A monoclonal antibody-defined adhesion-related antigen on bovine neutrophils is required for neutrophil aggregation

Surface adhesion molecules present on human leukocytes are known to regulate certain adhesion-related events, such as adhesion to endothelium, extravasation, and aggregation. We have used a mouse anti-human monoclonal antibody designated 60.3 (MAb 60.3) and indirect immunofluorescence technique to identify an antigen on bovine neutrophils (PMNs). MAb 60.3 bound to resting and stimulated bovine PMN in a surface-oriented pattern. Immunofluorescence flow cytometric analysis indicated that wanning the PMNs from 4C to 37C slightly increased (13.9%) expression of the antigen recognized by MAb 60.3. Zymosan-activated serum (ZAS, 10%) increased antigen expression by 12.4% over those PMNs in buffer alone, and phorbol 12-myristate 13-acetate (PMA; 100 ng/ml) by 65.6%. Bacterial lipopolysaccharide (LPS; 1g/ml) fromE. coli 0111:B4 did not enhance antigen expression. The functional nature of this antigen was demonstrated by use of MAb 60.3 and PMN aggregation. Preincubation of bovine PMN with MAb 60.3 for 10 min resulted in nearly complete inhibition of PMN-PMN aggregation upon subsequent stimulation with PMA (100 ng/ml); preincubation with a control antibody did not inhibit aggregation. These results indicate that bovine PMNs possess surface molecule(s) that may function in adhesion-related events, and surface expression may be enhanced by PMN stimulation.     

CD1 1b/CD18-Dependent Polymorphonuclear Leucocyte Interaction with Matrix Proteins in Adhesion and Migration

Adhesion of human polymorphonuclear leucocytes (PMN) stimulated with phorbol myristate acetate (PMA) to plastic dishes coated with the matrix proteins laminin (LM), libronectin (FN), collagen type I (CI) or collagen type IV (CIV) was inhibited by the monoclonal antibody 60.3 (MoAb 60.3; anti-CD18). The highest inhibitory effect was seen on adhesion to CI. PMN adhesion to CI was also effectively inhibited by Mol (anti-CD11b) but this antibody had only a minor effect on attachment of PMN to the other matrix proteins. In other experiments MoAb 60.3 inhibited LTB₄-induccd migration of PMN through polycarbonate filters (3 μm pores) coated with LM, FN, CI or CIV, with the most pronounced effect on migration through those filters coated with CI. By contrast, the antibody Mol had no effect on migration through any of the protein-coated filters tested. The results in this study suggest that the CD18 epitope, recognized by 60.3, mediates both adhesion and migration of PMN while theepitopeonCDl 1b recognized by the antibody Mol is restricted to adhesion. The results also indicate that CD11b/CD18 is the major receptor on human PMN for CI while interaction with LM, FN and CIV may in addition involve other mechanisms.     

Adhesion of polymorphonuclear cells to human endothelial cells. Adhesion-molecule-dependent, and Fc receptor-mediated adhesion-molecule-independent mechanisms.

Activation of human umbilical vein endothelial (HUVE) cells with the inflammatory mediators tumour necrosis factor-alpha (TNF), interleukin-1 (IL-1), lipopolysaccharide (LPS) and phorbol esters enhanced their adhesiveness for leucocytes. The appearance of an activation antigen ELAM-1, recognized by a monoclonal antibody (MoAb) ENA1, parallels the kinetics of the enhanced adherence of leucocytes to endothelial cells. Adhesion of polymorphonuclear cells (PMN) to activated HUVE cells could be blocked by F(ab')2 fragments of MoAb ENA1 up to 60%. An additive inhibition of the adhesion was established by pre-incubation of the PMN with anti-CD18 MoAb and/or leucocyte adhesion inhibitor (LAI), produced by endothelial cells. An opposite reaction, however, was observed when HUVE cells were pre-incubated with intact MoAb ENA1, resulting in an enhancement of the adhesion up to 200%. Apparently, the blocking effect of MoAb ENA1 could be bypassed by the strong interaction of the Fc part of the MoAb with the Fc receptor (FcR) on the PMN. Similarly, anti-CD18 MoAb and/or LAI reduced the adhesion observed if intact ENA1 were used, and Fc-FcR interaction took place. The results presented in this study indicate that adhesion via ELAM-1, the CD18 antigen and via the receptor for LAI are different mechanisms. These mechanisms may act in concert to strengthen the binding of PMN to HUVE cells. Moreover, a strong adhesion could be established via the Fc part of MoAbs directed against HUVE cells with the FcR on the PMN. The phenomenon described may play a role in graft rejection and in diseases where antibodies directed against endothelium are involved.     

Multiple receptors on human monocytes are involved in adhesion to cultured human endothelial cells

Monocytes exhibit significant basal (unstimulated) adherence to human umbilical vein endothelium (HUVE), which is only partially inhibited by an anti-CD18 monoclonal antibody (mAb) (60.3). We examined factors modulating the residual, CD18-independent monocyte binding to HUVE by pretreating monocytes with mAb 60.3 to eliminate CD18-dependent binding. Basal adherence was reduced from 32% +/- 2% to 14% +/- 2% with mAb 60.3 (means +/- SE of eight experiments; P less than 0.01). mAb 60.3-treated monocytes were incubated with tumor necrosis factor-gamma (TNF-alpha), interleukin-1 (IL-1), lipopolysaccharide (LPS), N-formylmethionyl-leucyl-phenylalamine (FMLP), or phorbol myristate acetate (PMA). Only PMA affected CD18-independent binding. Pretreatment with PMA alone reduced adherence to 21% +/- 2% (mean +/- SE of eight experiments; P less than 0.01). In conjunction with mAb 60.3, PMA virtually eliminated monocyte adherence to HUVE (7% +/- 1%, mean +/- SE of eight experiments; P less than 0.01). We also examined CD18-independent monocyte binding to endothelial-leukocyte adhesion molecules (E-LAMs) induced by pretreatment of HUVE with LPS. Monoclonal antibody 60.3-treated monocytes increased adherence from 14% +/- 2% with unstimulated HUVE to 37% +/- 2% with LPS-stimulated HUVE (mean +/- SE of four experiments; P less than 0.01). Monocytes pretreated with both mAb 60.3 and PMA increased adherence from 5% +/- 1% with the unstimulated HUVE to 18% +/- 1% with the LPS-stimulated HUVE (mean +/- SE of four experiments; P less than 0.01). This result implies the presence of a CD18-independent and PMA-insensitive receptor on human monocytes for an E-LAM induced by LPS. In summary, we have identified two CD18-independent mechanisms of monocyte adherence to HUVE; a PMA-sensitive mechanism mediating basal adherence and a PMA-insensitive mechanism involved in binding to E-LAMs.     

Inhibition of pulmonary neutrophil trafficking during endotoxemia is dependent on the stimulus for migration

In rat models of Gram-negative pneumonia, pulmonary emigration of neutrophils (polymorphonuclear leukocytes [PMNs]) is blocked when rats are made endotoxemic by an intravenous administration of endotoxin (lipopolysaccharide [LPS]). To test whether dysfunctional PMN migratory responses in the endotoxemic rat are specific for airway endotoxin, we gave rats intrapulmonary stimuli known to elicit different adhesion pathways for pulmonary PMN migration. Sprague-Dawley rats were treated intravenously with either saline or LPS and then instilled intratracheally with either sterile saline, LPS from Escherichia coli, interleukin (IL)-1, hydrochloric acid (HCl), zymosan-activated serum (ZAS), or lipoteichoic acid (LTA). Three hours later, accumulation of PMNs and protein in bronchoalveolar lavage fluid (BALF) were assessed. BALF PMN accumulation in response to intratracheal treatment with LPS (100%), IL-1 (100%), ZAS (40%), and LTA (58%) was inhibited by endotoxemia. In rats given intratracheal HCl, BALF PMN numbers were unaffected by intravenous LPS. The pattern of inhibition of migration suggests that intravenous LPS only inhibits migration in response to stimuli for which migration is CD18-dependent. In contrast to PMN migration, BALF protein accumulation was inhibited by intravenous LPS only when IL-1 or LPS was used as the intratracheal stimulus. To characterize further the differential responses to the various airway stimuli, the appearance in BALF of tumor necrosis factor-alpha (TNF-alpha) and the PMN chemokine macrophage inflammatory protein (MIP)-2 was measured. Accumulation of PMNs in BALF correlated with the BALF concentrations of MIP-2 (r = 0.846, P < 0.05) and TNF (r = 0.911; P < 0.05). The ability of intravenous LPS to inhibit pulmonary PMN migration correlated weakly with MIP-2 (r = 0.659; P < 0.05) and with TNF (r = 0.413; P > 0.05) concentrations in BALF. However, this correlation was strengthened for TNF (r = 0.752; P < 0.05) when data from IL-1-treated animals were excluded. Thus, the presence in BALF of inflammatory mediators that are known to promote CD18-mediated migration correlates with endotoxemia-related inhibition of PMN migration. Furthermore, the pattern of inhibition of pulmonary PMN migration during endotoxemia is consistent with the CD18 requirement of each migratory stimulus.     

Human polymorphonuclear leucocytes stimulated by tumour necrosis factor-alpha show increased adherence to extracellular matrix proteins which is mediated via the CD11b/18 complex.

The present study demonstrates that tumour necrosis factor (TNF) and FMLP, but not IL-1 or IL-8, enhanced the adherence of polymorphonuclear neutrophil (PMN) to fibronectin, an extracellular matrix protein. The adherence induced by FMLP was very rapid, within 5 min while the induction of adherence by TNF was much slower, reaching maximum at 60 min. TNF also enhanced an adhesion of PMN to other extracellular matrix proteins, such as laminin, collagen IV and gelatin II, but not to human serum albumin. Anti-CD18 MoAb completely inhibited the binding of TNF-stimulated PMN to fibronectin and partially inhibited the binding to laminin. Further investigation showed that adhesion of TNF-stimulated PMN to fibronectin and laminin was inhibited by anti-CD11b MoAb and to a lesser extent by CD11a MoAb. In contrast to TNF-stimulated PMN the binding of unstimulated PMN to fibronectin and laminin was only inhibited by anti-CD11a MoAb. Anti-CD11c had no effect on PMN adherence. These results suggest that unstimulated PMN adhere to extracellular proteins through the CD11a/18, while TNF-stimulated PMN adhere through the CD11b/18. These results suggest that TNF secreted at the site of inflammation may enhance the interaction of PMN with the extravascular environment through the CD11b/18 complex.     

Diminished priming of neonatal polymorphonuclear leukocytes by lipopolysaccharide is associated with reduced CD14 expression.

Previous research in our laboratory has shown that polymorphonuclear leukocytes (PMN) from neonates are not primed effectively in vitro with lipopolysaccharide (LPS) (from Escherichia coli 0111:B4) compared with priming of adult PMN. This finding led us to speculate that differences between neonatal and adult LPS receptors may account for the lower response by neonatal PMN to LPS. In these experiments, we investigated if CD14 or other LPS receptors contribute to the priming activity of PMN by LPS. We found that unprimed neonatal and adult PMN expressed similar numbers of CD14 (11.6 +/- 9.2 versus 18.6 +/- 2.7 fluorescence units [FlU]; P > 0.05) and LPS-binding sites (2.94 +/- 1.4 versus 4.94 +/- 0.79 FlU; P > 0.05). Monoclonal antibody against CD14 (MY4) did not significantly change the binding of LPS to adult unprimed PMN, suggesting that LPS receptors other than CD14 receptors are predominant on PMN. However, when PMN were pretreated with LPS (10 ng/ml) for 45 min at 37 degrees C, expression of CD14 on adult PMN increased to 33.8 +/- 4.9 FlU (P < 0.05 versus unprimed adult PMN) while that on neonatal PMN showed little change, increasing to 17.2 +/- 10.3 FlU (P > 0.05 versus unprimed neonatal PMN; P < 0.05 versus primed adult PMN). Furthermore, MY4 specifically blocked oxidative-radical production from PMN primed with LPS (10 ng/ml) compared with that from control PMN (P < 0.01). These studies suggest that LPS primes PMN by activating CD14 expression. We conclude that lower expression of CD14 or failure to up-regulate CD14 after LPS pretreatment contributes to the inability of neonatal PMN to be primed by LPS.     

Functional Fc-receptor defect of polymorphonuclear leukocytes in a patient with Sjögren's syndrome

Summary We investigated polymorphonuclear leukocyte (PMN) function in a 50-year-old woman with primary Sjögren's syndrome (SS). The respiratory burst of PMN was monitored by luminolenhanced chemiluminescence using zymosan, opsonized zymosan, zymosan-activated serum, and phorbol-myristate-acetate, as well as serial dilutions of aggregated immunoglobulin (aggIg) as Fcreceptor (FcR) stimulus. The effects of serum on the chemiluminescent response as well as the binding of aggIg to PMN were also compared. We found the patient's PMN not to respond to stimulation by aggIg, only the highest concentration (> 180 µg/ml) induced a marginal chemiluminescent response in the patient. By contrast, incubation of the patient's PMN with other stimuli resulted in responses similar to those in a healthy control. Binding of aggIg to PMN was higher in the patient (3.6% vs 1.5% of the radioactivity added in the control). Sera of patient and control induced similar chemiluminescence on PMN as did that of another human serum. Our data indicate a selective functional FcR defect of PMN despite unimpaired binding of aggIg in a patient with SS.Abkürzungsverzeichnis aggIg Aggregated immunoglobulin - DHD Dermatitis herpetiformis Duhring - FcR Fc-receptor - OpsZ Opsonized zymosan - PMA Phorbol-myristate-acetate - PMN Polymorphonuclear leukocytes - SS Sjögren's syndrome - SLE Systemic lupus erythematosus - ZAS Zymosan-activated serum - Z Zymosan Supported by grant Scho/273-2, Deutsche Forschungsgemeinschaft     

Direct and indirect effects ofE. Coli lipopolysaccharide on isolated human polymorphonuclear granulocytes and mixed leukocytes

Polymorphonuclear neutrophil granulocytes (PMN) may contribute to the lung injury induced by nonpulmonary infections with gram-negative bacteria. The direct effect ofE. coli lipopolysaccharide (LPS) on isolated human PMN or mixed leukocytes (ML), as well as the priming effect of preincubating cells with LPS, was examined in assays measuring the maximal rate of oxygen consumption (OC), cell chemiluminescence (CHML), and aggregation (AGG). LPS, 1–10/ml, caused no acute response in PMN or ML suspended in Fisher's-HEPES medium with BSA (FHA), but increased both CHML and AGG of cells suspended in autologous plasma. Preincubation in FHA with LPS, 1g/ml, for more than 15 min increased the OC of PMN activated with zymosan-activated plasma (ZAP) orn-formyl-methionyl-leucyl-phenylalanine (FMLP) by more than 100%. A similar increase in the CHML of such cells was seen after FMLP, but not after ZAP. ZAP, however, primed the CHML response of the cells to subsequent activation with FMLP more than did preincubation with LPS. Previous exposure to both agents had an additive effect. Preincubation of PMN with LPS decreased the time interval from addition of phorbol myristate acetate (PMA) to peak OC response, but less so than previous activation with FMLP. Neither agent affected the maximal rate of OC after addition of PMA. LPS also increased the PMN aggregation induced by ZAP and FMLP, but not by PMA. Cells preincubated with LPS, 0.01 g/ml, increased their CHML in response to FMLP if suspended in Krebs-Ringer balanced salt solution, but not if suspended in FHA. Such preincubation had no effect on OC of similarly activated cells in any of the media.     

In vitro stimulation of polymorphonuclear cell adhesion by ribomunyl and antibiotic + ribomunyl combinations: Effects on CD18, CD35 and CD16 expression

Several functions of polymorphonuclear cells (PMNs) require adhesion to occur. Various membrane proteins' functions such as CD18 (β₂ chain of integrin), CD35 (CR1) and CD16 (F c γ Receptor III) participate in adhesion. In vivo treatment with Ribomunyl (R), an immunomodulating agent, was shown to enhance adhesion and migration of PMNs. To explore the direct effect of R on PMNs, cells from healthy subjects were treated in vitro with R. A significant increase of PMN adhesion and expression of CD18 and CD35 molecules were observed with 50 and 100 μg/ml of R after 2 h incubation. However, R-treatment decreased the PMN reactivity towards anti-CD16 (F c γ RIII) monoclonal antibody. The effect of R on adhesion and membrane molecule expression was independent of the presence of serum and of polymixin B. Thus, this effect cannot be due to lipopolysaccharide (LPS) contaminants and does not require interactions with serum components.In previous studies, it was shown that in vitro amoxicillin increased some PMN functions whereas josamycin decreased them. The in vitro incubation of PMNs with R and amoxicillin (100 μg/ml) potentiated the positive effect of amoxicillin on adhesion and the antibiotic counterbalanced the negative of R on CD16 expression. In addition, R compensated the negative effect of josamycin (100 μg/ml) on PMN adhesion and on CD18 and CD35 expression.This study indicates: (1) the direct effect of R on PMN adhesion and on expression of molecules involved in adhesive-mediated functions, and (2) the beneficial effect of the association of R with antibiotics which can stimulate PMN activity.     

The inhibitory effect of dexamethasone on lymphocyte adhesion molecule expression and intercellular aggregation is not mediated by lipocortin 1

Glucocorticoids exert their anti-inflammatory activity through multiple pathways which include the inhibition of cell adhesion events. The glucocorticoid-induced protein lipocortin 1 (LC1) has reported anti-inflammatory properties and has been proposed as a putative mediator of the anti-inflammatory effects of glucocorticoids. The role of LC1 in mediating the glucocorticoid inhibition of lymphocyte adhesion and cell adhesion molecule (CAM) expression was investigated in vitro using a microaggregation assay, flow cytometry and confocal microscopy. Lymphocytes stimulated for 96 h with plastic-bound OKT3 antibody showed significant increases in LFA-1 and CD2 expression. Dexamethasone (DEX; 10(-6) M) inhibited this increase but the neutralizing anti-LC1 MoAb 1A (5 microg/ml) failed to reverse the DEX effect; neither was purified human LC1 (50 x 10(-9) M) able to inhibit CAM expression. The biological activity of the LC1 was confirmed by its ability to suppress monocyte phagocytosis and respiratory burst in response to bovine serum albumin (BSA)-anti-BSA complexes. OKT3 stimulation of cultured mononuclear cells resulted in intercellular aggregation, scored microscopically using a visual index. This aggregation was completely reversed by 10-6 M DEX but unaffected by LC1 (50 x 10(-9) M). Significant intracellular expression of lymphocyte LC1 was observed using the anti-LC1 MoAb 1B in saponin-permeabilized cells. Distribution of LC1 had a diffuse, cytoplasmic pattern. LC1 expression was reduced following 3 h treatment with 10(-6) M DEX. These findings indicate that the DEX effects on lymphocyte adhesion and CAM expression are not mediated by LC1. Thus the reported in vivo effects of LC1 on leucocyte adhesion and transmigration probably occur through functional/conformation changes of surface CAM, rather than by alteration in expression.     

Leukotriene B4-induced hyperadhesiveness of endothelial cells for neutrophils: relation to CD54.

Leukotriene B4 (LTB4) induced an in vitro transient state of hyperadhesiveness in cultured human umbilical vein endothelial cells (HUVEC), leading to a 2.2-fold increase in the binding of neutrophil granulocytes (PMN), which was less than that conferred by platelet activating factor (PAF) though more than thrombin did (3.4- or 2.0-fold increases, respectively). This study concerns the role of the adhesive molecules CD18 and CD54 for the LTB4- (as well as thrombin- and PAF-) induced endothelial hyperadhesiveness. The MoAbs 60.3 (to the CD18 molecule on PMN) and 84H10 (to one epitope of CD54 on the HUVEC) blocked the adherence of PMN to LTB4-treated HUVEC, whereas MoAb LB-2 (directed at another CD54 epitope) failed to do so. MoAb 84H10 blocked 43% of the thrombin-induced hyperadhesiveness, whereas the PAF response was unaffected. Thus, LTB4-induced HUVEC hyperadhesiveness may therefore be related to a specific domain on the CD54 (or on an antigenically related molecule) as well as being dependent on CD18, whereas the involvement of CD54 was much less or non-existent for the thrombin and PAF responses, respectively.     

Role of tumour necrosis factor and reactive oxygen intermediates in lipopolysaccharide-induced pulmonary oedema and lethality.

The purpose of this study was to characterize the role of tumour necrosis factor (TNF) and neutrophils (PMN) in the pathogenesis of pulmonary oedema induced by endotoxin (lipopolysaccharide (LPS)). Intraperitoneal administration to BALB/c mice of 0.6-1 mg of LPS caused pulmonary oedema and lethality. This was associated with production of TNF in serum and bronchoalveolar lavage fluid and with accumulation of PMN in the lung. In this experimental model, we could block TNF production by different means: pretreatment 30 min before LPS with 4 mg/kg of i.p. chlorpromazine (CPZ), 3 mg/kg of i.p. dexamethasone (DEX), 1 g/kg p.o. of N-acetylcysteine (NAC, an antioxidant precursor of glutathione), or an anti-TNF MoAb. CPZ, DEX and anti-TNF completely prevented LPS lethality but not pulmonary oedema or pulmonary PMN infiltration, indicating that: (i) lung oedema is not the main cause of death after LPS; and (ii) lung oedema induced by LPS is not mediated by TNF. Pretreatment with NAC not only inhibited TNF production but also protected against LPS-induced pulmonary oedema, indicating that reactive oxygen intermediates are implicated. NAC also blocked TNF production in blood and in bronchoalveolar lavage. We also tested the effect of PMN depletion induced with cyclophosphamide (CP) or 5-fluorouracil (5-FU). While no pulmonary PMN infiltrate was observed in PMN-depleted mice, neutropenia did not prevent LPS lethality or oedema, indicating PMN do not play an important role in the toxic effects of LPS in this experimental model.     

The contribution of LFA-1 (CD11a/CD18) and MAC-1 (CD11b/CD18) to the in vivo migration of polymorphonuclear leucocytes to inflammatory reactions in the rat.

Recently the critical requirement for the CD18 family of adhesion molecules on leucocytes for their adhesion and migration to inflammatory reactions has been recognized in humans and several animal models. The in vivo studies have mostly utilized antibodies to CD18, the common beta-subunit of CD11a,b,c/CD18 molecules and thus have blocked the function of all three family members, making evaluation of the role of individual subunits impossible. Furthermore, none of the reagents used were suitable for studies in rats. Here we report the effects on polymorphonuclear leucocyte (PMNL) adhesion and in vivo migration of a new monoclonal antibody (mAb) TA3, which recognizes and blocks rat CD11a/CD18 (LFA-1). These studies also evaluated mAb MRC OX42, which reacts with rat CD11b/CD18 (CR3, MAC-1). Neither antibody alone inhibited rat PMNL adhesion to interleukin-1 (IL-1)-activated rat endothelium, but the combination inhibited adhesion by 44%. OX42 treatment of rat PMNL inhibited phorbol myristate acetate (PMA) activated adhesion by 88%, while TA3 only inhibited this adhesion in combination with OX42, resulting in 99% inhibition of PMA-induced PMNL adhesion. Treatment of rats with TA3 alone partially inhibited 51Cr-labelled rat blood PMNL migration into zymosan-activated serum (C5adesArg; ZAS), but not IL-1, or endotoxin [lipopolysaccharide (LPS)] induced dermal inflammatory reactions. MAb OX42 had no such effect in vivo. However, treatment with both antibodies virtually eliminated any PMNL accumulation in all three types of inflammatory reactions. Ex vivo treatment of the 51Cr-labelled PMNL, prior to i.v. infusion showed that mAb TA3 again preferentially inhibited PMNL migration to ZAS. These results suggest that in the rat, CD11a/CD18 plays a major role in PMNL migration to C5a and that either CD11a or CD11b/CD18 can function to maintain normal PMNL migration to IL-1 or LPS dermal inflammatory reactions. More than one member of this adhesion family or their ligands may need to be targeted for effective modulation of PMNL infiltration, at least in this species.     

Rapid Adhesive Responses of Endothelial Cells and of Neutrophils Induced by Leukotriene B4 are Mediated by Leucocytic Adhesion Protein CD18

Controversy has existed as to the ability of leukotriene B4 (LTB4) to enhance adhesive properties of human neutrophils (PMN) and endothelial cells. We found that LTB4 induced a rapid but transient adhesion of PMN to an albumin-coated plastic surface and to cultured human umbilical vein endothelial cells (HUVEC). Although the adhesive response of PMN to the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) was longer lasting, peak hyperadherence was of similar magnitude as to LTB4 and was less susceptible to assay conditions. Adherence induced by either LTB4 or fMLP could be abrogated by the monoclonal antibody 60.3, indicating similar dependence on the leucocyte adhesion protein CD18. Lipoxin A did not induce PMN hyperadherence. Treating HUVEC with LTB4, but not with its omega-oxidized metabolites 20-OH- and 20-COOH-LTB4, lipoxin A, or with fMLP conferred a rapid, dose-related, enhanced adhesion of PMN. This effect was dependent on CD18 and on divalent cations. It disappeared with prolonged exposure to LTB4, required a metabolically active HUVEC, and was not due to passive binding of LTB4 to HUVEC. Thus, LTB4 induces a transient expression of hyperadhesiveness in HUVEC as well as in neutrophils, and both effects are dependent on expression of CD18.     

LPS的直接诱导对肺微血管内皮细胞ICAM-1的表达及对PMN黏附作用影响的研究

目的探讨内素毒(Endotoxin LPS)的直接诱导作用对肺微血管内皮细胞(PMVEC)ICAM-1表达的影响及诱导发生的PMVEC对多形核中性粒细胞(PMN)黏附作用的影响。方法100ng/m l LPS刺激PMVEC 0、2、4、6、8 h或10、50、100 ng/m l LPS刺激6 h,同时检测PMVEC ICAM-1的表达(免疫细胞化学法)、PMVEC-PMN黏附率及抗ICAM-1抗体对黏附作用的影响,并进行PMVEC与PMN黏附作用的扫描电镜观察。结果LPS的直接刺激可诱导PMVEC ICAM-1的表达增加,且表现为随LPS刺激的时间、剂量增加而增加;同时LPS直接刺激也促进了PMVEC对PMN的黏附率增加,其变化在时间与方式上几乎与ICAM-1的表达增加同步。Anti-ICAM-1抗体可以显著地抑制LPS诱导的PMVEC-PMN黏附(P<0.01)。扫描电镜可以直观地显示PMVEC-PMN黏附的超微结构表现,并且首次通过这种方式观察到了“间接系链”现象的存在。结论表明细菌致病因子LPS的直接诱导可以促进PMVEC表达ICAM-1,从而为PMVEC-PMN的黏附提供物质基础,而“间接系链”的发生更扩大和巩固了黏附效果。     

Porins and lipopolysaccharide (LPS) from Salmonella typhimurium induce leucocyte transmigration through human endothelial cells in vitro.

Bacteria or bacterial products may constitute important inducers of surface molecule expression on endothelial cells and leucocytes. This study was undertaken to determine the effects of the Salmonella typhimurium porins, LPS-S and LPS-R on the transendothelial migration of leucocytes through human umbilical vein endothelial cells (HUVEC). Treatment of the HUVEC with either porins or LPS-S or LPS-R increased the transmigration of different leucocyte populations, in particular that of neutrophils. The maximal increase occurred using LPS-S treatment, whereas porin stimulation fell between LPS-S and LPS-R. The transmigration increase was dose-dependent and reached its maximum at about 100-1000 ng/ml of stimulus. Optimal endothelial activation occurred after 2-4 h and 4-6 h using LPS and porin, respectively. Stimulation of leucocytes with either porins or LPS slightly increased their transmigration through non-activated endothelial cells. Transmigration increased remarkably during the simultaneous stimulation of endothelial cells by IL-1ss together with either porins or LPS. To assess participation of E-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and leucocyte adhesion complex (CD11/18) in porin- or LPS-mediated leucocyte migration, blocking MoAbs were used. Each blocking MoAb partially and selectively decreased leucocyte transmigration. The obtained results contribute to clarify some aspects of the inflammatory process at sites of infection.     

M5, a phosphoinositol-linked human myelomonocytic activation-associated antigen.

When investigating the previously described monoclonal antibody (MoAb) VIM-5, raised against THP1 cells and binding to human monocytes and granulocytes, we found that the antigen detected by this antibody, designated M5, becomes very strongly expressed on monocytes after overnight culture with phorbol myristate acetate (PMA) or lipopolysaccharide (LPS) but not with recombinant human interferon-gamma (rhIFN-gamma). Granulocytes stimulated with formyl-methionyl-leucyl-phenylalanine (FMLP) become negative for binding VIM-5. Immature granulocytes from bone marrow do not express M5, thus its expression on granulocytes is differentiation linked. The antigen bound by VIM-5 is sensitive to hydrolysis by phosphoinositol-specific phospholipase C (PI-PLC). The immunoprecipitated M5 antigen on monocytes is a broad band, with a peak of 50 kD (unreduced) and two bands of 53 kD and 44 kD (reduced). We have therefore detected an antigen that is upregulated on stimulated monocytes but, conversely, down-regulated on FMLP-stimulated granulocytes.     

Lipopolysaccharide binding proteins on polymorphonuclear leukocytes: comparison of adult and neonatal cells.

We have previously shown that polymorphonuclear leukocytes (PMN) from cord blood of normal full-term infants have a decreased priming response to lipopolysaccharide (LPS) compared with PMN of adults. Because the reason for this difference is poorly understood, we compared LPS binding on PMN from adults and newborns by using a photoactivatable iodinated LPS (from Escherichia coli O111:B4), coupled to 2-(p-azidosalicylamido)-1,3'-dithopropionate (LPS-ASD) to covalently link LPS to the PMN membrane. We incubated 2 x 10(4) adult or neonatal PMN with 125I-ASD-LPS (100 ng/ml) together with unlabelled LPS (0 to 100,000 ng/ml) for 20 min at 4 degrees C. The maximum total 125I-ASD-LPS binding to newborn PMN (1,004 +/- 103 cpm) was lower than that binding to adult PMN (3,583 +/- 444 cpm; P < 0.01 with respect to newborn PMN). However, the concentration of unlabelled LPS that displaced 50% of the maximum specifically bound 125I-ASD-LPS was similar for PMN from adult and newborn infants (-4.85 +/- 0.04 and -5.13 +/- 0.14 log g of LPS per ml, respectively; P > 0.05). We further assessed the membrane binding of 125I-ASD-LPS to PMN by using membrane extracts analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. LPS binding proteins were found at approximately 73, 55 to 57, and 25 kDa in both adult and neonatal PMN. However, PMN from newborn infants had markedly lower membrane-associated 125I-ASD-LPS at the 55- to 57- and 25-kDa protein bands as indicated by the intensity of the autoradiograph. Binding of LPS at these bands was specific for the lipid A portion of LPS, since purified unlabelled lipid A displaced 125I-ASD-LPS in a dose-dependent manner. Thus, PMN from newborn infants bind less LPS than do PMN from adults, even though the sites for LPS membrane binding appear to be the same.     

Cytomegalovirus induced PMN adherence in relation to an ELAM-1 antigen present on infected endothelial cell monolayers.

In human umbilical vein endothelial cells infected with cytomegalovirus (CMV), an activation antigen recognized by monoclonal antibody (mAb) ENA1 appeared. mAb ENA1 reacts with an inducible endothelial surface antigen which has characteristics similar to those of ELAM-1. Incubation with anti-IL-1 partly inhibited this appearance and, parallel to this, the virus-induced polymorphonuclear cell (PMN) adhesion was decreased. In addition, the adhesion of PMN to virus-infected endothelial cells could be reduced by F(ab)2 fragments of mAb ENA1 to almost control level. The results obtained after incubation of PMN with mAb IB4 (against CD18) suggest that the adhesion of PMN to uninfected endothelial cells is CD18 glycoprotein dependent, and virus infection up-regulates this glycoprotein-dependent mechanism. These results indicate that the virus-induced PMN adhesion is regulated by the following mechanism: virus infection of endothelial cells induces IL-1 production, and the autocrine IL-1 causes the expression of ELAM-1 on the surface of endothelial cells. In turn this activation antigen ELAM-1 binds with its putative ligand present on the PMN membrane. The virus-induced PMN adhesion occurs also through a CD18 glycoprotein-dependent mechanism.