagonist for Tie-2, our data presented here suggested the alternative role of Ang-2 in the relationship between endothelial cells and leukemia cells, at least in a subset of leukemia such as CD7(+)AML. These results were supported by the study using AML cell lines, KG-1 (CD7 negative) and its subline KG-1a (CD7 positive); KG-1 had mRNA expression profile of Ang-1(+)Ang-2(-)Tie-2(+), while KG-1a showed Ang-1(+)Ang-2(+)Tie-2(-). These difference in the expression profile of angiogenic factors between CD7(+)AML and t(8;21)AML may explain the characteristic morphological features of these leukemias (CD7(+)AML as blastic type and t(8;21)AML as differentiative type).

The aim of this work was to quantify by flow cytometry the main adhesion receptors on CD34+ cells. These cells were isolated from bone marrow (BM) or mobilized peripheral blood (PB). The proportions of CD34+/CD49d+ and CD34+/CD49e+ are weaker on PB cells, without quantitative expression variation. This phenotypic variation may induce CD34+ cells exist from BM into circulation, promoting the mobilization. The homing to the BM implicate the CD62L receptor, which expression was found more frequently and stronger on PB cells than on BM. The CD11b, CD18 and CD54 receptors are implicated in CD34+ cells adhesion to BM micro-environment. No significant variation in CD34+/CD11b+ and CD34+/CD18+ cells frequency was noted. Moreover, CD54 receptor was more frequently expressed on PB cells. Quantitative analysis revealed that CD18 was more strongly expressed on BM than on PB cells. This quantitative variation could promote progenitor adhesion by interacting with stromal cells. Finally, quantitative expression of the main receptors on CD34+ cells provides an original option for studying CD34+ cells during the mobilization, the homing or the adhesion to BM micro-environment.

Microbial pathogens must evade the human immune system to survive, disseminate and cause disease. By proteome analysis of the bacterium Group A Streptococcus (GAS), we identified a secreted protein with homology to the alpha-subunit of Mac-1, a leukocyte beta2 integrin required for innate immunity to invading microbes. The GAS Mac-1-like protein (Mac) was secreted by most pathogenic strains, produced in log-phase and controlled by the covR-covS two-component gene regulatory system, which also regulates transcription of other GAS virulence factors. Patients with GAS infection had titers of antibody specific to Mac that correlated with the course of disease, demonstrating that Mac was produced in vivo. Mac bound to CD16 (FcgammaRIIIB) on the surface of human polymorphonuclear leukocytes and inhibited opsonophagocytosis and production of reactive oxygen species, which resulted in significantly decreased pathogen killing. Thus, by mimicking a host-cell receptor required for an innate immune response, the GAS Mac protein inhibits professional phagocyte function by a novel strategy that enhances pathogen survival, establishment of infection and dissemination.

Overexpression of protein kinase C-zeta (PKC-zeta) in the leukemic myeloid cell line U937 (U937-PKC-zeta cells), previously shown to induce leukemic cell differentiation, resulted in nearly complete downregulation of leukocyte integrins CD11a, CD11b, CD11d, and CD18, but not CD11c from the cell surface. The steady-state level of mRNAs for the downregulated leukocyte integrins was not detectable by Northern analysis. Nuclear run-on analysis revealed that transcription of all the leukocyte integrin genes except CD11c was reduced 70-90% as compared to control U937-Vector cells [U937 cells transfected with the empty vector pSV2M(2)6]. Transfection analysis of CD11-promoter-luciferase constructs confirmed that transcription of the leukocyte integrin genes was drastically downregulated in U937-PKC-zeta cells. The two c-jun binding sites in the CD11c promoter were essential for continued expression of CD11c in U937-PKC-zeta cells. Additionally, the 3' untranslated region (3' UTR) from CD11b, when fused to the luciferase gene, lead to the destabilization of this chimeric mRNA in U937-PKC-zeta cells. This indicates that downregulation of CD11b expression in U937-PKC-zeta cells is also the result of reduced stability of CD11b mRNA. Thus, overexpression of PKC-zeta in U937 cells leads not only to leukemic cell differentiation, but also to differential regulation of the leukocyte integrins.

DNA immunization induces CD8(+) CTL responses by bone marrow-derived APCs, which are directly transfected with a plasmid DNA and/or acquire Ags from DNA-transfected non-APCs. To investigate the relative contribution of DNA-transfected APCs vs non-APCs to the initiation of CD8(+) T cell responses, we used tissue-specific promoter-directed gene expression and adoptive transfer systems in gene gun DNA immunization. In this study, we demonstrated that non-APC-specific gene expressions induced significant CD8(+) CTL and IFN-gamma-producing cells and Ab responses, whereas APC-specific gene expressions led to moderate CTL and IFN-gamma-producers, but no Ab responses. Interestingly, mice immunized with a non-APC-specific plasmid induced more rapid, vigorous, and prolonged proliferation of adoptively transferred Ag-specific CD8(+) T cells than APC-specific plasmid-immunized mice. In addition, the in vivo proliferative responses elicited by a non-APC-specific plasmid administration were dependent on TAP, but were independent of CD4(+) T cell help. Collectively, our results suggest that cross-priming, in which Ags expressed in non-APCs are taken up, processed, and presented by APCs, plays an important role in the initiation, magnitude, and maintenance of CD8(+) T cell responses in gene gun DNA immunization.

The leukocyte integrin Mac-1 (alphaMbeta2, CD11b/CD18) regulates important cell functions in inflammation, including adhesion, phagocytosis, and oxidative burst. Deficiency of Mac-1 reduces vessel wall inflammation and neointimal thickening after murine carotid artery injury. Although Mac-1 has been implicated in modulating AP-1 and NF-kappaB activity, the signal transduction pathways involved are undefined. cDNA array analysis of Mac-1-clustered compared with -nonclustered monocytic THP-1 cells showed increased expression of the signal transducer TRAF6 (TNF receptor-associated factor 6), leading us to consider the possibility that Mac-1 used a Toll/IL-1 receptor family-like signaling pathway. Mac-1-dependent activation of NF-kappaB was potentiated by wild-type, and attenuated by dominant negative, TRAF6- and TGF-beta-activated kinase (TAK1) constructs. IRAK1 (IL-1 receptor associated kinase), a kinase immediately upstream of TRAF6, coimmunoprecipitated with Mac-1. Taken together, these observations indicate that Mac-1 recruits a Toll/IL-1 receptor family-like cascade to modulate NF-kappaB activity. This represents a new pathway for integrin-dependent modulation of gene expression.

Leucocyte adhesion deficiency (LAD) is a hereditary disorder caused by mutations in the CD18 (beta2 integrin) gene. Four missense mutations have been identified in three patients. CD18(A270V) supports, at a diminished level, CD11b/CD18 (Mac-1, alphaMbeta2 integrin) and CD11c/CD18 (p150,95, alphaXbeta2 integrin) expression and function but not CD11a/CD18 (LFA-1, alphaLbeta2 integrin) expression. Conversely, CD18(A341P) supports a limited level of expression and function of CD11a/CD18, but not of the other two CD11/CD18 antigens. CD18(C590R) and CD18(R593C) show a decreasing capacity to associate with the CD11a, CD11c and CD11b subunits. Transfectants expressing the CD11a/CD18 with the C590R and R593C mutations are more adhesive than transfectants expressing wild-type LFA-1, and express the reporter epitope of the monoclonal antibody 24 constitutively. Thus, the four mutations affect CD18 differently in its capacities to support CD11/CD18 expression and adhesion. These results not only provide a biochemical account for the clinical diversity of patients with leucocyte adhesion deficiency, but also offer novel insights into the structural basis of interaction between the alpha and beta subunits, which is an integral component in our understanding of integrin-mediated adhesion and its regulation.

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.

Domoic acid (DA), a potent neurotoxin, administered intravenously (0.75 mg/kg body weight) in adult rats evoked seizures accompanied by nerve cell damage in the present study. Neuronal degeneration and microglial reaction in the hippocampus were investigated, and the temporal profile of bcl-2, bax, and caspase-3 genes in cell death or survival was assessed following the administration of DA. Nissl staining showed darkly stained degenerating neurons in the hippocampus following the administration of DA at 1-21 days, the degeneration being most severe at 5 days. Ultrastructural study in CA1 and CA3 regions of hippocampus revealed two types of neuronal degeneration, cells that exhibited swollen morphology and shrunken electron-dense cells. Immunoreactivity of Bcl-2 and Bax was increased considerably at 16 hr and 24 hr in the neurons of the hippocampus following DA administration. No significant change was observed in the immunoreactivity of caspase-3 in the controls and DA-treated rats at any time interval. Microglial cells in the hippocampus showed intense immunoreaction with the antibodies OX-42 and OX-6 at 1-21 days after DA administration, indicating the up-regulation of complement type 3 receptors and major histocompatibility complex type II antigens for increased phagocytic activity and antigen presentation, respectively. Terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL) showed occasional positive neurons in the CA1 and CA3 regions at 5 days after DA administration, with no positive cells in the controls. RT-PCR analysis revealed that bcl-2 and bax mRNA transcripts in the hippocampus were significantly increased at 16 hr and gradually decreased at 24 hr following the administration of DA. Although bax and bcl-2 mRNA expression is rapidly induced at early stages, in situ hybridization analysis revealed complete loss of bcl-2, bax, and caspase-3 mRNA at 24 hr after DA administration in the region of neuronal degeneration in the hippocampus. These results indicate that the pattern of neuronal degeneration observed during DA-induced excitotoxic damage is mostly necrotic., (Copyright 2001 Wiley-Liss, Inc.)

The urokinase receptor (uPAR) is linked to cellular migration through its capacity to promote pericellular proteolysis, regulate integrin function, and mediate cell signaling in response to urokinase (uPA) binding. The mechanisms for these activities remain incompletely defined, although uPAR was recently identified as a cis-acting ligand for the beta2 integrin CD11b/CD18 (Mac-1). Here we show that a major beta1 integrin partner for uPAR/uPA signaling is alpha3. In uPAR-transfected 293 cells uPAR complexed (>90%) with alpha3beta1 and antibodies to alpha3 blocked uPAR-dependent vitronectin (Vn) adhesion. Soluble uPAR bound to recombinant alpha3beta1 in a uPA-dependent manner (K(d) < 20 nM) and binding was blocked by a 17-mer alpha3beta1 integrin peptide (alpha325) homologous to the CD11b uPAR-binding site. uPAR colocalized with alpha3beta1 in MDA-MB-231 cells and uPA (1 nM) enhanced spreading and focal adhesion kinase phosphorylation on fibronectin (Fn) or collagen type I (Col) in a pertussis toxin- and alpha325-sensitive manner. A critical role of alpha3beta1 in uPA signaling was verified by studies of epithelial cells from alpha3-deficient mice. Thus, uPAR preferentially complexes with alpha3beta1, promoting direct (Vn) and indirect (Fn, Col) pathways of cell adhesion, the latter a heterotrimeric G protein-dependent mechanism of signaling between alpha3beta1 and other beta1 integrins.

Mac-1 (CD11b/CD18) is an important adhesion molecule involved in the migration of leukocytes, cell signaling, and subsequent secretory responses. Its precise role in eosinophil recruitment and activation in vivo is not entirely clear. We wished to directly examine the role of Mac-1 in eosinophil migration in a murine model of allergic pulmonary inflammation. Briefly, wild-type (C57Bl/6) and Mac-1-deficient/knockout (Mac-1 KO) mice were intraperitoneally sensitized with ovalbumin (OVA) and alum (AlOH) on Days 0 and 14, and intranasally challenged with OVA either once on Day 14 or five times on Days 14 and 25 through 28. Control animals were challenged with saline. Bronchial hyperresponsiveness was measured, bronchoalveolar lavage (BAL) fluid was collected, and lungs were harvested for histology 24 h after the last challenge. The data demonstrate that wild-type (WT) mice do not respond to one OVA challenge but do develop bronchial hyperreactivity and airway and tissue eosinophilia after five OVA challenges. Conversely, Mac-1 KO mice develop significant airway eosinophilia after one OVA challenge, and the degree of airway inflammation is comparable to that observed in allergic WT mice after five challenges. In Mac-1 KO mice, after five challenges, bronchial hyperreactivity and airway inflammation was significantly enhanced compared with their wild-type counterparts. Administration of an anti-Mac-1 antibody to WT mice, before each of five intranasal OVA challenges, significantly reduces the airway eosinophilia but has no effect on tissue eosinophilia or bronchial hyperresponsiveness. Intravenous injection of interleukin-5 induced a significant blood eosinophilia in both WT and Mac-1 KO mice. Intranasal eotaxin administration induced similar levels of eosinophil migration into the lung tissues and airways of both WT and Mac-1 KO mice. In conclusion, Mac-1-deficient mice develop enhanced eosinophilic inflammation in the lung in response to allergic antigen challenge.

Acute severe (necrotizing) pancreatitis is often associated with pancreatic or peripancreatic infection. Decreased bacterial clearance due to impaired immune defense may cause local infection. We investigated expressions of surface opsonin receptors (CD11b, complement receptor 3; CD32/CD16, immunoglobulin G Fc receptor) on local and circulatory neutrophils, in murine acute pancreatitis. The mild and severe forms of acute pancreatitis were induced by seven and 13 subcutaneous injections of caerulein, respectively. Peritoneal exudative and circulatory neutrophils were counted and assayed for receptor expressions by flow cytometry, serially at 1-72 hours after pancreatitis induction. Histologically, mild and severe forms showed edematous and necrotizing pancreatitis, respectively. The peritoneal exudative neutrophil count was greater in mild than in severe pancreatitis. Expressions of CD11b and CD32/CD16 on local neutrophils were upregulated early in mild pancreatitis. This upregulation was attenuated in severe pancreatitis. The circulatory neutrophil count was elevated in severe pancreatitis but was unchanged in mild pancreatitis. Opsonin receptor expression on circulatory neutrophils showed a transient, modest upregulation in the early phase of mild pancreatitis. Receptor-positive circulatory neutrophils showed a marked elevation that persisted throughout the course of severe pancreatitis. In conclusion, severe (necrotizing) pancreatitis is associated with reduced opsonin receptor expression on local neutrophils and enhanced expression on circulatory neutrophils, as compared with mild (edematous) pancreatitis. These changes may contribute to local infectious complications and multiple organ failure, in severe pancreatitis.

Mice deficient in CD18, which lack all four CD11 integrins, have leukocytosis and increased susceptibility to bacterial infection. To determine the effect of deficiencies in LFA-1 (CD11a/CD18) or Mac-1 (CD11b/CD18) on host defense against systemic bacterial infection, knockout mice were inoculated i.p. with Streptococcus pneumoniae. Increased mortality occurred in both LFA-1(-/-) (15 of 17 vs 13 of 35 in wild type (WT), p < 0.01) and Mac-1(-/-) (17 of 34 vs 6 of 25, p < 0.01) mice. All deaths in LFA-1(-/-) mice occurred after 72 h, whereas most deaths in Mac-1(-/-) mice occurred within 24-48 h. At 24 h, 21 of 27 Mac-1(-/-) mice were bacteremic, vs 15 of 25 WT (p = 0.05); no difference was observed between LFA-1(-/-) and WT. Increased bacteria were recovered from Mac-1(-/-) spleens at 2 h (p = 0.03) and 6 h (p = 0.002) and from livers (p = 0.001) by 6 h. No difference was observed at 2 h in LFA-1(-/-) mice, but by 6 h increased bacteria were recovered from spleens (p = 0.008) and livers (p = 0.04). Baseline and peak leukocyte counts were similar between Mac-1(-/-) and WT, but elevated in LFA-1(-/-). At 8 h, peritoneal neutrophils were increased in Mac-1(-/-), but not significantly different in LFA-1(-/-). Histopathologically, at 24 h Mac-1(-/-) animals had bacteremia and lymphoid depletion, consistent with sepsis. LFA-1(-/-) mice had increased incidence of otitis media and meningitis/encephalitis vs WT at 72 and 96 h. Both Mac-1 and LFA-1 play important but distinct roles in host defense to S. pneumoniae.

Granulocyte (neutrophil) antibodies can cause febrile and pulmonary (TRALI) transfusion reactions as well as immune neutropenias. In the last decade enormous efforts have been made to characterize the implicated alloantigens. The NA1, NA2, and SH antigens could be identified as polymorphic forms of the neutrophil Fc gammaReceptor IIIb encoded by three alleles. The antigens MART and OND have been located on the leucocyte adhesion molecules (beta2 integrins) and found to be caused by single nucleotide mutations in the alphaM (CD11b) and alphaL (CD11a) subunits. We have succeeded in throwing light on the primary structure of the NB1 antigen, which has recently been clustered as CD177. Based on these findings, the Granulocyte Antigen Working Party of the ISBT introduced in 1998 a new nomenclature for human neutrophil alloantigens (HNA nomenclature) based on the antigen's glycoprotein location. Elucidation of the molecular nature of granulocyte antigens now allows antigen identification by glycoprotein-specific immunoassays and genotyping by DNA techniques. Thus, considerable progress has been made in the characterization of granulocyte antigens. Further studies will improve our diagnostic tools and facilitate the prevention and management of transfusion reactions and immune neutropenias.

Bacterial lipoprotein (BLP) is the most abundant protein in gram-negative bacterial cell walls, heavily outweighing lipopolysaccharide (LPS). Herein we present findings demonstrating the potent in vitro effects of BLP on neutrophil (PMN) activation status, function, and capacity to transmigrate an endothelial monolayer. PMNs are the principal effectors of the initial host response to injury or infection and constitute a significant threat to invading bacterial pathogens. The systemic inflammatory response syndrome (SIRS) is characterised by significant host tissue injury mediated, in part, by uncontrolled regulation of PMN cytotoxic activity. We found that BLP-activated human PMN as evidenced by increased CD11b/CD18 (Mac-1) expression. Up-regulation of PMN Mac-1 in response to BLP occurred independently of membrane-bound CD14 (mCD14). A similar up-regulation of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells was observed whilst E-Selectin expression was unaffected. PMN transmigration across a human umbilical vein endothelial cell (HUVEC) monolayer was markedly increased after treating either PMN's or HUVEC independently with BLP. This increased transmigration did not occur as a result of any direct effect of BLP on HUVEC monolayer permeability, assessed objectively using the passage of FITC-labeled Dextran-70. BLP primed PMN for enhanced respiratory burst and superoxide anion production in response to PMA, but did not influence phagocytosis of opsonized Escherichia coli. BLP far exceeds LPS as a gram-negative bacterial wall component, these findings therefore implicate BLP as an additional putative mediator of SIRS arising from gram-negative infection.

Leukocyte migration is the hallmark of inflammation, and integrin alpha(M)beta(2) and its ligand fibrinogen (Fg) are key participants in this cellular response. Cells expressing wild-type or mutant alpha(M)beta(2) and Fg or its derivatives have been used to dissect the molecular requirements for this receptor-ligand pair to mediate cell migration. The major conclusions are that (a) Fg, its D fragment, and its P1 and P2 alpha(M)beta(2) recognition peptides support a chemotactic response; (b) when the I domain of alpha(L) was replaced with the I domain of alpha(M), the chimeric receptor supported cell migration to Fg; however, the alpha(M) subunit, containing the I domain but lacking the beta(2) subunit, supported migration poorly, thus, the alpha(M)I domain is necessary but not sufficient to support chemotaxis, and efficient migration requires the beta(2) subunit and alpha(M)I domain; and (c) in addition to supporting cell migration, P2 enhanced alpha(M)beta(2)-mediated chemotaxis to Fg and the P1 peptide. This activation was associated with exposure of the activation-dependent epitope recognized by monoclonal antibody 7E3 and was observed also with human neutrophils. Taken together, these data define specific molecular requirements for alpha(M)beta(2) to mediate cell migration to Fg derivatives and assign a novel proinflammatory activity to the P2 peptide.

Oral infections of mice with Trichinella spiralis induce activation of peritoneal exudate cells to transiently express and secrete a crystallizable protein Ym1. Purification of Ym1 to homogeneity was achieved. It is a single chain polypeptide (45 kDa) with a strong tendency to crystallize at its isoelectric point (pI 5.7). Co-expression of Ym1 with Mac-1 and scavenger receptor pinpoints macrophages as its main producer. Protein microsequencing data provide information required for full-length cDNA cloning from libraries constructed from activated peritoneal exudate cells. A single open reading frame of 398 amino acids with a leader peptide (21 residues) typical of secretory protein was deduced and later deposited in GenBank (accession number M94584) in 1992. By means of surface plasmon resonance analyses, Ym1 has been shown to exhibit binding specificity to saccharides with a free amine group, such as GlcN, GalN, or GlcN polymers, but it failed to bind to other saccharides. The interaction is pH-dependent but Ca2+ and Mg2+ ion-independent. The binding avidity of Ym1 to GlcN oligosaccharides was enhanced by more than 1000-fold due to the clustering effect. Specific binding of Ym1 to heparin suggests that heparin/heparan sulfate may be its physiological ligand in vivo during inflammation and/or tissue remodeling. Although it shares approximately 30% homology with microbial chitinases, no chitinase activity was found associated with Ym1. Genomic Southern blot analyses suggest that Ym1 may represent a member of a novel lectin gene family.

The adenylate cyclase toxin (CyaA) of Bordetella pertussis is a major virulence factor required for the early phases of lung colonization. It can invade eukaryotic cells where, upon activation by endogenous calmodulin, it catalyzes the formation of unregulated cAMP levels. CyaA intoxication leads to evident toxic effects on macrophages and neutrophils. Here, we demonstrate that CyaA uses the alpha(M)beta(2) integrin (CD11b/CD18) as a cell receptor. Indeed, the saturable binding of CyaA to the surface of various hematopoietic cell lines correlated with the presence of the alpha(M)beta(2) integrin on these cells. Moreover, binding of CyaA to various murine cell lines and human neutrophils was specifically blocked by anti-CD11b monoclonal antibodies. The increase of intracellular cAMP level and cell death triggered by CyaA intoxication was also specifically blocked by anti-CD11b monoclonal antibodies. In addition, CyaA bound efficiently and triggered intracellular cAMP increase and cell death in Chinese hamster ovary cells transfected with alpha(M)beta(2) (CD11b/CD18) but not in cells transfected with the vector alone or with the alpha(X)beta(2) (CD11c/CD18) integrin. Thus, the cellular distribution of CD11b, mostly on neutrophils, macrophages, and dendritic and natural killer cells, supports a role for CyaA in disrupting the early, innate antibacterial immune response.

The cysteine-rich repeats in the stalk region of integrin beta subunits appear to convey signals impinging on the cytoplasmic domains to the ligand-binding headpiece of integrins. We have examined the functional properties of mAbs to the stalk region and mapped their epitopes, providing a structure-function map. Among a panel of 14 mAbs to the beta(2) subunit, one, KIM127, preferentially bound to alpha(L)beta(2) that was activated by mutations in the cytoplasmic domains, and by Mn(2+). KIM127 also bound preferentially to the free beta(2) subunit compared with resting alpha(L)beta(2). Activating beta(2) mutations also greatly enhanced binding of KIM127 to integrins alpha(M)beta(2) and alpha(X)beta(2). Thus, the KIM127 epitope is shielded by the alpha subunit, and becomes reexposed upon receptor activation. Three other mAbs, CBR LFA-1/2, MEM48, and KIM185, activated alpha(L)beta(2) and bound equally well to resting and activated alpha(L)beta(2), differentially recognized resting alpha(M)beta(2) and alpha(X)beta(2), and bound fully to activated alpha(M)beta(2) and alpha(X)beta(2). The KIM127 epitope localizes within cysteine-rich repeat 2, to residues 504, 506, and 508. By contrast, the two activating mAbs CBR LFA-1/2 and MEM48 bind to overlapping epitopes involving residues 534, 536, 541, 543, and 546 in cysteine-rich repeat 3, and the activating mAb KIM185 maps near the end of cysteine-rich repeat 4. The nonactivating mAbs, 6.7 and CBR LFA-1/7, map more N-terminal, to subregions 344-432 and 432-487, respectively. We thus define five different beta(2) stalk subregions, mAb binding to which correlates with effect on activation, and define regions in an interface that becomes exposed upon integrin activation.

The leukocyte integrin alpha(M)beta(2) (Mac-1, CD11b/CD18) is a cell surface adhesion receptor for fibrinogen. The interaction between fibrinogen and alpha(M)beta(2) mediates a range of adhesive reactions during the immune-inflammatory response. The sequence gamma(383)TMKIIPFNRLTIG(395), P2-C, within the gamma-module of the D-domain of fibrinogen, is a recognition site for alpha(M)beta(2) and alpha(X)beta(2). We have now identified the complementary sequences within the alpha(M)I-domain of the receptor responsible for recognition of P2-C. The strategy to localize the binding site for P2-C was based on distinct P2-C binding properties of the three structurally similar I-domains of alpha(M)beta(2), alpha(X)beta(2), and alpha(L)beta(2), i.e. the alpha(M)I- and alpha(X)I-domains bind P2-C, and the alpha(L)I-domain did not bind this ligand. The Lys(245)-Arg(261) sequence, which forms a loop betaD-alpha5 and an adjacent helix alpha5 in the three-dimensional structure of the alpha(M)I-domain, was identified as the binding site for P2-C. This conclusion is supported by the following data: 1) mutant cell lines in which the alpha(M)I-domain segments (245)KFG and Glu(253)-Arg(261) were switched to the homologous alpha(L)I-domain segments failed to support adhesion to P2-C; 2) synthetic peptides duplicating the Lys(245)-Tyr(252) and Glu(253)-Arg(261) sequences directly bound the D fragment and P2-C derivative, gamma384-402, and this interaction was blocked efficiently by the P2-C peptide; 3) mutation of three amino acid residues within the Lys(245)-Arg(261) segment, Phe(246), Asp(254), and Pro(257), resulted in the loss of the binding function of the recombinant alpha(M)I-domains; and 4) grafting the alpha(M)(Lys(245)-Arg(261)) segment into the alpha(L)I-domain converted it to a P2-C-binding protein. These results demonstrate that the alpha(M)(Lys(245)-Arg(261)) segment, a site of the major sequence and structure difference among alpha(M)I-, alpha(X)I-, and alpha(L)I-domains, is responsible for recognition of a small segment of fibrinogen, gammaThr(383)-Gly(395), by serving as ligand binding site.

Leukostasis and tissue infiltration by leukemic cells are poorly understood life-threatening complications of acute leukemia. This study has tested the hypothesis that adhesion receptors and cytokines secreted by blast cells play central roles in these reactions. Immunophenotypic studies showed that acute myeloid leukemia (AML) cells (n = 78) of the M0 to M5 subtypes of the French-American-British Cooperative Group expressed various amounts of adhesion receptors, including CD11a, b, c/CD18, CD49d, e, f/CD29, CD54, sCD15, and L-selectin. The presence of functional adhesion receptors was evaluated using a nonstatic adhesion assay. The number of blast cells attached to unactivated endothelium increased by 7 to 31 times after a 6-hour exposure of endothelium to tumor necrosis factor (TNF)-alpha. Inhibition studies showed that multiple adhesion receptors--including L-selectin, E-selectin, VCAM-1, and CD11/CD18--were involved in blast cell adhesion to TNF-alpha-activated endothelium. Leukemic cells were then cocultured at 37 degrees C on unactivated endothelial cell monolayers for time periods up to 24 hours. A time-dependent increase in the number of blasts attached to the endothelium and a concomitant induction of ICAM-1, VCAM-1, and E-selectin were observed. Additional experiments revealed that endothelial cell activation by leukemic myeloblasts was caused by cytokine secretion by blast cells, in particular TNF-alpha and IL-1 beta, and direct contacts between adhesion receptors expressed by blast cells and endothelial cells. Thus, leukemic cells have the ability to generate conditions that promote their own adhesion to vascular endothelium, a property that may have important implications for the pathophysiology of leukostasis and tissue infiltration by leukemic blast cells. (Blood. 2001;97:2121-2129)

In the absence of opsonizing antibodies, Bordetella pertussis, the causative agent of pertussis, readily binds to phagocytes via complement receptor 3 (CR3). After opsonization with antibodies, binding is mediated by IgG receptors (FcgammaR). The effect of targeting B. pertussis to either FcgammaR or CR3 was studied. The fate of unopsonized B. pertussis, IgG-opsonized B. pertussis, and B. pertussis opsonized with bispecific antibodies (BsAbs) directed to CR3 or FcgammaRII/-III was compared. IgG antibodies mediated binding and phagocytosis of B. pertussis via FcgammaR by polymorphonuclear leukocytes (PMNL) in vitro. Opsonization of B. pertussis with BsAbs directed against either CR3 or FcgammaRII/-III facilitated PMNL phagocytosis; however, in vivo studies with BsAb revealed that FcgammaR-mediated uptake facilitates B. pertussis clearance, in contrast to uptake via CR3. Targeting of B. pertussis to FcgammaRII/-III in mice deficient in FcgammaRII or FcgammaRIII indicated that the protective effect is attributable to FcgammaRIII. Competition between uptake via CR3 or FcgammaR may determine the outcome of natural infection.

In this study, 2 distinct populations of mature dendritic cells (DCs) were identified in the human thymus. The major population is CD11b-, CD11c+, and CD45RO(low) and does not express myeloid-related markers. It displays all the characteristics of mature DCs with a typical dendritic morphology, high surface levels of HLA-DR, CD40, CD83, and CD86, and expression of DC-lysosome-associated membrane glycoprotein messenger RNA (mRNA). In addition, CD11b- thymic DCs do not express macrophage inflammatory protein-1alpha (MIP-1alpha) mRNA, but express thymus-expressed chemokine (TECK) mRNA and are able to secrete bioactive interleukin 12 (IL-12) upon stimulation. In contrast, the minor and variable thymic DC population is CD11b+, CD11c(high), and CD45RO(high) and comprises CD83+CD14- mature and CD83- CD14+ immature DCs. It expresses macrophage-colony stimulating factor receptor, MIP-1alpha mRNA and high amounts of decysin mRNA after CD40 activation, but does not express TECK and is a weak bioactive IL-12 producer. Also identified were the IL-3Ralpha(high) plasmacytoid cells, which are present in the thymic cortex and medulla. Upon culture with IL-3, granulocyte/macrophage-colony stimulating factor, and CD40 ligand, the plasmacytoid cells can adopt a phenotype resembling that of freshly isolated CD11b- thymic DCs. However, these plasmacytoid-derived DCs fail to secrete bioactive IL-12; therefore, conclusions cannot be made about a direct relation between thymic plasmacytoid cells and CD11b- DCs. Whereas CD11b+ thymic DCs appear to be related to tonsillar germinal-center DCs, the major CD11b- IL-12-secreting human thymus DC population has similarities to mouse CD11b- CD8+ DCs.

C-reactive protein (CRP) and complement are hypothesized to be major mediators of inflammation in atherosclerotic plaques. We used the reverse transcriptase-polymerase chain reaction technique to detect the mRNAs for CRP and the classical complement components C1 to C9 in both normal arterial and plaque tissue, establishing that they can be endogenously generated by arteries. When the CRP mRNA levels of plaque tissue, normal artery, and liver were compared in the same cases, plaque levels were 10.2-fold higher than normal artery and 7.2-fold higher than liver. By Western blotting, we showed that the protein levels of CRP and complement proteins were also up-regulated in plaque tissue and that there was full activation of the classical complement pathway. By in situ hybridization, we detected intense signals for CRP and C4 mRNAs in smooth muscle-like cells and macrophages in the thickened intima of plaques. By immunohistochemistry we showed co-localization of CRP and the membrane attack complex of complement. We also detected up-regulation in plaque tissue of the mRNAs for the macrophage markers CD11b and HLA-DR, as well as their protein products. We showed by immunohistochemistry macrophage infiltration of plaque tissue. Because CRP is a complement activator, and activated complement attacks cells in plaque tissue, these data provide evidence of a self-sustaining autotoxic mechanism operating within the plaques as a precursor to thrombotic events.

Fibrin plays important roles in the wound healing processes, including blood clotting and platelet aggregation. Additional activities of fibrin were found in this study, which utilizes human THP-1 cells treated 1,25-(OH)2 vitamin D3 and plasminolytic fragments derived from fibrin. Coated fibrin fragment E on culture plates induced cell adhesions and morphological changes of the THP-1 cells, being resembled to tissue macrophages. Morphological changes of the THP-1 cells were caused by microfilament reorganization. IL-1beta production was increased in the THP-1 cells by adherent fibrin fragment E, but not by fibrin fragment D or by fibrinogen fragment E. The elevation of IL-1beta production is caused by transcriptional activation. Incubation with cytochalacin D, an actin polymerization inhibitor, prevents both microfilament reorganization and morphological changes, but has no effect on the IL-1beta production stimulated by fibrin fragment E. This data suggests that the IL-1beta production in the THP-1 cells do not require microfilament reorganization and integrin aggregation. Taken together, these results indicate that fibrin matrix plays an additional role in the stimulation of monocytes for production of IL-1beta, morphological changes and cell adhesion, resulting in the facilitation of the wound healing processes.