Transendothelial migration enables subsequent transmigration of neutrophils through underlying pericytes.

During acute inflammation, neutrophil recruitment into extravascular tissue requires neutrophil tethering and rolling on cytokine-activated endothelial cells (ECs), tight adhesion, crawling towards EC junctions and transendothelial migration (TEM). Following TEM, neutrophils must still traverse the subendothelial basement membrane and network of pericytes (PCs). Until recently, the contribution of the PC layer to neutrophil recruitment was largely ignored. Here we analyze human neutrophil interactions with interleukin (IL)-1ß-activated human EC monolayers, PC monolayers and EC/PC bilayers in vitro. Compared to EC, PC support much lower levels of neutrophil binding (54.6% vs. 7.1%, respectively) and transmigration (63.7 vs. 8.8%, respectively) despite comparable levels of IL-8 (CXCL8) synthesis and display. Remarkably, EC/PC bilayers support intermediate levels of transmigration (37.7%). Neutrophil adhesion to both cell types is Mac-1-dependent and while ICAM-1 transduction of PCs increases neutrophil adhesion to (41.4%), it does not increase transmigration through PC monolayers. TEM, which increases neutrophil Mac-1 surface expression, concomitantly increases the ability of neutrophils to traverse PCs (19.2%). These data indicate that contributions from both PCs and ECs must be considered in evaluation of microvasculature function in acute inflammation.

Inflammation-induced secretion of CCL21 in lymphatic endothelium is a key regulator of integrin-mediated dendritic cell transmigration.

Tissue inflammation induces rapid mobilization of antigen-charged dendritic cells (DCs), which migrate to draining lymph nodes via afferent lymphatics to elicit the immune response. This increase in DC trafficking has been shown to require integrin-dependent adhesion to ICAM-1 and VCAM-1, expressed on inflamed lymphatic endothelium. In addition, both constitutive- and inflammation-induced DC migration involves the chemokine CCL21, which most likely triggers integrin activation on DC via its receptor CCR7. Recently, however, conflicting evidence has suggested that DC entry occurs independently of integrins, implying that the role of CCL21 in lymphatics is purely chemotactic. Hence, while CCL21 is reported to be inducible during inflammation, the details of this induction and the role of CCL21 during initial DC trafficking are unclear. Here, we have characterized both the production of CCL21 and the mechanism of its action in DC transmigration using primary human dermal lymphatic endothelial cells (HDLECs) and a mouse model of skin contact hypersensitivity. We showed that CCL21 is constitutively expressed intracellularly but rapidly secreted after exposure to the inflammatory cytokine tumour necrosis factor (TNF) α following de novo RNA and protein synthesis. Furthermore, using in vitro transmigration assays, we showed that endogenous HDLEC-derived CCL21 stimulates DC translymphatic migration by a predominantly chemotactic mechanism in resting HDLEC and by a ß2 integrin-mediated mechanism in TNFα-stimulated HDLEC. These results imply a direct role for CCL21 in lymphatic transmigration that involves the selective use of integrin activation in inflammation.

Endotoxin-neutralizing peptides as gram-negative sepsis therapeutics.

Bacterial endotoxin [e.g. lipopolysaccharide (LPS)] can trigger systemic hyper-inflammatory that subsequently leads to multiple organ failure and lethality (gram-negative sepsis). This paper describes the development of endotoxin-neutralizing peptides that potentially treat sepsis. These peptides have been derived from bactericidal/permeability-increasing protein (BPIP), anti-microbial peptides, and leukocyte CD18 antigen and some of these peptides have been tested in clinical studies.

FcgammaRIIA and FcgammaRIIIB mediate nuclear factor activation through separate signaling pathways in human neutrophils.

Receptors for IgG Abs (Fcgamma receptors) are capable of triggering diverse cell responses in leukocytes. In neutrophils, two Fcgamma receptors, namely FcgammaRIIA and FcgammaRIIIB, are constitutively expressed. The signaling pathways that regulate FcgammaRIIA-mediated phagocytosis have been relatively well described. However, the different signaling pathways that lead to NF activation after engagement of each Fcgamma receptor have only been partially described. To address this problem, neutrophils were stimulated by cross-linking selectively each type of Fcgamma receptor with specific mAbs, and NF activation was then analyzed. FcgammaRIIIB, but not FcgammaRIIA, promoted a robust increase in phosphorylated ERK in the nucleus, and also efficient phosphorylation of the NF Elk-1. Complete mAb 3G8 (anti-FcgammaRIIIB) induced a higher response than did F(ab')(2) fragments of mAb 3G8, suggesting a possible synergistic effect of both FcgammaR receptors. However, mAb IV.3 (anti-FcgammaRIIA) alone did not cause an increase of phosphorylated ERK in the nucleus. FcgammaRIIIB-induced nuclear phosphorylation of ERK, and of Elk-1, was not affected by Syk, PI3K, or MEK inhibitors. In contrast, FcgammaRIIA- or FcgammaRIIIB-mediated phosphorylation of cytoplasmic ERK depended on Syk, PI3K, and MEK. Also, ERK, but not MEK, was constitutively present in the nucleus, and FcgammaRIIIB cross-linking did not increase the levels of nuclear ERK or MEK. These data clearly show that different neutrophil Fcgamma receptors possess different signaling capabilities. FcgammaRIIIB, but not FcgammaRIIA, activates a unique signaling pathway leading to the nuclear-restricted phosphorylation of ERK and Elk-1, independently of Syk, PI3K, or MEK.

The effect of fever-like temperatures on neutrophil signaling.

The effect of fever on neutrophils has not been explored. We tested the hypothesis that fever-like temperature spikes affect neutrophil signaling and function. Prior 60 min, 42 degrees C heat exposure inhibited p38 MAPK, ERK, PI3-Kinase/Akt, and NF-kappaB activation in TNF-alpha-challenged suspended neutrophils. Using pharmacological inhibitors and an inhibitory peptide transduced into neutrophils by a HIV-TAT sequence, we found that p38 MAPK and NF-kappaB mediate TNF-alpha-mediated delayed apoptosis in suspended neutrophils. Heat exposure (39-42 degrees C) did not affect constitutive apoptosis but abrogated TNF-alpha-delayed apoptosis in these suspended cells. In contrast, adhesion-dependent functions were not inhibited. Furthermore, we found that heat exposure neither blocked p38 MAPK, ERK, and NF-kappaB activation in neutrophils on fibronectin nor prevented delayed apoptosis by TNF-alpha when cells interacted with fibronectin. Above and beyond apoptosis, TNF-alpha initiated NF-kappaB-dependent gene transcription. Heat exposure blocked this effect in suspended neutrophils but not in neutrophils on fibronectin. Finally, we show that beta2-integrins, which are not necessary for TNF-alpha-induced NF-kappaB activation at 37 degrees C, transduce costimulatory signals allowing NF-kappaB activation after heat exposure. The effect could protect circulating neutrophils from TNF-alpha activation, while not interfering with activation of adherent neutrophils. Fever could make neutrophils more parsimonious.

Beta1 and beta2 integrins mediate adhesion during macrophage fusion and multinucleated foreign body giant cell formation.

An in vitro system of interleukin (IL)-4-induced human monocyte-derived macrophage fusion was used to investigate the cell/substrate adhesive mechanisms that support multinucleated foreign body giant cell (FBGC) formation. Monocytes were cultured for 3 days and IL-4 was added to induce macrophage fusion and FBGC formation by day 7. Functionally defined anti-integrin antibodies demonstrated that initial monocyte adhesion is mediated by beta2 integrins, whereas during the induction of macrophage fusion by IL-4, an additional dependence on beta1 integrins is acquired. The combination of anti-beta1 plus anti-beta2 was most effective, reducing macrophage/FBGC adhesion to 10% of controls. Consistent with integrin-mediated signaling, the tyrosine kinase inhibitor genistein and the phosphatidylinositol-3-kinase inhibitors wortmannin and LY294002 also attenuated macrophage/FBGC adhesion. Confocal microscopic analysis revealed that beta2 integrins are present on monocytes after initial adhesion and are strongly expressed on fusing macrophages, particularly in peripheral cell areas, and on FBGCs. In contrast, beta1 integrins are not detected on monocytes but begin to appear during macrophage development and are strongly expressed on fusing macrophages and FBGCs. For the first time, these results demonstrate the IL-4-induced acquisition of cooperation between beta1 and beta2 integrins in the cell/substrate adhesive interactions that are required for multinucleated FBGC formation.

Platelet/polymorphonuclear leukocyte adhesion: a new role for SRC kinases in Mac-1 adhesive function triggered by P-selectin.

Adhesion of polymorphonuclear leukocytes (PMNLs) to activated platelets requires a P-selectin-triggered, tyrosine kinase-dependent adhesiveness of Mac-1 and is accompanied by tyrosine phosphorylation of a 110-kd protein (P-110) in PMNLs. Inhibitors of SRC tyrosine kinases were found to inhibit PMNL adhesion to activated platelets or to P-selectin expressing Chinese hamster ovary (CHO-P) cells and the tyrosine phosphorylation of P-110. Adhesion of PMNLs to activated platelets or to CHO-P cells stimulated activity of LYN and HCK. Monoclonal antibody blockade of P-selectin or beta2-integrins reduced the activation of both kinases. In PMNLs either adherent to platelets or aggregated by P-selectin-IgG chimera, Mac-1 was rapidly redistributed to the Triton X-100-insoluble cytoskeletal fraction, and large clusters of Mac-1 colocalized with patches of F-actin at the sites of cell-cell contact. In PMNLs stimulated by P-selectin-IgG chimera, SRC kinase inhibition impaired Mac-1 clustering, F-actin accumulation, and CD18 redistribution to the cytoskeleton. Disruption of the actin filament network by cytochalasin D prevented PMNL-platelet adhesion and P-selectin-induced PMNL aggregation and impaired the clustering of Mac-1. In agreement with the requirement for the beta2-integrin in the functional up-regulation of LYN and HCK, integrin blockade by monoclonal antibodies resulted in a complete inhibition of P-selectin-induced Mac-1 clustering and F-actin accumulation. Taken together, the results indicate that, after an initial P-selectin-triggered beta2-integrin interaction with the ligand, SRC kinases are activated and allow the remodeling of cytoskeleton-integrin linkages and integrin clustering that finally strengthen cell-cell adhesion. This model highlights a new role for SRC kinases in a regulatory loop by which the Mac-1 promotes its own adhesive function.

Mechanisms of granulocytosis in the absence of CD18.

Genetic deficiency in CD18 leads to disease characterized by myeloid hyperplasia, including profound granulocytosis and splenomegaly. Myeloid hyperplasia could directly result from the disruption of CD18 functions essential to granulopoiesis or basal leukocyte trafficking. Alternatively, myeloid hyperplasia could be reactive in nature, due to disruption of essential roles of CD18 in leukocyte responses to microbial challenge. To distinguish between these mechanisms, the hematopoietic systems of lethally irradiated wild-type (WT) mice were reconstituted with either WT fetal liver cells or CD18-deficient fetal liver cells, or an equal mixture of both types of cells. Granulocytosis and splenomegaly developed in mice that received CD18-deficient fetal liver cells. Splenomegaly was prevented and granulocytosis was inhibited by more than 95% in mice that had received both CD18-deficient and WT fetal liver cells, suggesting that myeloid hyperplasia was largely reactive in nature. Consistent with this postulate, the circulating life spans in the blood and the fraction of neutrophils that incorporated BrdU in the bone marrow were not increased for CD18-deficient neutrophils compared with the WT. However, these animals did develop mild granulocytosis compared with mice reconstituted with WT cells alone, and a higher percentage of CD18-deficient leukocytes were neutrophils compared with the WT leukocytes. These observations suggest that the granulocytosis observed in the absence of CD18 occurs through at least 2 mechanisms: one that is dramatically improved by the presence of WT cells, likely reactive in nature, and a second that is independent of the WT hematopoietic cells, involving an alteration in the lineage distribution of blood leukocytes.

Synergistic mobilization of hemopoietic progenitor cells using concurrent beta1 and beta2 integrin blockade or beta2-deficient mice.

The hierarchy of cytoadhesion molecules involved in hematopoietic/stem progenitor cell mobilization has not yet been delineated. Previous studies have suggested an important role for alpha4beta1 integrin in this process. To test whether mobilization involves dynamic interactions of alpha4beta1 with other integrins on hematopoietic cells, especially the beta2 integrins, mice and primates were treated with anti-beta1 or anti-beta2 antibodies alone or in combination. A single injection of anti-alpha4beta1 antibody elicited reproducible mobilization in contrast to other antibodies, and 3 injections yielded higher mobilization efficiency than each of the other antibodies. When the anti-beta2 (anti-CD11a or anti-CD18) or anti-alpha5/beta1 integrin antibody was combined with anti-alpha4, an augmentation in mobilization was seen that was either additive or synergistic, depending on the potency of the antibody used. Synergy between anti-alpha4 and anti-CD18 (beta(2)) antibody blockade was seen in primates and confirmed in anti-alpha4-treated CD18-deficient mice. In the latter, there was a 49-fold increase in mobilization with anti-alpha4, much higher than in littermate control animals, in CD18 hypomorphic mice, or in other strains of mice tested. Data from both the antibody blockade and gene-targeted mice suggest that the cooperativity of alpha4beta1 with beta2 integrins becomes evident when they are concurrently inhibited. It is unclear whether this cooperativity is exerted at the stage of reversible adhesion versus migration, and enhancement of and whether the 2 integrins work in a sequential or parallel manner. Whatever the mechanism, the data provide a novel example of beta1 and beta2 integrin crosstalk in stem/progenitor cell mobilization.

Inhibitory effects of synthetic beta peptide on invasion and metastasis of liver cancer.

PURPOSE: To study the inhibitory effects of synthetic beta peptide on invasion and metastasis of liver cancer. METHODS: Membrane-type intercellular adhesion molecule-1 (ICAM-1) expression of SMMC-7721 cultured hepatoma cells (7721 cells) was detected by immunofluorescence cell flowmeter. The adhesion of 7721 cells to fibronectin (FN) was assayed by the MTT method. The adhesion of 7721 cells to 7721 cells, 7721 cells to endothelial cells, and 7721 cells to lymphocyte cells was detected by adhesion assay. LCI-D20 human liver cancer metastasis model in nude mice was used in this experiment. One hundred micrograms of beta peptide per mouse were injected subcutaneously after tumor was resected premetastatically or postmetastatically to observe its effect on liver cancer metastasis after hepatectomy. RESULTS: Membrane-type ICAM-1 expression of SMMC-7721 cells treated by beta peptide was lower than that of the untreated cells. The adhesion of 7721 cells to FN, 7721 cells to 7721 cells, 7721 cells to endothelial cells, and 7721 cells to lymphocyte cells was also lower in the beta peptide group than in the untreated group. CONCLUSIONS: beta Peptide can block the adhesion of 7721 cells to FN, 7721 cells to some host cells in vitro, and inhibit HCC metastasis of LCI-D20 model posthepatectomy in vivo, so it could potentially act as an antimetastasis drug.

Rapid and prominent up-regulation of high-affinity receptor for immunoglobulin G (Fc gamma RI) by cross-linking of beta 2 integrins on polymorphonuclear leukocytes.

Receptors for the Fc region (FcR) of immunoglobulin (Ig)G play essential roles in effector functions of polymorphonuclear leukocytes (PMNs) including the antibody-mediated clearance of microbes. In contrast to the constitutive expression of the low-affinity receptors for IgG (Fc gamma RII [CD32] and Fc gamma RIII [CD16]), the high-affinity receptor Fc gamma RI (CD64) is barely detectable on unactivated PMNs. CD64 expression is induced in a slow kinetic manner by interferon (IFN)-gamma and granulocyte colony-stimulating factor (G-CSF) after 12 to 24 hours of exposure to these agents. We found that the cross-linking of CD11b as well as of CD18 induced comparable rapid increases in CD64 expression on the surface of PMNs, occurring within 15 minutes of exposure. Cross-linking of neither CD11a nor CD11c induced CD64 expression. In contrast to slow induction by IFN-gamma and G-CSF, the integrin-induced rapid CD64 expression did not require RNA synthesis. Genistein, herbimycin A, and 1,2-bis(o-aminophenoxy)ethan-N,N-N',N'-tetraacetic acid blocked the immediate expression of CD64 in a dose-dependent manner, suggesting that the signal is mediated through calcium mobilization and protein tyrosine kinase(s). Such rapid modulation of the high-affinity Fc gamma RI receptor by integrin cross-linking may reflect the requirement for rapid up-regulation of PMN effector functions, after interaction with endothelial cells, platelets or bacteria.

The hepatic inflammatory response after acetaminophen overdose: role of neutrophils.

Acetaminophen overdose induces severe liver injury and hepatic failure. There is evidence that inflammatory cells may be involved in the pathophysiology. Thus, the aim of this investigation was to characterize the neutrophilic inflammatory response after treatment of C3Heb/FeJ mice with 300 mg/kg acetaminophen. A time course study showed that neutrophils accumulate in the liver parallel to or slightly after the development of liver injury. The number of neutrophils in the liver was substantial (209 +/- 64 PMN/50 high-power fields at 12 h) compared to baseline levels (7 +/- 1). Serum levels of TNF-alpha and the C-X-C chemokines KC and MIP-2 increased by 28-, 14-, and 295-fold, respectively, over levels found in controls during the injury process. In addition, mRNA expression of MIP-2 and KC were upregulated in livers of acetaminophen-treated animals as determined by ribonuclease protection assay. However, none of these mediators were generated in large enough quantities to account for neutrophil sequestration in the liver. There was no upregulation of Mac-1 (CD11b/ CD18) or shedding of L-selectin on circulating neutrophils. Moreover, an anti-CD18 antibody had no protective effect against acetaminophen overdose during the first 24 h. These results indicate that there is a local inflammatory response after acetaminophen overdose, including a substantial accumulation of neutrophils in the liver. Because of the critical importance of beta2 integrins for neutrophil cytotoxicity, these results suggest that neutrophils do not contribute to the initiation or progression of AAP-induced liver. The inflammation observed after acetaminophen overdose may be characteristic for a response sufficient to recruit neutrophils for the purpose of removing necrotic cells but is not severe enough to cause additional damage.

Effects of roughness, fibronectin and vitronectin on attachment, spreading, and proliferation of human osteoblast-like cells (Saos-2) on titanium surfaces.

The influence of surface roughness and the presence of adhesion molecules in the culture medium were studied regarding cell adhesion, shape, and proliferation of osteoblast-like cells grown on two types of titanium disk. Type I disks were acid etched and type II disks were sandblasted and acid etched. Surface roughness was determined by contact profilometry and scanning electron microscopy. Chemical composition and oxide thickness of the superficial titanium layer were established with energy dispersive X-ray spectrometry, electron spectroscopy for chemical analysis and auger electron spectroscopy. Titanium release in the culture medium was assessed by inductively coupled plasma-optical emission spectrometry. Osteoblast-like cells (Saos-2) were cultured on both types of titanium disks (1) in standard conditions (DMEM culture medium supplemented with fetal calf serum), (FCS), (2) with the culture medium alone (DMEM alone), (3) in the presence of fibronectin or vitronectin (DMEM supplemented with fibronectin or vitronectin). Cultures were also performed in the presence of monoclonal anti-integrin (beta1, alphav) to test the cell adhesion molecules involved in the cell binding to the titanium surface. We found that sandblasting does not modify the chemical surface composition and that titanium represents only 5-6% (in the atom percentage) of surface elements. Release of titanium in the culture medium was found to increase from 24 to 72 hours. In the absence of FCS, fibronectin, or vitronectin, cells appeared scanty and packed in clusters. On the contrary, cells cultured in the presence of FCS, fibronectin, or vitronectin were flattened with large and thin cytoplasmic expansions. The addition of anti beta1 or alphav integrin subunit monoclonal antibody in the culture medium decreased adhesion and spreading of cells, particularly in the presence of fibronectin. Cell proliferation was significantly higher on culture plastic than on both types of disks, but was increased on rough but not on smooth surfaces. These results indicate that a high surface roughness and presence of fibronectin or vitronectin are critical elements for adhesion, spreading, and proliferation of cells on titanium surfaces.

[The effect of PMN adhesion mediated by CD11b/CD18 on the increasing permeability of microvascular endothelial monolayer after severe burn injury].

OBJECTIVE: To detect the effect of burn-activated PMN adhesion and its adhesion molecule CD11b/CD18 on microvascular endothelial permeability using an experimental model of endothelial monolayer on polycarbonate microporous filters. METHODS: An experimental model for in vitro study of endothelal monolayer for permeability analysis was established. Seven groups were divided into according to the treatment of microvascular endothelial monolayer. Fluid filtration coeffecient(Kf) and albumin reflection coeffecient(delta) were measured after endothelial monolayer was perfused with albumin labelled by FITC. RESULTS: Burn-activated PMN could increase the level of fluid filtration coeffecient(Kf) and decrease the albumin reflection coeffecient(delta). Monoclonal antibody sealing off CD11b/CD18 on PMN provented the change of delta induced by burn-activated PMN. Another microporous filter interposed between PMNs and endothelial monolayer corrected the changes of Kf and delta. CONCLUSION: The permeability enhancing effect of PMNs may be attributed mainly to the PMN-EC adhesion mediated by CD11b/CD18. Blocking the PMN-EC over-adhesion in moderation may be helpful in reducing the lung injury due to severe burn injury.

[Effects of blocking CD18-mediated leukocyte adhesion on the survival of the island flap].

OBJECTIVE: This study was to investigate the role of leukocyte and leukocyte adhesion in tissue injury from ischemia and reperfusion. METHODS: The experiment utilized the monoclonal antibody (mAb) directed to the leukocyte adhesion glycoprotein CD18 to block leukocyte adhesion and aggregation in an island flap model in rats. Tissue content of myeloperoxidase (MPO) and malondialdehyde (MDA) were detected after transient treatment with either saline or mAb directed to CD18. Flap survival was assessed 7 days afterwards. RESULTS: The content of both MPO and MDA was significantly increased with 8 h ischemia and 1 h reperfusion of the flap. The treatment with anti-CD18 mAb significantly decreased the levels of MPO and MDA and also significantly improved the survival of flaps compared with the saline-treated controls. CONCLUSION: CD18-mediated leukocyte adhesion plays an important role in tissue injury from ischemia and reperfusion. Blocking leukocyte adhesion can attenuate leukocyte-mediated injury, providing protective effects on island flaps.

Cell adhesion molecules of experimental otitis media in the rat.

We examined the expression of cell adhesion molecules (CAM) in immune-mediated otitis media using keyhole limpet haemocyanin (KLH) in the rat, as well as the regulation of these CAM in peripheral blood polymorphonuclear leucocytes (PMN) and lymphocytes upon exposure to middle ear effusion (MEE). After general immunization, a topical antigen was introduced into the middle ear cavity. One day after exposure, CD18+ cells, primarily PMN, had maximally invaded the middle ear mucosa and mucosal epithelium. Mucosal epithelium strongly expressed intercellular adhesion molecule-1 (ICAM-1), only on the first day. The total number of cells in MEE reached a peak on day 3. On day 3, ICAM-1+ cells had reached a peak of 24.5% of the total cells. On day 2. CD18+ cells had reached a peak at 75.3% of the total cells. We examined the regulation of CAM in peripheral blood upon exposure to MEE. The percentage of fluorescent CD18+ PMN increased with MEE compared to those incubated in its absence, but those of L-selectin-positive PMN significantly decreased CAM on the surface lymphocytes did not change when incubated with MEE. The expression of CAM (CD18, ICAM-1) appears important for the initiation of otitis media. Moreover, it was thought that the interaction between the infiltrated PMN and MEE may modify the expression of CAM during the inflammatory process in the middle ear cavity.

Soluble intercellular adhesion molecule-1 provokes polymorphonuclear leukocyte elastase release by CD18.

BACKGROUND: Elevated levels of soluble intercellular adhesion molecule-1 (sICAM-1) correlate with the development of postinjury multiple organ failure. Soluble ICAM-1 secretion is known to be induced in endothelial cells and monocytes by diverse inflammatory stimuli. We have found that incubation of quiescent polymorphonuclear leukocytes (PMNs) with sICAM-1 elicits elastase release and, more recently, that cross-linking CD18 receptors on PMNs also produces elastase release. Consequently, our study hypothesis was that sICAM-1 provokes PMN elastase release through its interaction with CD18. METHODS: To obtain sICAM-1, Chinese hamster ovarian cells transfected with human ICAM-1 were lysed and centrifuged at 150,000 g for 1 hour; the supernatant was passed over an ICAM-1 affinity column, eluted with 0.1 mmol/L glycine HCl, and concentrated with dialysis filter. Human PMNs (2.5 x 10(5)) were saturated with specific monoclonal antibodies for the beta 2 subunits (CD11a, CD11b, CD18) or nonspecific monoclonal antibodies for 30 minutes on ice before a 1-hour incubation with sICAM-1 (75 ng/ml) at 37 degrees C. Elastase activity was measured by the cleavage of n-methoxysuccinyl-A-A-P-V-p-nitroanilide. RESULTS: Neutrophil incubation with sICAM-1 resulted in 19.2% +/- 2.8% of total PMN elastase, compared with 2.4% +/- 0.5% in the controls. Blockade of CD18 abrogated sICAM-1 provoked elastase release with monoclonal antibodies to CD18 (TS1/18, 31H8) resulting in 4.3% +/- 1.0% and 5.5% +/- 1.4% elastase release, respectively. Blockade of CD11a, CD11b, and nonspecific antibody controls had no effect on sICAM-1 induced elastase release. CONCLUSIONS: In vitro, sICAM-1 provokes PMN elastase release through CD18. This may represent a mechanism by which elevated levels of circulating sICAM-1, released from local injury sites, provoke distal organ dysfunction.

Apoptosis following interleukin-2 withdrawal from T cells: evidence for a regulatory role of CD18 (beta 2-integrin) molecules.

Following a successful immune response against invading microorganisms, the majority of activated T cells is eliminated, while a minor fraction survives as memory T cells. A decline in T lymphocyte growth factors such as interleukin-2 (IL-2) appears to play a role in the elimination of previously activated T cells. Thus, removal of IL-2 from proliferating T cells not only induces growth arrest, but triggers a massive cell death due to apoptosis. While the apoptotic response involves a series of well-described events, it remains less clear how apoptosis is regulated following IL-2 withdrawal. Here, we provide evidence that CD18 molecules (beta 2-integrins) play a regulatory role in the apoptotic response following removal of IL-2 from previously activated, antigen specific CD4+ T cell lines. Thus, CD18 mAb inhibited the apoptotic response to IL-2 deprivation, whereas mAb against other adhesion molecules (CD28, CD29, CD49d, CD80, CD86) did not. Secondly, IL-2 withdrawal resulted in a retarded apoptotic response in LFA-1 (CD11a/CD18) negative T cells obtained from a leukocyte adhesion deficiency (LAD) patient, as compared to LFA-1 positive T cell lines. Thirdly, co-culture of LFA-1 positive- and negative-T cells at different ratios induced apoptotic responses that were higher than expected, had the two lymphocyte populations not been interacting and significantly higher than that seen in pure LFA-1 negative T cells. Supernatants from LFA-1 positive T cell cultures undergoing apoptosis did not induce an enhanced apoptotic responses in LFA-1 negative T cells, and, reversely, culture supernatants from LFA-1 negative T cells did not rescue LFA-1 positive cells from undergoing apoptosis. The apoptotic response was partly blocked by IL-15, a newly identified T cell growth factor. Taken together, these findings suggest that CD18 molecules (beta 2-integrins) play a regulatory role in the apoptotic response following cytokine withdrawal, and that the regulation is mediated, at least partly, through T-T cell interactions. Thus, apoptotic death following IL-2 deprivation appears to be under "social" control by surrounding T cells.

L- and P-selectins, but not CD49d (VLA-4) integrins, mediate monocyte initial attachment to TNF-alpha-activated vascular endothelium under flow in vitro.

Monocyte adhesion to the vascular endothelium is a pivotal step during their egress to tissues at sites of inflammation and immune reactions, and during atherogenesis. In this study, an in vitro flow model and blocking mAb were used to define the role of adhesion molecules in monocyte interactions with activated HUVEC under flow conditions. By videomicroscopy, freely flowing monocytes abruptly halted (initial attachment) on 6-h TNF-alpha-activated HUVEC under flow via L- and P-selectin, whereas E-selectin was not involved. CD49d/CD29 integrin (VLA-4), which can mediate initial attachment of certain T cells to VCAM-1 under flow, did not support monocyte initial attachment. Once initially attached, a small number of monocytes began rolling at 9 microns/s through a mechanism involving L-selectin, as well as CD49d and CD11/CD18 integrins, while the remaining monocytes became firmly adherent, or released to the flow stream. Monocyte stable arrest and subsequent transendothelial migration occurred rapidly and efficiently through either CD49d or CD18 integrin adhesion pathways. Transendothelial passage was also dependent on PECAM-1 (CD31). These data reveal monocytes initially attach to activated endothelium via an L-selectin-dependent mechanism, with a smaller contribution from P-selectin and no contribution by CD49d. Subsequent monocyte rolling, arrest, and transmigration require overlapping functions between multiple members of the selectin, integrin, and Ig gene families.

Kupffer cell-mediated cytotoxicity against hepatoma cells occurs through production of nitric oxide and adhesion via ICAM-1/CD18.

Rat Kupffer cell (KC)-mediated cytotoxicity against both the syngeneic hepatoma cell line AH70 and hepatocytes was evaluated by changes in mitochondrial function, and the possible role of ICAM-1/CD18 in the interaction between the cells was studied. Rhodamine 123 fluorescence, a marker of the mitochondrial membrane potential, decreased in AH70 cells after co-culture with CK, while that in hepatocytes was unchanged by co-culture. This decrease was blocked by anti-ICAM-1 anti-CD18 and the inhibition of nitric oxide synthesis. Cytometric studies demonstrated that ICAM-1 expression on AH70 cells increased after addition of IFN-gamma, IL-1beta, tumor necrosis factor (TNF)-alpha or KC, while in hepatocytes ICAM-1 was not increased. Anti-ICAM-1 pretreatment inhibited the increase in ICAM-1 expression and the decrease in rhodamine 123 fluorescence on AH70 cells after co-culture with KC. CD18 on KC was increased only after co-culture with AH70. TNF-alpha but not IFN-gamma was detected in the supernatant of co-culture between KC and AH70 cells, and this production was partially inhibited by anti-ICAM-1 and anti-CD18. The activity of inducible nitric oxide synthase in Kupffer cells and the levels of nitrites and nitrates in the co-culture supernatant increased over time, and this increase was attenuated either by addition of NO synthesis inhibitors, anti-ICAM-1 or anti-CD18. These results indicate that the rat KC causes mitochondrial dysfunction in cancer cells via the production of NO and cell-to-cell adhesion via ICAM-1/CD18 has an important role in this cytotoxic process.