Platelet/polymorphonuclear leukocyte interaction in dynamic conditions: evidence of adhesion cascade and cross talk between P-selectin and the beta 2 integrin CD11b/CD18

Adhesion between platelets and polymorphonuclear leukocytes (PMN) is a key event in thrombosis and inflammation. Double color fluorescence-activated cell sorter (FACS) analysis was used to determine the extent and kinetics of adhesion of thrombin-activated platelets to resting or activated PMN when mixed cell populations were incubated in dynamic conditions. Activated platelets bound very rapidly to PMN. Mixed cell conjugates reached a maximum at 1 minute and were reversible within 10 minutes. Platelet/PMN adhesion required both Ca2+ and Mg2+ and was markedly increased by the presence of Mn2+. The latter made mixed cell conjugates stable up to 10 minutes. Adhesion of platelets required metabolic activity of PMN and was abolished by tyrosine kinase inhibitors. Furthermore, adhesion of platelets to PMN resulted in binding of a monoclonal antibody (MoAb 24) known as beta 2 integrins "activation reporter." When PMN were activated by exogenous stimuli, the adhesion of platelets was markedly increased: fMLP induced a rapid and transient effect, while PMA resulted in a slower, but stable, increase in mixed conjugates formation. The hypothesis that activated PMN beta 2 integrins are able to bind a counter-receptor on platelets was directly demonstrated by the increase of mixed cell conjugates following PMN treatment with KIM127 and KIM185, two anti-CD18 antibodies able to induce the active conformation of beta 2 integrins. Consistently, two other anti-CD18, as well as an anti-CD11b inhibitory antibody abolished platelet/PMN adhesion. PMN beta 2 integrin activation was not the only mechanism for activated platelet/PMN adhesion to occur: indeed, this phenomenon could also be inhibited by two anti-P-selectin antibodies. Resting platelets did not adhere to resting PMN, but markedly adhered to fMLP- or PMA-activated PMN. Resting platelet/fMLP-activated PMN adhesion was abolished by anti-CD18 antibodies, but not by anti-P-selectin antibodies. In conclusion, activated platelet/PMN interaction can be modeled as an adhesion cascade involving a P-selectin-dependent recognition step and a functional signal. The latter proceeds through tyrosine kinase activation and enables a beta 2 integrin-dependent adhesion to a not yet identified counter-receptor constitutively expressed on platelet surface.     

Alterations in adhesion molecule (CD11b/CD18 and CD62L) expression on granulocytes after chemotherapy

The evaluation of brain tumor recurrence and therapy-induced benign changes following surgery and/or irradiation is a diagnostic challenge for imaging methods based on either morphology (cCT/MRI) or function (SPECT/PET). Current literature and the present data of our own patients demonstrate the diagnostic efficiency of IMT-SPECT and FDG-PET in the detection of recurrence and in-vivo grading. Thirty-nine patients suspected of brain tumor recurrence at follow-up were studied by FDG-PET and IMT-SPECT. Thirty-four of 39 patients showed recurrences; in 12 cases even a change in the grade of malignancy was observed. All high-grade recurrences could be confirmed by either methods. IMT-SPECT showed a higher sensitivity in detecting low-grade tumors at recurrence. In contrast to IMT-SPECT, FDG-PET supports sufficient in-vivo grading. Both methods can be used to differentiate between tumor recurrence and radionecrosis. In conclusion the results of our study demonstrate the efficiency of IMT-SPECT and FDG-PET in confirming recurrences and determining the actual tumor grade.     

A natural glycoprotein inhibitor (NIF) of CD11b/CD18 reduces leukocyte adhesion in the liver after hemorrhagic shock

Legume seeds and fibre rich plant foods usually improve aspects of human diabetes control as they are potential sources of "delayed release" carbohydrates. A regional bakery mixture of soybean and cereals, interesting for its palatability and high content in non starch polysaccharides was chemically and nutritionally evaluated. Comparisons were made with the usual commercial laboratory chow and with a cafeteria mixture. Each one of the three diets was offered ad libitum to adult rats of line IIMb/Fm beta (beta), affected by obesity, hypertriglyceridemia and glucose intolerance or diabetes. Treatments lasted three months and were performed on two groups of male rats: (a) From 100 days old growing significantly. (b) From 200 days old. Meals had similar carbohydrate and calorie contents but acid followed by enzymatica hydrolysis was required to free monosaccharides from the soybean mixture. Cafeteria mixture lacked in fibre, was rich in saturated fats and sodium, and it caused hyperphagia. Each group of rats showed similar food intakes in both ages although weight gain was significantly higher in the younger animals. In the latter, values of glycemic response showed no difference between diets. Cafeteria mixture caused significant hyperglycemia to the elder rats, while the soybean bakery mixture produced a remarkably lower glycemic response; in one case it was even lower than the one produced by the commercial chow. Differential response showed more clearly with age. The results of the feces analysis demonstrated an increased proportion of fecal water for the bakery mixture group, probably due to the amount of undigestible fibre, inducing beneficial effects on large bowel functionality.     

Human polymorphonuclear leukocytes adhere to complement factor H through an interaction that involves alphaMbeta2 (CD11b/CD18)

The work presented here demonstrates that human complement factor H is an adhesion ligand for human neutrophils but not for eosinophils. The adherence of polymorphonuclear leukocytes (PMNs) to plastic wells coated with factor H depended on divalent metal ions and was augmented by C5a and TNF-alpha. PMN adhesion to factor H in the presence or absence of C5a was blocked specifically by mAbs against CD11b or CD18. Affinity purification using factor H Sepharose followed by immunoprecipitation using mAbs to various integrin chains identified Mac-1 (CD11b/CD18) as a factor H binding receptor. The presence of surface bound factor H enhanced neutrophil activation resulting in a two- to fivefold increase in the generation of hydrogen peroxide by PMNs stimulated by C5a or TNF-alpha. When factor H was mixed with PMNs, 1.4 to 3.8-fold more cells adhered to immobilized heparin or chondroitin A. In addition, augmented adhesion of PMNs was measured when factor H, but not HSA or C9, was absorbed to wells that were first coated with heparin or chondroitin A. The adhesion of PMNs to glycosaminoglycan-factor H was blocked by mAbs to CD11b and CD18. These studies demonstrate that factor H is an adhesion molecule for human neutrophils and suggest that the interaction of factor H with glycosaminoglycans may facilitate the tethering of this protein in tissues allowing factor H to serve as a neutrophil adhesion ligand in vivo.     

Expression of the adhesion molecule CD11b and polymerization of actin by polymorphonuclear granulocytes of patients endangered by sepsis

Attributable risks (ARs) for bladder cancer were computed in relationship to cigarette smoking, coffee consumption, low intake of vegetables, history of cystitis, and occupation using data from a case-control study conducted in northern Italy between 1985 and 1993. Cases were 431 patients with histologically confirmed bladder cancer, and controls were 491 patients admitted to the same network of hospitals for acute, nonneoplastic, and non-urinary-tract diseases. Overall, the AR estimates were 49% for cigarette smoking, 23% for coffee consumption, 16% for low intake of vegetables, 12% for history of cystitis, and 4% for occupation. These five factors together explained more than 70% of bladder cancer cases in this population. The AR for cigarette smoking was significantly higher among men (56%) than women (17%), whereas coffee consumption, low vegetable intake, and cystitis were more important (but not significantly so) among women. These results suggest that more than 2500 of the 5400 deaths due to bladder cancer in Italy in 1990 could have been prevented by the elimination of cigarette smoking. With some appropriate dietary modification and intervention to prevent urinary tract infections and occupational exposures, this figure could approach 4000 avoidable deaths. Thus, bladder cancer could become a rare cause of death in this population.     

The urokinase receptor (CD87) facilitates CD11b/CD18-mediated adhesion of human monocytes

Urokinase receptors (uPAR; CD87) from complexes with complement receptor 3 (CR3) (CD11b/CD18), a beta2 integrin. In this study, we sought to determine if this association modulates the adhesive function of CR3. Both CR3 and uPAR concentrate at the ventral surface of fibrinogen-adherent human monocytes, and CR3-uPAR coupling increases substantially upon adhesion to fibrinogen. Pretreatment with anti-uPAR monoclonal antibody reduced adhesion to CR3 counterligands (fibrinogen and keyhole limpet hemocyanin) by 50%, but did not affect adhesion to fibronectin, a beta1 integrin counterligand. Antisense (AS) oligonucleotides were used to determine if selectively suppressing uPAR expression also modulates CR3 adhesive function. AS-uPAR oligo reduced CR3-dependent adhesion by 43+/-9% (P<0.01), but did not affect CR3-independent adhesion. To determine if the effects of uPAR are mediated through its ligand, monocytes were pre-treated with AS oligo to block uPA expression. Unlike the effects of blocking uPAR expression, AS-uPA oligo increased adhesion by 46% (P<0.005), and exogenous intact uPA, but not uPA fragments, reversed this effect. We conclude that complex formation with uPAR facilitates the adhesive functions of CR3. This function of uPAR is not dependent upon its occupancy with uPA, which negatively influences adhesion.     

Increased expression of intercellular adhesion molecule 1, CD11/CD18 cell surface adhesion glycoproteins and alpha 4 beta 1 integrin in a rat model of chronic interstitial lung fibrosis

The expression of the intercellular adhesion molecule 1 (ICAM-1), and the integrins CD49, CD11b/c, and CD11a (LFA-1 alpha chain) was analyzed in an experimental model of pulmonary fibrosis. Adult rats were exposed to 75% oxygen during 10 weeks, and to 2.0 mg/kg of paraquat twice weekly. Rats were sacrificed at 2 days, and at 2 and 10 weeks after the first injection of paraquat. Lungs were fixed in 4% paraformaldehyde and used for histology and immunohistochemistry. At 2 days the lungs showed a diffuse inflammation composed of a mixed polymorphonuclear and mononuclear cell infiltrate. Afterwards, the inflammatory process was predominantly mononuclear, and an increasing fibroblast proliferation was observed. Early inflammatory events (48 h) correlated with a moderate increased expression of ICAM-1, LFA, and CD11b/c in epithelial cells as well as a pronounced expression of ICAM-1 and CD11b/c in macrophages. At 2 and 10 weeks, there was a progressive increased expression of CD11b/c and ICAM-1 by macrophages, as well as of LFA in epithelial cells, and of ICAM-1 and CD49 by epithelial and interstitial cells. Lymphocytes showed a slight increased expression of LFA at 2 weeks, and of CD49 at 2 and 10 weeks. These results suggest that macrophages expressing ICAM-1, CD11b/c, and CD49 are involved in the earlier and late phases of the disease whereas fibroblast and epithelial cells expressing ICAM-1 and CD49 might play a role in the cell interactions involved in the fibrotic phase.     

CD11/CD18 and CD14 share a common lipid A signaling pathway

The activation of phagocytes by the lipid A moiety of LPS has been implicated in the pathogenesis of Gram-negative sepsis. While two LPS receptors, CD14 and CD11/CD18, have been associated with cell signaling, details of the LPS signal transduction cascade remain obscure. CD14, which exists as a GPI-anchored and a soluble protein, lacks cytoplasmic-signaling domains, suggesting that an ancillary molecule is required to activate cells. The CD11/CD18 integrins are transmembrane proteins. Like CD14, they are capable of mediating LPS-induced cellular activation when expressed on the surface of hamster fibroblasts Chinese hamster ovary (CHO)-K1. The observation that a cytoplasmic deletion mutant is still capable of activating transfected CHO-K1 argues that CD11/CD18 also utilizes an associated signal transducer. We sought to identify further similarities between the signaling systems utilized by CD14 and CD11/CD18. LPS-binding protein, which transfers LPS to CD14, enhanced both LPS-induced cellular activation and binding of Gram-negative bacteria in CD11/CD18-transfected CHO-K1, thus implying that LPS-binding protein can also transfer LPS to CD11/CD18. When synthetic lipid A analogues were analyzed for their ability to function as LPS agonists, or antagonists, in the CHO transfectants, we found the effects were identical regardless of which LPS receptor was expressed. This supports the hypothesis that a receptor distinct from CD14 and CD11/CD18 is responsible for discriminating between the lipid A of LPS and the LPS antagonists. We propose that this receptor, which is the target of the LPS antagonists, functions as the true signal transducer in LPS-induced cellular activation for both CD14 and CD11/CD18.     

Functional mapping of CD11b/CD18 epitopes important in neutrophil-epithelial interactions: a central role of the I domain

In the intestine, lung, and urinary tract, neutrophil (polymorphonuclear leukocyte, PMN) transepithelial migration is dependent on the leukocyte beta2 integrin CD11b/CD18. While the regions of CD11b involved in recognition of several soluble ligands are known, those that mediate PMN-epithelial interactions have not been investigated. In this study, mAbs reactive with four extracellular regions on CD11b, the NH2-terminal region, I (inserted) domain, cation-binding region, and region proximal to the transmembrane domain (C domain), were analyzed for the ability to block CD11b/CD18-mediated interactions with T84 intestinal epithelial cells. In such a manner, epitope mapping was applied to the complex interactions between CD11b/CD18 and a cell-based ligand system. I domain Abs strongly inhibited both adhesion of PMN to epithelial cells and PMN migration across T84 epithelial monolayers. However, the profile of inhibition was distinct from that of other known ligands of CD11b/CD18. CBRM1/32, an Ab to a discontinuous epitope residing within the NH2- and cation-binding domains, strongly inhibited both adhesion and transmigration responses. C domain Abs had minimal effects on adhesion and transmigration. These findings appear applicable to other epithelia, since similar results were obtained in transmigration experiments with CF15 human airway epithelial cells. Finally, Ab inhibition profiles were confirmed with adhesion assays of isolated epithelial cells to purified CD11b/CD18. These findings demonstrate the central role of the I domain and the participation of a discontinuous region shared by the NH2- and cation-binding domains in mediating PMN-adhesive interactions with epithelial cells.     

The role of LFA-1 (CD11a/CD18) cytoplasmic domains in binding to intercellular adhesion molecule-1 (CD54) and in postreceptor cell spreading

We have investigated the role of the cytoplasmic domains of LFA-1 in binding to ICAM-1 and in postadhesion events. Various truncated and chimeric forms of LFA-1 alpha (CD11a) and beta (CD18) chains were generated and transfected into murine fibroblast TNR-2 cells. Transfected fibroblasts expressing wild-type LFA-1 adhered only weakly to ICAM-1 immobilized on plastic, and phorbol ester pretreatment enhanced this adhesion significantly. In contrast, transfected cells expressing LFA-1 lacking both the alpha and the beta cytoplasmic domains, the beta cytoplasmic domain alone, or GPI-anchored LFA-1 adhered to immobilized ICAM-1 without prior activation. Truncation of the alpha cytoplasmic domain alone resulted in much reduced cell adhesion which could be only weakly upregulated by PMA. The presence of manganese dramatically enhanced the binding to ICAM-1 of LFA-1 lacking the alpha cytoplasmic domain or both cytoplasmic domains, whereas it had relatively little effect on wild-type LFA-1 or the mutant lacking the beta cytoplasmic domain. Soluble LFA-1, generated by phosphatidylinositol-specific phospholipase-C treatment of GPI-anchored LFA-1, was capable of binding ICAM-1+ cells. Although doubly truncated or GPI-anchored LFA-1 mediated cell adhesion to immobilized ICAM-1, cells expressing these mutants, as well as those expressing individual alpha and beta chain truncations, failed to spread out following this adhesion, whereas the wild-type transfectants did so readily. Manganese had no effect on cell spreading. Fluorescent staining of these cells indicated no significant variation in the distribution of LFA-1 on the cell surface. From these results we conclude that (1) cells expressing LFA-1 lacking both the alpha and the beta cytoplasmic domains adhere to ICAM-1 without prior stimulation, indicating the importance of LFA-1 cytoplasmic domains in inside-out signaling, (2) truncation of the alpha cytoplasmic domain alone inhibits cell adhesion by making LFA-1 nonresponsive to inside-out signaling, and (3) both cytoplasmic domains are required for cell spreading following adhesion to immobilized ICAM-1.     

Contribution of CD11a/CD18, CD11b/CD18, ICAM-1 (CD54) and -2 (CD102) to human monocyte migration through endothelium and connective tissue fibroblast barriers

Recently we reported that monocyte migration through a barrier of human synovial fibroblasts (HSF) is mediated by the CD11/CD18 (beta2) integrins, and the beta1 integrins VLA-4 and VLA-5 on monocytes. Here we investigated in parallel the role of beta2 integrin family members, LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) on monocytes, and the immunoglobulin supergene family members, ICAM-1 and ICAM-2 on HSF and on human umbilical vein endothelial cells (HUVEC), in monocyte migration through HSF and HUVEC monolayers. Using function blocking monoclonal antibodies (mAb), when both VLA-4 and VLA-5 on monocytes were blocked, treatment of monocytes with mAb to both LFA-1 and to Mac-1 completely inhibited monocyte migration across HSF barriers, although blocking either of these beta2 integrins alone had no effect on migration, even when VLA-4 and VLA-5 were blocked. This indicates that optimal beta2 integrin-dependent monocyte migration in synovial connective tissue may be mediated by either LFA-1 or Mac-1. Both ICAM-1 and ICAM-2 were constitutively expressed on HSF and on HUVEC, although ICAM-2 was only minimally expressed on HSF. Based on results of mAb blockade, ICAM-1 appeared to be the major ligand for LFA-1-dependent migration through the HSF. In contrast, both ICAM-1 and ICAM-2 mediated LFA-1-dependent monocyte migration through HUVEC. However, neither ICAM-1 nor ICAM-2 was required for Mac-1 -dependent monocyte migration through either cell barrier, indicating that Mac-1 can utilize ligands distinct from ICAM-1 and ICAM-2 on HSF and on HUVEC during monocyte transmigration.     

Porphyromonas gingivalis fimbriae use beta2 integrin (CD11/CD18) on mouse peritoneal macrophages as a cellular receptor, and the CD18 beta chain plays a functional role in fimbrial signaling

In this study, we demonstrate that Porphyromonas gingivalis fimbriae use molecules of beta2 integrin (CD11/CD18) on mouse peritoneal macrophages as cellular receptors and also show that the beta chain (CD18) may play a functional role in signalling for the fimbria-induced expression of interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) genes in the cells. Using a binding assay with 125I-labeled fimbriae, we observed that fimbrial binding to the macrophages was inhibited by treatment with CD11a, CD11b, CD11c, or CD18 antibody but not by that with CD29 antibody. Western blot assays showed that the fimbriae bound to molecules of beta2 integrin (CD11/CD18) on the macrophages. Furthermore, Northern blot analyses showed that the fimbria-induced expression of IL-1beta and TNF-alpha genes in the cells was inhibited strongly by CD18 antibody treatment and slightly by CD11a, CD11b, or CD11c antibody treatment. Interestingly, intracellular adhesion molecule 1 (ICAM-1), a ligand of CD11/CD18, inhibited fimbrial binding to the cells in a dose-dependent manner. In addition, ICAM-1 clearly inhibited the fimbria-induced expression of IL-1beta and TNF-alpha genes in the cells. However, such inhibitory action was not observed with laminin treatment. These results suggest the importance of beta2 integrin (CD11/CD18) as a cellular receptor of P. gingivalis fimbriae in the initiation stage of the pathogenic mechanism of the organism in periodontal disease.     

Increased expression of CD11b/CD18 on phagocytes in ischaemic disease: a bridge between inflammation and coagulation

The aim of this study was to assess the expression of CD11b/CD18 integrin adhesion molecules on the phagocytes of patients with ischaemic diseases, and to evaluate the concentration of soluble adhesion molecules that are released from endothelium (sICAM-1) and from phagocytes (sL-selectin). A total of 370 patients were enrolled: 120 with coronary artery disease (CAD); 50 with peripheral artery occlusive disease (PAOD); and 200 control subjects with no clinical manifestations of ischaemic disease. CD11b/CD18 integrin was detected by flow cytometry, whereas sL-selectin and sICAM-1 concentrations were detected using a sandwich-type immunoassay. CD11b/CD18 integrin expression was found to be higher in the patients with ischaemic disease than in the control subjects (P < 0.001). The PAOD patients had higher values of CD11b/CD18 integrin than the CAD ones (P < 0.01). The concentration of soluble adhesion molecules did not show any significant differences within the three groups (P = NS). The high expression of CD11b/CD18 integrin in ischaemic disease patients may depend on the increased, but probably stable, cytokine network that has been demonstrated to occur in chronic ischaemic diseases: the difference observed between PAOD and CAD patients could be the consequence of higher inflammatory activation probably resulting from the greater extent of the atherosclerotic process in PAOD, or of the more localized ischaemic area in CAD patients. CD11b/CD18 can therefore be considered a marker of chronic phagocyte activation during ischaemic disease. On the other hand, sICAM and sL-selectin concentrations were found to be within the normal range; they have recently been considered as a marker for acute ischaemic events and acute inflammatory process activation. Our results confirm that in uncomplicated atherosclerosis no acute inflammatory process activation should occur.     

The NF-kappa B inhibitor, tepoxalin, suppresses surface expression of the cell adhesion molecules CD62E, CD11b/CD18 and CD106

Tepoxalin, a dual enzyme inhibitor of cyclooxygenase and 5-lipoxygenase has been shown to inhibit T-cell activation. Its immunosuppressive property is distinct from cyclosporin because only tepoxalin, but not cyclosporin, suppresses NF-kappa B activation. Here we report that tepoxalin selectively inhibits intercellular adhesion molecule-1 (ICAM-1, CD54)/MAC-1 (CD11b/CD18) dependent adhesion of polymorphonuclear cells to IL-1 activated human umbilical vein endothelial cells. The mechanism of inhibition is related to the surface expression of several cell adhesion molecules. Flow cytometry analyses on cultured cells that were treated with tepoxalin or antisense oligonucleotides to the P65/p50 subunit of NF-kappa B, and then stimulated with PMA, revealed a reduced expression of CD11b/CD18 on monocytic HL60 cells, and endothelial adhesion molecule-1 (CD62E) and vascular adhesion molecule-1 (CD106) on human umbilical vein endothelial cells. Expression of other adhesion molecules such as lymphocyte function associated-antigen-1 (CD11a/CD18) and CD54 were unaffected. Tepoxalin also inhibited the secretion of a NF-kappa B regulated chemokine, IL-8, a known inducer of CD11b/CD18 expression. Thus the suppression of CD11b/CD18 expression by tepoxalin may involve IL-8. Our results suggest that by inhibiting NF-kappa B activation, surface expression of several adhesion molecules can be modulated and that tepoxalin may be useful in treating selected adhesion mediated events such as leukocyte migration or atherosclerotic plaque formation.     

Regulation of CD11b/CD18 expression in human neutrophils by phospholipase A2

Recent evidence suggests that phospholipase A2 (PLA2)-derived lipid mediators may regulate a number of neutrophil responses including degranulation and adhesion. In view of the potential role of PLA2 in stimulus-secretion coupling, we examined the relationship between PLA2 activation and the surface expression of CD11b/CD18 (MAC-1) in human polymorphonuclear leukocytes (hPMNL), including the functional consequences of PLA2 inactivation on MAC-1-dependent adhesion. The selective inhibition of PLA2 by the marine natural products manoalide (MLD) and scalaradial (SLD) blocks [3H]arachidonic acid (AA) release in calcium ionophore A23187-stimulated neutrophils, and also inhibits secretion of specific and azurophilic granule constituents. Additional studies demonstrate that MLD, SLD, and other less potent PLA2 inhibitors such as 4-bromophenacylbromide and nordihydroguiaretic acid inhibit the surface expression of MAC-1 (IC50: MLD, 0.33 microM; SLD, 0.23 microM; 4-bromophenacylbromide, 2.8 microM; NDGA, 3.5 microM) at concentrations similar to those at which they inhibit [3H]AA release. Inhibitors of cyclooxygenase, 5-lipoxygenase, protein kinase C, or calcium channel antagonists have no effect on MAC-1 expression. PLA2 inactivation also prevents MAC-1 up-regulation in hPMNL stimulated with FMLP, IL-8, TNF-alpha, PMA, or platelet activating factor. In FMLP-stimulated hPMNL, under conditions in which no secondary granule constituents are secreted, MAC-1 and alkaline phosphatase up-regulation from intracellular granules is inhibited by MLD and SLD. Functional assays also demonstrate that MLD and SLD block MAC-1-dependent adhesion of activated neutrophils to keyhole limpet hemocyanin at concentrations that block the surface expression of MAC-1. [3H]AA release and MAC-1 expression in MLD and SLD-treated hPMNL could be recovered in the presence of 1 mM hydroxylamine in a time-dependent fashion, consistent with reported data that MLD and SLD inactivate PLA2 through Schiff base formation. In summary, these data emphasize the role of PLA2 as a key regulator of MAC-1 expression in models of neutrophil adhesion.     

Mice deficient in Mac-1 (CD11b/CD18) are less susceptible to cerebral ischemia/reperfusion injury

Many children with esophagitis demonstrate histological changes without gross evidence of esophagitis by esophagoscopy. The effect of omeprazole on the histological healing of esophagitis in children is unknown. Therefore, the aim of this study was to determine the effect of omeprazole on refractory histological esophagitis in pediatric patients. Eighteen patients with histological evidence of esophagitis and recurrent symptoms despite therapy with H2-receptor antagonists and prokinetic agents were prospectively treated with omeprazole. Dosing was adjusted by monitoring intragastric pH, and esophagoscopy was repeated after 8-12 weeks of omeprazole treatment. Two patients did not complete the study due to either worsening symptoms or hypergastrinemia. Of the remaining patients, 76% were asymptomatic with omeprazole treatment and 24% reported improvement in their symptoms. Approximately 40% demonstrated complete histological healing of their esophagitis. Three patients (17%) had persistent elevations in serum gastrin levels while on omeprazole treatment, which was associated with both younger patient age and higher omeprazole dosing; however, all elevated gastrin levels returned to normal after discontinuation of the medication. All patients had recurrence of their symptoms after completing a course of omeprazole, even patients with complete histological healing. Omeprazole is efficacious in treating children with esophagitis refractory to H2-receptor antagonist and prokinetic agents. However, none of the patients were able to discontinue acid suppressive therapy even after documented healing of their esophagitis.     

CD11b/CD18 mediates the neutrophil chemotactic activity of fibrin degradation product D domain

Coagulation and fibrinolysis universally accompany tissue injury and repair. The accumulation of regionally generated fibrin degradation products (FDP) may modify the local inflammatory response. We have found FDP to be potent neutrophil chemotaxins. We separated plasmin FDP by chromatofocusing and found chemotactic activity limited to fractions containing the fibrinogen D domain (D-D dimer and D monomer). The bioactivity of the D-D dimer did not require an intact cross link site as removal of this sequence with puff adder venom or hypocalcemic plasmic digestion did not decrease chemotaxis. Peptide inhibition studies confirmed that the chemotactic region did not involve terminal gamma chain sequences or alpha chain RGD motifs. The internal gamma chain peptide KYGWTVFQKRLDGSV (P1), known to bind CD11b/CD18, exhibited concentration dependent chemotactic activity. Similarly, monoclonal antibodies directed against CD11b/CD18 blocked PMN migration to FDP without similar inhibition of chemotaxis to IL-8 or LTB4. Thus, neutrophil chemotaxis to FDP is mediated by interactions between the fibrinogen D domain and CD11b/CD18.     

Reversible inactivation of purified leukocyte integrin CR3 (CD11b/CD18, alpha m beta 2) by removal of divalent cations from a cryptic site

Integrins exhibit reversible changes in their ability to bind ligands and these changes enable transient cell adhesion. We recently showed that leukocyte integrin CR3 (complement receptor type three, CD11b/CD18, alpha m beta 2) may be purified in a form that is either capable or incapable of binding soluble, monomeric ligand and that "inactive" CR3 may be rendered capable of binding ligand by addition of an anti-CR3 mAb known as KIM-127 (Cai and Wright, JBC. 270: 14358, 1995). Here, we demonstrate that active CR3 may be rendered inactive by treatment of immobilized receptor with EDTA. EDTA-treated CR3 failed to bind ligand even in the presence of mM Ca2+ and Mg2+, suggesting that EDTA-treatment caused a change in the receptor that is not readily reversed. EDTA-treated receptor did, however, bind ligand upon addition of KIM-127 plus Mg2+ with an affinity (17.8 +/- 4.5 nM) similar to untreated, active receptor (12.5 +/- 4.7 nM). EDTA-treated CR3 thus exhibits the properties of inactive CR3, in which the ligand binding site is cryptic but subject to exposure by KIM-127. A candidate for the cryptic ligand binding site is the I-domain, a Mg2+-binding region in the alpha chain of CR3. We found that monomeric C3bi binds directly to recombinant I-domain in a Mg(2+)-dependent fashion with an affinity of 300 +/- 113 nM. These results thus suggest that CR3 may be inactivated by removing tightly bound divalent cation from a cryptic site in CR3.     

Cooperation between CD44 and LFA-1/CD11a adhesion receptors in lymphokine-activated killer cell cytotoxicity

IL-2-activated NK cells exhibit cytotoxic activity against a wide variety of tumor cells in a non-MHC-restricted fashion and in the absence of prior sensitization. The molecular mechanisms that regulate the cytotoxicity and attachment of activated killer cells to tumor target cells are not known. We provide genetic evidence in CD44(-/-) and LFA-1(-/-) mice that the cell adhesion receptors LFA-1 and CD44 regulate the cytotoxic activity of IL-2-activated NK cells against a variety of different tumor cells. This defect in cytotoxicity was significantly enhanced in mice that carried a double mutation of both CD44 and LFA-1. In vitro differentiation, TNF-alpha and IFN-gamma production, and expression of the cytolytic effector molecules perforin and Fas-L were comparable among IL-2-activated NK cells from LFA-1(-/-), CD44(-/-), CD44(-/-)LFA-1(-/-), and control mice. However, CD44(-/-), LFA-1(-/-), and CD44(-/-)LFA-1(-/-) IL-2-activated NK cells showed impaired binding and conjugate formation with target cells. We also show that hyaluronic acid is the principal ligand on tumor cells for CD44-mediated cytotoxicity of IL-2-activated NK cells. These results provide the first genetic evidence of the role of adhesion receptors in IL-2-activated NK killing. These data also indicate that distinct adhesion receptors cooperate to mediate binding between effector and target cells required for the initiation of "natural" cytotoxicity.