Circulating antiphospholipid antibodies (aPL) are associated with a syndrome of thrombosis, recurrent fetal loss, and thrombocytopenia. We have demonstrated the activation of cultured human umbilical vein endothelial cells (HUVEC) by IgG from patients with anticardiolipin antibodies (aCL).Incubation of HUVEC for 4 h with purified IgG (100 jig/ ml) from patients with high-titer aCL induced a 2*3-fold increase in monocyte adhesion over that seen in HUVEC incubated with IgG's from normal subjects. The effect of aCL was not attributable to LPS contamination, Fc receptors, or immune complexes. Monocyte adhesion was not induced when the aCL were added in serum-free media but was restored by the addition of purified (32GP1, previously described as a necessary cofactor for aCL reactivity. Purified rabbit polyclonal IgG raised against f2GP1 also induced monocyte adhesion when incubated with HUVEC. Preadsorption of patient serum with cardiolipin reduced monocyte adhesion by 60%. Immunofluorescent microscopy demonstrated that endothelial cells incubated with patient IgG expressed cell adhesion molecules, including E-selectin, vascular cell adhesion molecule-i, and intracellular adhesion molecule-i. These data support the hypothesis that aPL activate vascular endothelial cells, thereby leading to a prothrombotic state. (J. Clin. Invest 1995. 96:2211-2219

Neutrophil-activating protein 1/interleukin 8 (NAP-1/IL-8)' was first identified as a 72 amino acid peptide secreted by monocytes in response to bacterial LPS, with the properties of activating and attracting polymorphonuclear leukocytes (PMN) in vitro (1). Subsequently several different types ofcells, including macrophages and endothelial cells, were found to synthesize NAP-1/IL-8 upon stimulation with TNF or IL-1. Sequence data indicate that NAP-1/IL-8 is homologous to a group of peptides including platelet basic protein, platelet factor 4, and macrophage inflammatory protein 2 . In vivo accumulation of PMN at sites of injection of NAP-1/IL-8 suggests that it may participate in the recruitment of PMN into inflamed tissue, and NAP-1/IL-8 has been identified in skin lesions of patients with psoriasis (1).Several lines of evidence indicate that the CD11/CD18 complex ofleukocyte adhesion receptors may participate in the adhesion of PMN to the vascular endothelium which is necessary for extravasation . mAbs specific for CD18 are effective in blocking the adherence of stimulated PMN to endothelium in vivo (2) and in vitro (3, 4), and patients genetically deficient in the CDII/CD18 complex exhibit poor recruitment of PMN to Rebuck skin windows (5). Here we describe the response of CD11/CD18 on PMN to NAP-1/IL-8, with particular emphasis on the numerically dominant member, CD11b/CD18 . This receptor not only recognizes ligands on endothelium but also binds to complement protein C3bi (6), fibrinogen (7), and LPS (8).

SummaryTwo classes of adhesion molecules have well-defined roles in the attachment of unstimulated polymorphonuclear leukocytes (PMN) to cytokine-treated endothelial cells. Endothelial-leukocyte adhesion molecule 1(ELAM-1) on endothelial cells interacts with specific carbohydrate residues on the PMN, and the leukocyte integrins (CD18 antigens) on PMN interact with intracellular adhesion molecule 1 and other structures on endothelium . Here we show that these two classes of molecules can act sequentially in an "adhesion cascade". Interaction of PMN with SLAM-1-bearing endothelial cells causes PMN to express enhanced adhesive activity of the integrin CR3 (CD11b/CD18) . Expression of ELAM-1 on the cytokine-treated endothelium appears both necessary and sufficient for the stimulation of CR3 activity since blockade of ELAM-1 with mAbs prevents the activation of CR3 by cytokine-treated endothelium, and immobilized recombinant ELAM-1 activates CR3 . The ability to activate CR3 is shared by chemattractants, suggesting that ELAM-1 may serve as a "tethered chemattractants" This hypothesis is strengthened by the observation that recombinant soluble ELAM-1 directs movement of PMN in chemotaxis chambers. These results suggest a mechanism by which multiple adhesive molecules may function together in diapedesis. ELAM-1 serves both as an adhesin and as a trigger that recruits the participation of additional adhesion molecules. Our results also suggest that ligands for adhesion molecules may also be "receptors" capable of generating intracellular signals.

Circulating polymorphonuclear leukocytes (PMN)' exhibit negligible binding affinity for unstimulated vascular endothelium (EC). After intravenous administration ofa chemotactic factor such as C5a, PMN attach to EC as evidenced by a rapid (<1 min) neuropenia (1, 2), but they soon detach and normal levels of circulating PMN are restored in 15-20 min. Thus PMN possess the means oftransiently making then breaking adhesions to EC. Transient adhesion of PMN to EC appears necessary for extravasation in response to a chemotactic signal . A gradient ofchemotactic agent emanating from the surrounding tissues causes PMN first to bind to the lu minal surface of the EC, then to break that adhesion as they move out of the vascular space .There is strong evidence that members of the CD11/CD18 complex of leukocyte receptors mediate adhesion of stimulated PMN to unstimulated EC . mAbs against CDll/CD18 block adhesion of PMN to EC both in vitro (3, 4) and in vivo (5, 6) .In addition, PMN from patients deficient in CDll/CD18 fail to adhere to EC both in vitro (3) and in vivo (7) . The CDll/CD18 molecules are ideal candidates for mediating transient adhesion of PMN to EC in vivo, because their capacity to bind ligands in vitro can be transiently enabled . CD11b/CD18, the most abundant member of the CD11/CD18 complex on PMN, functions as a receptor for surface-bound C3bi (8) . We have previously shown that the ability of this receptor to bind to erythrocytes coated with C3bi is dramatically stimulated by phorbol esters and that this stimulation is short in duration (9) . Here show that CDll/CD18-dependent adhesion of phorbol-treated PMN to EC increases and subsequently decreases with a time course identical with that shown for the effect of phorbol esters on the binding activity of CDllb/CD18 for C3bi . We further show that adhesion of PMN to EC may be transiently stimulated with the physiological mediators TNF or C5a .Expression of CDllb/CD18 on the surface of PMN is increased upon stimulation with chemotactic factors (9-11) . We found that cytoplasts, which are depleted of inThis work was supported by grants AI-22003 and AI-24775 from the U . S . Public Health Service and by a Grant-in-Aid from the American Heart Association/New York City Affiliate . Materials and MethodsReagents . Recombinant human TNF was a gift of Dr. A. Cerami (The Rockefeller University). TNF was stored sterilely at 4'C before use. Human recombinant complement fragment C5a was a gift of Dr. M. Springer (Merck Research Laboratories, Rahway, NJ) and was dissolved in HAP buffer (Dulbeccds PBS containing 0.5 mg/ml human serum albumin, 3 mM glucose, and 0.3 U/ml aprotinin) that contained (10 -4 M) Plumber's inhibitor (DL-2-mercaptomethyl-3-guanidinoethyl-thiopropanoic acid; CalBiochem-Behring Corp., San Diego, CA) to inhibit the activity of carboxypeptidases . Phorbol dibutyrate (PDB) was obtained from Sigma Chemical Co. (St . Louis, MO) . Collagenase was purchased from Worthington Biochemical Corp. (Freehold, NJ) . Human fibronectin was supplied from the New Yor...

Reactive oxygen radicals (ROS) generated by phagocytes promote human polymorphonuclear leukocyte (PMN) adhesion to human umbilical vein endothelial cells (EC). We determined the effects of hydrogen peroxide (H2O2), a phagocyte-derived ROS, on EC adhesiveness by determining steady-state intracellular adhesion molecule 1 (ICAM-1) mRNA and ICAM-1 protein expression. The adhesion of PMN to H2O2-treated EC was concentration dependent with maximal adhesion achieved at 0.1 mM H2O2. PMN adhesion occurred rapidly, reaching its maximum value within a 1-h treatment time. The PMN adhesion was dependent on the interaction between CD11/CD18 integrins on PMN and ICAM-1 on EC, since either anti-CD18 or anti-ICAM-1 monoclonal antibodies (MAbs) inhibited (by > 90%) the adhesive interaction between PMN and EC. In parallel with the increases in EC adhesivity, we detected a two- to threefold increase in EC surface expression of ICAM-1 between 0.5 and 1 h after H2O2 challenge. Northern analysis revealed an increase in the steady-state ICAM-1 mRNA signal within 0.5 h after H2O2 exposure, and the response was sustained up to 2 h. Inhibition of intracellular catalase in H2O2-treated EC by 3-amino-1,2,3-triazole augmented the PMN adhesion by 20%, whereas enhancement of EC H2O2-scavenging activity by addition of catalase abrogated the H2O2-induced PMN adhesion, indicating that oxidant-antioxidant balance at the EC interface is a critical factor modulating PMN-EC adhesive interactions. The results suggest that H2O2-induced PMN adhesion is dependent on the rapid induction of the ICAM-1 mRNA signal and the surface expression of ICAM-1 on EC.(ABSTRACT TRUNCATED AT 250 WORDS)