The P2Y1 receptor-mediated leukocyte adhesion to endothelial cells is inhibited by melatonin.

Extracellular ATP (released by endothelial and immune cells) and its metabolite ADP are important pro-inflammatory mediators via the activation of purinergic P2 receptors (P2Y and P2X), which represent potential new targets for anti-inflammatory therapy. Endothelial P2Y1 receptor (P2Y1R) induces endothelial cell activation triggering leukocyte adhesion. A number of data have implicated melatonin as a modulator of immunity, inflammation, and endothelial cell function, but to date no studies have investigated whether melatonin modulates endothelial P2YR signaling. Here, we evaluated the putative effect of melatonin on P2Y1R-mediated leukocyte adhesion to endothelial cells and TNF-α production, using mesenteric endothelial cells and fresh peripheral blood mononuclear cells isolated from rats. Endothelial cells were treated with the P2Y1R agonist 2MeSATP, alone or in combination with melatonin, and then exposed to mononuclear cells. 2MeSATP increased leukocyte adhesion to endothelial cells and TNF-α production in vitro, and melatonin inhibited both effects without altering P2Y1R protein expression. In addition, assays with the Ca2+ chelator BAPTA-AM indicate that the effect of melatonin on 2MeSATP-stimulated leukocyte adhesion depends on intracellular Ca2+ modulation. P2Y1R is considered a potential target to control chronic inflammation. Therefore, our data unveiled a new endothelial cell modulator of purinergic P2Y1 receptor signaling.

Intracellular signalling during neutrophil recruitment.

Recruitment of leucocytes such as neutrophils to the extravascular space is a critical step of the inflammation process and plays a major role in the development of various diseases including several cardiovascular diseases. Neutrophils themselves play a very active role in that process by sensing their environment and responding to the extracellular cues by adhesion and de-adhesion, cellular shape changes, chemotactic migration, and other effector functions of cell activation. Those responses are co-ordinated by a number of cell surface receptors and their complex intracellular signal transduction pathways. Here, we review neutrophil signal transduction processes critical for recruitment to the site of inflammation. The two key requirements for neutrophil recruitment are the establishment of appropriate chemoattractant gradients and the intrinsic ability of the cells to migrate along those gradients. We will first discuss signalling steps required for sensing extracellular chemoattractants such as chemokines and lipid mediators and the processes (e.g. PI3-kinase pathways) leading to the translation of extracellular chemoattractant gradients to polarized cellular responses. We will then discuss signal transduction by leucocyte adhesion receptors (e.g. tyrosine kinase pathways) which are critical for adhesion to, and migration through the vessel wall. Finally, additional neutrophil signalling pathways with an indirect effect on the neutrophil recruitment process, e.g. through modulation of the inflammatory environment, will be discussed. Mechanistic understanding of these pathways provide better understanding of the inflammation process and may point to novel therapeutic strategies for controlling excessive inflammation during infection or tissue damage.

Bighorn sheep beta2-integrin LFA-1 serves as a receptor for Mannheimia haemolytica leukotoxin.

Mannheimia haemolytica is an important cause of pneumonia in bighorn sheep (BHS; Ovis canadensis). Leukotoxin (Lkt), the primary virulence determinant of M. haemolytica, induces cytolysis of all subsets of leukocytes. Previously, we have shown that CD18, the beta subunit of beta2-integrins, mediates Lkt-induced cytolysis. However, it is not clear whether CD18 of all three beta2-integrins, LFA-1, Mac-1, and CR4, mediates Lkt-induced cytolysis. The objective of this study was to determine whether BHS LFA-1 (CD11a/CD18) serves as a receptor for Lkt. Plasmids encoding cDNA for BHS CD11a and CD18 were cotransfected into Lkt-resistant HEK-293 cells. Flow cytometric analysis of transfectants confirmed cell surface expression of BHS LFA- 1, Lkt-LFA-1 binding and Lkt-induced intra-cellular calcium elevation. More importantly, the transfectants were efficiently lysed by Lkt in a concentration-dependent manner. Collectively, these results indicate that BHS LFA-1 serves as a functional receptor for M. haemolytica Lkt.

Surface engineering of quantum dots for in vivo vascular imaging.

Quantum dot-antibody bioconjugates (QD-mAb) were synthesized incorporating PEG cross-linkers and Fc-shielding mAb fragments to increase in vivo circulation times and targeting efficiency. Microscopy of endothelial cell cultures incubated with QD-mAb directed against cell adhesion molecules (CAMs), when shielded to reduce Fc-mediated interactions, were more specific for their molecular targets. In vitro flow cytometry indicated that surface engineered QD-mAb labeled leukocyte subsets with minimal Fc-mediated binding. Nontargeted QD-mAb nanoparticles with Fc-blockade featured 64% (endothelial cells) and 53% (leukocytes) lower nonspecific binding than non-Fc-blocked nanoparticles. Spectrally distinct QD-mAb targeted to the cell adhesion molecules (CAMs) PECAM-1, ICAM-1, and VCAM-1 on the retinal endothelium in a rat model of diabetes were imaged in vivo using fluorescence angiography. Endogenously labeled circulating and adherent leukocyte subsets were imaged in rat models of diabetes and uveitis using QD-mAb targeted to RP-1 and CD45. Diabetic rats exhibited increased fluorescence in the retinal vasculature from QD bioconjugates to ICAM-1 and VCAM-1 but not PECAM-1. Both animal models exhibited leukocyte rolling and leukostasis in capillaries. Examination of retinal whole mounts prepared after in vivo imaging confirmed the fluorescence patterns seen in vivo. Comparison of the timecourse of retinal fluorescence from Fc-shielded and non-Fc-shielded bioconjugates indicated nonspecific uptake and increased clearance of the non-Fc-shielded QD-mAb. This combination of QD surface design elements offers a promising new in vivo approach to specifically label vascular cells and biomolecules of interest.

Mannheimia (Pasteurella) haemolytica leukotoxin utilizes CD18 as its receptor on bighorn sheep leukocytes.

Pneumonia caused by Mannheimia (Pasteurella) haemolytica is a highly fatal disease of bighorn sheep (Ovis canadensis). Leukotoxin (Lkt), secreted by M. haemolytica, is an important virulence factor of this organism, and is cytolytic to bighorn sheep leukocytes. Previously, we have shown that CD18, the beta subunit of beta2 integrins, serves as the receptor for Lkt on bovine leukocytes. Furthermore, anti-CD18 antibodies inhibit Lkt-induced cytotoxicity of bighorn sheep leukocytes. Therefore, we hypothesized that Lkt utilizes CD18 as its receptor on bighorn sheep leukocytes. Confirmation of bighorn sheep CD18 as a receptor for Lkt requires the demonstration that the recombinant expression of bighorn sheep CD18 in Lkt-nonsusceptible cells renders them susceptible to Lkt. Therefore, we transfected cDNA encoding CD18 of bighorn sheep into a Lkt-nonsusceptible murine cell line. Cell surface expression of bighorn sheep CD18 on the transfectants was tested by flow cytometry with anti-CD18 antibodies. Transfectants stably expressing bighorn sheep CD18 on their surface were subjected to flow cytometric analysis for detection of Lkt binding, and cytotoxicity assays for detection of Lkt-induced cytotoxicity. Leukotoxin bound to the transfectants. More importantly, the transfectants were effectively lysed by Lkt in a concentration-dependent manner, whereas the parent cells were not. These results clearly indicate that M. haemolytica Lkt utilizes CD18 as a receptor on bighorn sheep leukocytes. Identification of CD18 as a receptor for Lkt on bighorn sheep leukocytes should enhance our understanding of the pathogenesis of pneumonia, which in turn should help in the development of control measures against this fatal disease of bighorn sheep.

Bioactive hydrogel substrates: probing leukocyte receptor-ligand interactions in parallel plate flow chamber studies.

The binding of activated integrins on the surface of leukocytes facilitates the adhesion of leukocytes to vascular endothelium during inflammation. Interactions between selectins and their ligands mediate rolling, and are believed to play an important role in leukocyte adhesion, though the minimal recognition motif required for physiologic interactions is not known. We have developed a novel system using poly(ethylene glycol) (PEG) hydrogels modified with either integrin-binding peptide sequences or the selectin ligand sialyl Lewis X (SLe(X)) within a parallel plate flow chamber to examine the dynamics of leukocyte adhesion to specific ligands. The adhesive peptide sequences arginine-glycine-aspartic acid-serine (RGDS) and leucine-aspartic acid-valine (LDV) as well as sialyl Lewis X were bound to the surface of photopolymerized PEG diacrylate hydrogels. Leukocytes perfused over these gels in a parallel plate flow chamber at physiological shear rates demonstrate both rolling and firm adhesion, depending on the identity and concentration of ligand bound to the hydrogel substrate. This new system provides a unique polymer-based model for the study of interactions between leukocytes and endothelium as well as a platform to develop improved scaffolds for cardiovascular tissue engineering.

Promotion of leukocyte adhesion by a novel interaction between vitronectin and the beta2 integrin Mac-1 (alphaMbeta2, CD11b/CD18).

OBJECTIVE: The leukocyte integrin Mac-1 (alphaMbeta2, CD11b/CD18) binds a number of ligands and counter-receptors and thereby is a major determinant in regulation of leukocyte adhesion and extravasation. Vitronectin (VN) is an adhesion-promoting factor that is abundantly present as matrix molecule in vascular diseases such as atherosclerosis. Until now, only an indirect interaction between Mac-1 and VN via the urokinase receptor (urokinase plasminogen activator receptor) was known. We now propose that Mac-1 and VN can directly interact with each other. METHODS AND RESULTS: In an in vitro system with purified components, Mac-1 specifically bound the multimeric matrix form of VN but not the monomeric plasma form. Using various competitors, the interaction domains in Mac-1 and VN were localized. Mac-1-expressing but not untransfected Chinese hamster ovary cells adhered strongly on VN. Introduction of a GFFKR deletion in the alphaM subunit of Mac-1, which increases the constitutive activation of the integrin, led to increased adhesion on VN. Peripheral human blood neutrophils adhered and migrated on multimeric VN in a Mac-1-dependent manner. CONCLUSIONS: These results show that there is a specific integrin-affinity-regulated interaction between Mac-1 and the matrix form but not the plasma form of VN that may significantly participate in leukocyte adhesion and extravasation.

FTY720 exerts differential effects on CD4+ and CD8+ T-lymphocyte subpopulations expressing chemokine and adhesion receptors.

BACKGROUND: FTY720 (FTY), a novel immunomodulator with the potential to improve immunosuppressive therapy after organ transplantation, is currently under clinical investigation. Previous experimental animal studies have shown that FTY has a unique mechanism of action associated with altered lymphocyte recirculation. METHODS: Participating in a phase I clinical trial, we studied the pharmacodynamic effects of FTY in stable renal allograft recipients. We analysed the effect of FTY on surface marker expression on T-cell subpopulations by flow cytometry. RESULTS: A single oral dose of FTY (0.25-3.5 mg) significantly reduced peripheral lymphocyte counts by 30-70%. FTY reduced all T-lymphocyte subsets, CD4(+) cells more than CD8(+) cells. However, we observed that lower doses of FTY (0.25-2 mg, n = 11) did not affect peripheral CD4(+)CCR5(+) T-lymphocyte counts, while the highest FTY dose of 3.5 mg (n = 2) exerted a rapid reduction of CD4(+)CCR5(+) cells. Peripheral CD8(+)CCR5(+) T-lymphocyte counts were reduced by either low (0.25-2 mg) or high (3.5 mg) doses of FTY. In contrast to CCR5(+) cells, cells expressing CD62L were preferentially reduced after administration of FTY. In particular, CD4(+)CD62L(+) T cells declined after treatment. CD4(+) and CD8(+) T-lymphocyte subpopulations expressing the other chemokine and adhesion receptors (CXCR4, CD11a and CD49d) were reduced to a similar extent as compared with overall CD4(+) or CD8(+) T-lymphocyte counts. CONCLUSIONS: Despite the limited number of patients, especially in the placebo (n = 3) and the high-dose groups (n = 2), our observations suggest that FTY exerts differential effects on T-cell subpopulations. FTY predominantly reduces CD4(+)CD62L(+) cells in the peripheral blood suggesting increased migration into lymph nodes. It seems that only FTY doses above 2 mg are able to reduce peripheral CD4(+)CCR5(+) T lymphocytes, which are potentially capable of infiltrating into the allograft during rejection.

Neutrophil airway influx by platelet-activating factor in asthma: role of adhesion molecules and LTB4 expression.

Platelet-activating factor (PAF)-induced neutrophil lung sequestration may require cell surface adhesion molecules (macrophage-1 antigen (MAC-1) and lymphocyte function-associated antigen-1 (LFA-1)). In this randomised, double-blinded, crossover study, the neutrophil kinetics after PAF and Lyso-PAF (L-PAF) airway challenge were investigated in nine mild-intermittent asthmatics. Neutrophils were measured in peripheral blood (PB) before and at 5, 15, 45 and 240 min after bronchoprovocation, and in induced sputum before and at 240 min after challenge. MAC-1 and LFA-1 expression were assessed by immunocytochemistry, and leukotriene B4 (LTB4) was measured by enzyme-immunoassay in induced-sputum supernatants. Compared with baseline, neutrophils in PB decreased 5 min after PAF, while at 240 min neutrophils in induced sputum increased. Compared with baseline and L-PAF, PAF decreased the percentages of MAC-1- and LFA-1-positive neutrophils in PB at 5 min, but increased the percentages of MAC-1 and LFA-1 in neutrophil-induced sputum. Moreover, compared with baseline and L-PAF, PAF-induced sputum revealed higher LTB4 levels, a finding that correlated with the elevated number of neutrophils in induced sputum. These findings suggest that macrophage-1 antigen and lymphocyte function-associated antigen-1 are involved in platelet-activating factor-induced neutrophil lung traffic, and that this process is modulated by enhanced leukotriene B4 release within the airways.

Receptor-ligand binding: 'catch' bonds finally caught.

Leukocyte recruitment to sites of inflammation is initiated by the selectin family of adhesion receptors. Recent research reveals that P-selectin binding to its ligand exhibits 'catch' to 'slip' bond transition that may help explain the shear threshold phenomenon.

Leukocyte trans-endothelial migration: JAMs add new pieces to the puzzle.

The molecular details of leukocyte transmigration through the endothelial barrier (also called diapedesis), which is the final step of leukocyte extravasation from the circulation to a given site of inflammation, are by far not well understood. The present review will focus on the different mechanisms potentially involved in leukocyte trans-endothelial migration. Both homophilic and heterophilic interactions between leukocyte and endothelial cell receptors will be covered, with a particular focus on the growing gene family of junctional adhesion molecules (JAM). Deciphering their mechanisms of interaction will also allow to unravel novel strategies for therapeutic intervention in inflammatory or atherothrombotic diseases.

Nonsteroidal anti-inflammatory drugs prevent early diabetic retinopathy via TNF-alpha suppression.

Leukocyte adhesion to the diabetic retinal vasculature results in blood-retinal barrier breakdown, capillary nonperfusion, and endothelial cell injury and death. Intercellular adhesion molecule-1 (ICAM-1) and the leukocyte integrin CD18 are required for these processes. Diabetes was induced in Long Evans rats, resulting in a two- to threefold increase in retinal leukocyte adhesion. Following one week of diabetes, neutrophil CD11a, CD11b, and CD18 expression was increased significantly, as were retinal ICAM-1 levels. Animals were treated with aspirin, a cyclooxygenase 2 (COX-2) inhibitor (meloxicam), or a soluble tumor necrosis factor alpha (TNF-alpha) receptor/Fc construct (TNFR-Fc, etanercept). High-dose aspirin, etanercept, and high-dose meloxicam each reduced leukocyte adhesion and suppressed blood-retinal barrier breakdown. High-dose aspirin also reduced the expression of CD11a, CD11b, and CD18, whereas meloxicam and etanercept did not. High-dose aspirin, etanercept, and high-dose meloxicam each reduced retinal ICAM-1 expression. Aspirin and meloxicam both lowered retinal TNF-alpha levels. Notably, aspirin, meloxicam, and etanercept did not change retinal vascular endothelial growth factor levels. High-dose aspirin, etanercept and high-dose meloxicam, each suppressed the retinal expression of eNOS and the DNA-binding capacity of retinal nuclear factor-kappaB. High-dose aspirin also suppressed Erk kinase activity, which is involved in CD18 up-regulation. Taken together, these data identify COX-2 and TNF-alpha as operative in the early signature pathologies of diabetic retinopathy, a newly recognized inflammatory disease.

Elements of cerebral microvascular ischaemia.

Although neuronal cells have long been thought to be the prime target of ischaemic insults, events which occur at the blood-vascular-parenchymal interface are necessary for the initiation of ischaemic tissue injury. This cascade of microvascular events includes fibrin accumulation, endothelium expression of leukocyte adhesion receptors, breakdown of the basal laminae with loss of astrocyte and endothelial cell contacts leading to blood-brain barrier disruption and consequently oedema formation and haemorrhagic transformation. Potential stroke treatments have been studied in the clinic and many have not been particularly successful, probably due to the delicate balance between improved outcome and adverse reactions as well as the window of opportunity for drug treatment after symptom onset. The only acute intervention trial demonstrating any benefit in patients was that of intravenous tissue plasminogen activator (tPA), administered within 3 h of the onset of symptoms of ischaemic stroke. Such treatment improved clinical outcome at 3 months, although there was an increased incidence of symptomatic haemorrhage [New Engl. J. Med. 333 (1995) 1581]. The recent progress made in defining the mechanisms involved in the initiation of ischaemic events, as described in this review, may lead to the identification of new strategies for intervention in the ischaemic cascade.

Effect of LFA-1beta antibody on leukocyte adherence in response to hemorrhagic shock in rats.

The activation and adherence of leukocytes to the venular endothelium are critical steps in the pathogenesis of generalized microvascular injury following hemorrhagic shock. Previous studies have shown that the integrins CD11/CD18 play a significant role in this interaction. The purpose of this study is to examine the efficacy of anti-LFA-1beta, an antibody to CD11a/CD18, in attenuating leukocyte adherence before, during, and after hemorrhagic shock. Following a control period, blood was withdrawn to reduce the mean arterial pressure to 40 mm Hg for 30 min in urethane-anesthetized rats. Mesenteric venules in a transilluminated segment of the small intestines were examined to quantitate leukocyte adherence using intravital microscopy. In sham-operated rats (control), there was minimal to no leukocyte adherence throughout the experiment. Hemorrhagic shock resulted in significant leukocyte adherence during resuscitation (10.8 +/- 1.7 cells/100 microm, P < 0.01) when compared to control. Anti-LFA-1beta, when given before hemorrhagic shock, significantly attenuated leukocyte adherence during resuscitation (1.1 +/- 0.8, P < 0.01) when compared with hemorrhagic shock alone. This protective effect of anti-LFA-1beta on leukocyte adherence was even demonstrated when it was given during (1.6 +/- 0.3, P < 0.01) and 10 min after hemorrhagic shock (5.8 +/- 0.4, P < 0.05). These results suggest that anti-LFA-1beta may be of potential therapeutic benefit against microvascular injury caused by hemorrhagic shock.

Functional relevance of activated beta1 integrins in mercury-induced nephritis.

Cell adhesion through different adhesion molecules is a crucial event in the inflammatory response. Integrins can only bind and mediate cellular adhesion after their activation by different specific stimuli. The state of beta1 integrin activation can be assessed by a group of monoclonal antibodies (HUTS) that selectively recognize beta1 integrins in their active form. A similar activated epitope in the rat was defined using the anti-human monoclonal antibody HUTS-21, which recognizes an activation-dependent epitope on the beta1 chain. It was found that the divalent cations Mn(2+) and Hg(2+) were able to induce in vitro the activation of beta1 integrins on rat lymphocytes. The Hg(2+) cation induces an autoimmune disease in the Brown Norway rat characterized by synthesis and glomerular deposits of anti-glomerular basement membrane antibodies, proteinuria, and interstitial nephritis. Using the mercury model of nephritis, it was found that the expression of HUTS-21 epitope is induced in vivo in rat lymphocytes, and its appearance is correlated with the other parameters at the onset of the disease. In addition, the administration of HUTS-21 monoclonal antibody to HgCl(2)-treated rats offered evidence of its protective effects (1) against infiltration of renal interstitium by leukocytes, and (2) in the reduction of anti-glomerular basement membrane synthesis and glomerular deposition. Nevertheless, urinary protein values remained unaffected. These results demonstrate a key role of beta1-activated integrins in both leukocyte cell-cell interactions and leukocyte infiltration pathway mechanism, and also indicate that leukocyte migration may have less importance in the development of this disease than previously thought.

Integrin-mediated neutrophil adhesion and retinal leukostasis in diabetes.

PURPOSE: A critical early event in the pathogenesis of diabetic retinopathy is leukocyte adhesion to the diabetic retinal vasculature. The process is mediated, in part, by intercellular adhesion molecule-1 (ICAM-1) and results in blood-retinal barrier breakdown and capillary nonperfusion. This study evaluated the expression and function of the corresponding ICAM-1-binding leukocyte beta2-integrins in experimental diabetes. METHODS: Diabetes was induced in Long Evans rats with streptozotocin. The expression of the surface integrin subunits CD11a, CD11b, and CD18 on rat neutrophils isolated from peripheral blood was quantitated with flow cytometry. In vitro neutrophil adhesion was studied using quantitative endothelial cell-neutrophil adhesion assays. The adhesive role of the integrin subunits CD11a, CD11b, and CD18 was tested using specific neutralizing monoclonal antibodies. CD18 bioactivity was blocked in vivo with anti-CD18 F(ab')2 fragments, and the effect on retinal leukocyte adhesion was quantitated with acridine orange leukocyte fluorography. RESULTS: Neutrophil CD11a, CD11b, and CD18 surface integrin levels were 62% (n = 5, P = 0.006), 54% (n = 5, P = 0.045), and 38% (n = 5, P = 0.009) greater in diabetic versus nondiabetic animals, respectively. Seventy-five percent more neutrophils from diabetic versus nondiabetic animals adhered to rat endothelial cell monolayers (n = 6, P = 0.02). Pretreatment of leukocytes with either anti-CD11b or anti-CD18 antibodies lowered the proportion of adherent diabetic neutrophils by 41% (n = 6, P = 0.01 for each treatment), whereas anti-CD11a antibodies had no significant effect (n = 6, P = 0.5). In vivo, systemic administration of anti-CD18 F(ab')2 fragments decreased diabetic retinal leukostasis by 62% (n = 5, P = 0.001). CONCLUSIONS: Neutrophils from diabetic animals exhibit higher levels of surface integrin expression and integrin-mediated adhesion. In vivo, CD18 blockade significantly decreases leukostasis in the diabetic retinal microvasculature. Integrin adhesion molecules may serve as therapeutic targets for the treatment and/or prevention of early diabetic retinopathy.

Nitric oxide synthase inhibitor augments post-ischemic leukocyte adhesion in the cerebral microcirculation in vivo.

The objective was to examine the effect of the nitric oxide synthase inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME) on leukocyte adhesion in the cerebral microcirculation during reperfusion following partial forebrain ischemia in the rat. Intravital fluorescence video-microscopy through a closed cranial window was used to visualize leukocyte-endothelium interaction in small pial veins of 15-100 microns diameter. Forebrain ischemia was produced by the ligation of both common carotid arteries plus elevation of the intracranial pressure to 20 mmHg for 60 min. The number of leukocytes adhering to the endothelium for longer than 3 sec was determined during ischemia (5 min and 60 min) and during reperfusion (5 min and 60 min). Two experimental groups were treated with either L-NAME or its inactive enantiomer D-NAME (20 mg kg-1 i.v.) 30 min prior to reperfusion. In a third group, also treated with D-NAME, post-ischemic hyperemia was prevented by lowering the ICP without removing the occlusion of common carotid arteries (partial reperfusion). The velocity of flow adjacent to the endothelial surface of pial veins was measured by tracking the movement of fluorescently labeled red blood cells as flow markers before and after ischemia. During ischemia, the number of adhering leukocytes increased approximately two-fold at 5 min, and three-fold at 60 min. In the D-NAME-treated group with complete reperfusion, leukocyte adhesion returned to the baseline level by 60 min of reperfusion. However, in the L-NAME-treated group, leukocyte adhesion remained elevated at 60 min of reperfusion. Post-ischemic flow velocity was significantly decreased (-66%) from control after L-NAME treatment whereas it was increased (+53%) in the D-NAME-treated group. In the partial reperfusion group, leukocyte adhesion continued to increase after the first hour of ischemia and reached a level 2.7-fold over baseline at 60 min reperfusion. Flow velocity remained below control (-26%) at 60 min reperfusion. Leukocyte adhesion was absent in pial arteries and no plugging by leukocytes was observed in cortical capillaries. The results suggest that leukocyte adhesion in small pial veins increases during 1 h forebrain ischemia and continues to increase during reperfusion if the velocity of flow or shear rate is low. The increase in leukocyte adhesion is reversible if flow velocity is elevated during reperfusion. L-NAME prevents post-ischemic hyperemia and augments leukocyte adhesion principally via a decrease in velocity or shear rate.

Beta 2 (CD11/CD18) integrins can serve as signaling partners for other leukocyte receptors.

Fig. 1 depicts our current thinking about the ways in which Mo1 and p150,95 form cis interactions with other leukocyte receptors. With respect to the associations of Mo1 with Fc gamma RIIIB and uPAR, the inhibitory effect of saccharides such as NADG suggests a lectin-carbohydrate interaction that may involve the recognition of Mo1's beta-glucan site for N-linked carbohydrates4 that are expressed by both Fc gamma RIIIB and uPAR. This hypothesis is supported by the results of Stockl et al., who showed that the binding of C-terminal-specific mAb VIM12 to Mo1, which enhances the phospholipase C-mediated release of Fc gamma RIIIB, was inhibited by NADG. However, unlike the sample lectin-carbohydrate interaction that appears to govern the association between Mo1 and Fc gamma RIIIB, effective Mo1-dependent uPAR signaling also depends on the binding of intact uPA to uPAR (the receptor-binding ATF of uPA proving insufficient to prime neutrophils for an enhanced burst response to FMLP). We speculate that ATF (residues 6-135) binds to uPAR while the carboxyl terminal fragment (residues 136-411), which includes a glycosylation site at residue 144, binds to the lectinlike site of Mo1, thus fostering the linkage between the two receptors. In support of this model is the fact that exposure of neutrophils to ATF reduced the degree of molecular proximity between Mo1 and uPAR (the latter probably occupied by endogenous intact uPA) and increased the molecular association between Mo1 and Fc gamma RIIIB (both as detected by quantitative RET). This hypothesis is analogous to the concept proposed by Nykjaer et al in which plasminogen activator inhibitor-1 initially binds to uPA to form a complex that secondarily binds to the alpha 2 macroglobulin receptor, leading to internalization of the complex. Whereas the contribution of intact uPA to the interaction between Mo1 and uPAR remains speculative (based on the indirect data available), no such ambiguity exists for the role of the LPS/LBP ligand in regulating the association between Mo1 and CD14. In this circumstance, no physical linkage exists between the two receptors without the ligand complex. This observation is consistent with the previously described affinity of the beta 2 integrins for LPS, leading to the notion that the LPS portion of the LPS/LPB complex binds to Mo1, serving to link it with LPS/LBP bound to CD14. The observed reversibility of the interactions between the integrin glycoproteins and uPAR or CD14 illustrates the fact that these associations can be highly dynamic and tied to cellular processes that include directed motility (Mo1-uPAR), adherence to substrates (Mo1-CD14), and energy metabolism (p150,95-uPAR). We speculate that the GPI-anchored receptor proteins serve as rapidly diffusible, expendable "scouts" for the beta 2 integrins, which serve to expand their ligand binding repertoire in a cis-acting fashion.

Animal membrane receptors and adhesive molecules.

This review summarizes some important data, principles, opinions, commentaries, and modern methodology concerning the receptor structure, interactions, signaling and receptor-mediated cell functions. Three sections give a brief overview of the signaling synergy, multivariant signaling, and some reactions in phosphorylation networks. A concise report about the cytotoxic reaction of NK cells represents an example of multistage recognition reaction, involving differently acting receptors, changes in affinities of cell-cell interactions, and secretion of regulatory and cytotoxic molecules. Some interesting trends in receptor engineering, including antibody molecules as a special receptor phenomenon are mentioned in the final section.

DNA-fragmentation and apoptosis-related proteins of muscle cells in motor neuron disorders.

Apoptosis has been described as one of the mechanisms of muscle fiber loss in infantile spinal muscular atrophy. In order to investigate if muscle fiber-apoptosis plays a role in other denervating disorders as well, we studied DNA-fragmentation, a hallmark of apoptosis, by the TUNEL-method and, moreover, the expression patterns of apoptosis-related proteins in 2 patients suffering from ALS and in 6 patients with polyneuropathy. We identified DNA-cleavage in muscle fibers of all these patients. Furthermore, we found strong expression of bax and ICE promoting apoptosis in muscle fibers. However, also strong expression of the anti-apoptotic factor bcl-2 was found. Our findings indicate that defective innervation may prompt muscle fibers to activate an intrinsic "suicide" programme which is promoted by the proapoptotic factors bax and ICE, which seems to induce formation of apoptotic bodies by cleavage of actin. Nevertheless, there are also anti-apoptotic strategies in muscle fibers manifested by expression of the bax-antagonist bcl-2 which is able to neutralize high bax levels.