Homotypic Neutrophil Aggregation Research Articles

The Aminopeptidase CD13 Induces Homotypic Aggregation in Neutrophils and Impairs Collagen Invasion.

Aminopeptidase N (CD13) is a widely expressed cell surface metallopeptidase involved in the migration of cancer and endothelial cells. Apart from our demonstration that CD13 modulates the efficacy of tumor necrosis factor-α-induced apoptosis in neutrophils, no other function for CD13 has been ascribed in this cell. We hypothesized that CD13 may be involved in neutrophil migration and/or homotypic aggregation. Using purified human blood neutrophils we confirmed the expression of CD13 on neutrophils and its up-regulation by pro-inflammatory agonists. However, using the anti-CD13 monoclonal antibody WM-15 and the aminopeptidase enzymatic inhibitor bestatin we were unable to demonstrate any direct involvement of CD13 in neutrophil polarisation or chemotaxis. In contrast, IL-8-mediated neutrophil migration in type I collagen gels was significantly impaired by the anti-CD13 monoclonal antibodies WM-15 and MY7. Notably, these antibodies also induced significant homotypic aggregation of neutrophils, which was dependent on CD13 cross-linking and was attenuated by phosphoinositide 3-kinase and extracellular signal-related kinase 1/2 inhibition. Live imaging demonstrated that in WM-15-treated neutrophils, where homotypic aggregation was evident, the number of cells entering IL-8 impregnated collagen I gels was significantly reduced. These data reveal a novel role for CD13 in inducing homotypic aggregation in neutrophils, which results in a transmigration deficiency; this mechanism may be relevant to neutrophil micro-aggregation in vivo.

S55 The Aminopeptidase CD13 Regulates Homotypic Aggregation of Neutrophils

Neutrophils are critical effector cells of the innate immune response and are recruited to sites of tissue injury in response to locally generated chemoattractants. Neutrophil recruitment is a highly regulated...

Signaling molecules differentiate single versus dual ligation of complement receptor 3

Complement Receptor 3 (CR3), an integrin found on neutrophils, regulates firm cell adhesion to the extracellular matrix and serves as a pattern recognition receptor for the fungal Pathogen Associated Molecular Pattern (PAMP) β‐glucan. Coincident ligation of CR3 with fibronectin and β‐glucan is possible due to spatially distinct ligand binding domains. Current work demonstrates that dual ligation of CR3 with fibronectin at its I‐domain and β‐glucan at its lectin‐like site causes homotypic aggregation of primed human neutrophils. The downstream signaling pathways resulting from differential CR3 ligation are unknown. Using phosphoproteomics, we have identified several proteins that are significantly phosphorylated when ligated with fibronectin and β‐Glucan but not either ligand alone. Phosphoinositide 3 Kinase (PI3K), Protein Kinase Cδ (PKCδ), Glycogen Synthase Kinase 3β (GSK3β), and ERK show increased phosphorylation and each may play a role in the homotypic aggregation of neutrophils via CR3. Western Blotting has validated our initial phosphoproteomic findings for each protein. Use of inhibitors obviated homotypic aggregation of neutrophils for all aforementioned proteins except for GSK3β. Thus PI3K, PKCδ, and ERK are all components of the signaling pathway of dually ligated CR3 that are also relevant to homotypic aggregation.

Depletion of tissue plasminogen activator attenuates lung ischemia-reperfusion injury via inhibition of neutrophil extravasation

Ischemia-reperfusion (IR) injury following lung transplantation remains a major source of early morbidity and mortality. Histologically, this inflammatory process is characterized by neutrophil infiltration and activation. We previously reported that lung IR injury was significantly attenuated in plasminogen activator inhibitor-1-deficient mice. In this study, we explored the potential role of tissue plasminogen activator (tPA) in a mouse lung IR injury model. As a result, tPA knockout (KO) mice were significantly protected from lung IR injury through several mechanisms. At the cellular level, tPA KO specifically blocked neutrophil extravasation into the interstitium, and abundant homotypic neutrophil aggregation (HNA) was detected in the lung microvasculature of tPA KO mice after IR. At the molecular level, inhibition of neutrophil extravasation was associated with reduced expression of platelet endothelial cell adhesion molecule-1 mediated through the tPA/ LDL receptor-related protein/NF-κB signaling pathway, whereas increased P-selectin triggered HNA. At the functional level, tPA KO mice incurred significantly decreased vascular permeability and improved lung function following IR. Protection from lung IR injury in tPA KO mice occurs through a fibrinolysis-independent mechanism. These results suggest that tPA could serve as an important therapeutic target for the prevention and treatment of acute IR injury after lung transplantation.

Differential Regulation of Neutrophil CD18 Integrin Function by Di- and Tri-Valent Cations: Manganese vs. Gadolinium

Affinity regulation of integrin function plays an important role during both leukocyte-endothelial and leukocyte-leukocyte interactions. We compared the roles of Mn(2+) (Manganese) and Gd(3+) (Gadolinium) in regulating leukocyte CD18-integrin function. We observed that: (i) Both cations prolonged neutrophil homotypic aggregation following chemoattractant IL-8 stimulation, with Gd(3+) being effective at doses two orders of magnitude (10 microM range) lower that Mn(2+). (ii) While both Gd(3+) and Mn(2+) mediate homotypic cell aggregation via L: -selectin and CD18 integrins, their effects on the integrin subunits, LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18), was different. Gd(3+) altered both LFA-1 and Mac-1 function, while the dominant effect of Mn(2+) was on Mac-1. This effect of Gd(3+) on LFA-1 function was confirmed in cell-free studies that measured the binding of recombinant ICAM-1 to LFA-1 immobilized on beads. (iii) Both ions augmented the binding of 327C, an antibody that recognizes active CD18 on human neutrophils, both in the presence and absence of exogenous IL-8. The effects of Mn(2+) was more pronounced since it caused 3-4-fold increase in mAb 327C binding to neutrophils compared to Gd(3+) which increased antibody binding by only approximately 80%. 327C binding was partially reduced by Ca(2+). Further, 327C binding induced by Mn(2+) did not correlate tightly with cell adhesion function. (iv) In studies that monitored intracellular Ca(2+) ([Ca(2+)](i)), the addition of Mn(2+) but not Gd(3+) to neutrophils altered [Ca(2+)](i) levels. Overall, while both Gd(3+) and Mn(2+) stabilize high affinity CD18 mediated cell adhesion, Gd(3+) affects integrin conformation while Mn(2+) may also trigger other effects.

YC-1 attenuates homotypic human neutrophil aggregation through inhibition of phosphodiesterase activity

This study was undertaken to assess the effects of 3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole (YC-1), a known activator of soluble guanylyl cyclase, on formyl- l-methionyl- l-leucyl- l-phenylalanine (FMLP) and complement component 5a (C5a)-induced homotypic human neutrophil aggregation. YC-1 as well as the phosphodiesterase (PDE)4 inhibitors rolipram and Ro 20-1724, but not the PDE3 inhibitor milrinone, inhibited the aggregation responses stimulated by FMLP and C5a. In contrast, sodium nitroprusside (SNP) had no effect on FMLP- or C5a-induced neutrophil aggregation. Moreover, SNP together with YC-1 failed to modify the YC-1-induced responses. In addition, YC-1 and rolipram, but not milrinone, induced substantial increases in cAMP levels, which occurred through the inhibition of PDE activity but not an increase in adenylate cyclase function. Interestingly, adenosine deaminase abolished the inhibitory effects and cAMP levels of YC-1, rolipram, and Ro 20-1724. In conclusion, these results indicate that the inhibitory effect of YC-1 on homotypic neutrophil aggregation is attributed to an elevation in the cAMP concentration through inhibition of the activity of PDE, which may potentiate the autocrine functions of endogenous adenosine.

Valvular interstitial cell mechanobiology: effects of substrate stiffness

During inflammation, neutrophil capture by vascular endothelial cells is dependent on L-selectin and β2-integrin adhesion receptors. One of us (S.I.S.) previously demonstrated that homotypic neutrophil aggregation is analogous to this process in that it is also mediated by these receptors, thus providing a model for studying the dynamics of neutrophil adhesion. In the present work, we set out to confirm the hypothesis that cell–cell adhesion via selectins serves to increase the lifetimes of neutrophil doublets formed through shear-induced two-body collisions. In turn, this would facilitate the engagement of more stable β2-integrin bonds and thus increase the two-body collision efficiency (fraction of collisions resulting in the formation of nonseparating doublets). To this end, suspensions of unstimulated neutrophils were subjected to a uniform shear field in a transparent counter-rotating cone and plate rheoscope, and the formation of doublets and growth of aggregates recorded using high-speed videomicroscopy. The dependence of neutrophil doublet lifetime and two-body collision-capture efficiency on shear rate, G, from 14 to 220 s−1 was investigated. Bond formation during a two-body collision was indicated by doublets rotating well past the orientation predicted for break-up of doublets of inert spheres. A striking dependence of doublet lifetime on shear rate was observed. At low shear (G = 14 s−1), no collision capture occurred, and doublet lifetimes were no different from those of neutrophils pretreated with a blocking antibody to L-selectin, or in Ca++-depleted EDTA buffers. At G ≥ 66 s−1, doublet lifetimes increased, with increasing G reaching values twice those for the L-selectin-blocked controls. This correlated with capture efficiencies in excess of 20%, and, at G ≥ 110 s−1, led to the rapid formation of large aggregates, and this in the absence of exogenous chemotactic stimuli. Moreover, the aggregates almost completely broke up when the shear rate was reduced below 66 s−1. Partial inhibition of aggregate formation was achieved by blocking β2-integrin receptors with antibody. By direct observation of the shear-induced interactions between neutrophils, these data reveal that steady application of a threshold level of shear rate is sufficient to support homotypic neutrophil aggregation.

Effects of a snake venom metalloproteinase, triflamp, on platelet aggregation, platelet-neutrophil and neutrophilneutrophil interactions: involvement of platelet GPIbα. and neutrophil PSGL-1

The biologically active components from Viperidae venoms specifically affect cell-matrix interactions, and have been utilized for developing anti-adhesive therapy as anti-thrombotic and anti-angiogenic agents. Utilizing platelet aggregometry coupled with flow cytometry, we found that a metalloproteinase isolated from Trimeresurus flavoviridis, termed triflamp, inhibited heterotypic adhesion between platelets and neutrophils in whole blood samples. Triflamp is a monomeric glycoprotein with a molecular weight of approximately 28 kDa. Triflamp has a N-terminal amino acid sequence homologous to other venom metalloproteinases isolated from T. flavoviridis. The enzymatic activity of triflamp was inhibited by EDTA and phenanthroline but not by PMSF. Moreover, triflamp is a pure alpha-fibrinogenase. Studies aimed at determining the nature of triflamp in affecting platelets or neutrophils revealed a selective inhibitory activity to glycoprotein (GP) Ibalpha-dependent platelet aggregation and PSGL-1-dependent neutrophil homotypic aggregation, indicating that its effects are rather specific. As judged by Western blotting, GPIbalpha on platelets and PSGL-1 on neutrophils are the substrates of triflamp. In conclusion, we suggest the novel role of venom metalloproteinase from Viperidae affecting the blood cell-cell interactions, thus offering a potential approach for further exploration of anti-inflammatory agents.

Effects of heparin and related molecules upon neutrophil aggregation and elastase release in vitro.

1 Neutrophil-derived elastase is an enzyme implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). Heparin inhibits the enzymatic activity of elastase and here we provide evidence for the first time that heparin can inhibit the release of elastase from human neutrophils. 2 Unfractionated and low molecular weight heparins (UH and LMWH, 0.01-1000 U ml(-1)) and corresponding concentrations (0.06-6000 micro g ml(-1)) of nonanticoagulant O-desulphated heparin (ODH), dextran sulphate (DS) and nonsulphated poly-L-glutamic acid (PGA) were compared for their effects on both elastase release from and aggregation of neutrophils. 3 UH, ODH and LMWH inhibited (P<0.05) the homotypic aggregation of neutrophils, in response to both N-formyl-methionyl-leucyl-phenylalanine (fMLP, 10(-6) M) and platelet-activating factor (PAF, 10(-6) M), as well as elastase release in response to these stimuli, in the absence and presence of the priming agent tumour necrosis factor-alpha (TNF-alpha, 100 U ml(-1)). 4 DS inhibited elastase release under all the conditions of cellular activation tested (P<0.05) but had no effect on aggregation. PGA lacked efficacy in either assay, suggesting general sulphation to be important in both effects of heparin on neutrophil function and specific patterns of sulphation to be required for inhibition of aggregation. 5 Further investigation of the structural requirements for inhibition of elastase release confirmed the nonsulphated GAG hyaluronic acid and neutral dextran, respectively, to be without effect, whereas the IP(3) receptor antagonist 2-aminoethoxydiphenylborate (2-APB) mimicked the effects of heparin, itself an established IP(3) receptor antagonist, suggesting this to be a possible mechanism of action.

Dynamics of Neutrophil Aggregation in Couette Flow Revealed by Videomicroscopy: Effect of Shear Rate on Two-Body Collision Efficiency and Doublet Lifetime

During inflammation, neutrophil capture by vascular endothelial cells is dependent on L-selectin and β 2-integrin adhesion receptors. One of us (S.I.S.) previously demonstrated that homotypic neutrophil aggregation is analogous to this process in that it is also mediated by these receptors, thus providing a model for studying the dynamics of neutrophil adhesion. In the present work, we set out to confirm the hypothesis that cell–cell adhesion via selectins serves to increase the lifetimes of neutrophil doublets formed through shear-induced two-body collisions. In turn, this would facilitate the engagement of more stable β 2-integrin bonds and thus increase the two-body collision efficiency (fraction of collisions resulting in the formation of nonseparating doublets). To this end, suspensions of unstimulated neutrophils were subjected to a uniform shear field in a transparent counter-rotating cone and plate rheoscope, and the formation of doublets and growth of aggregates recorded using high-speed videomicroscopy. The dependence of neutrophil doublet lifetime and two-body collision-capture efficiency on shear rate, G, from 14 to 220 s −1 was investigated. Bond formation during a two-body collision was indicated by doublets rotating well past the orientation predicted for break-up of doublets of inert spheres. A striking dependence of doublet lifetime on shear rate was observed. At low shear ( G = 14 s −1), no collision capture occurred, and doublet lifetimes were no different from those of neutrophils pretreated with a blocking antibody to L-selectin, or in Ca ++-depleted EDTA buffers. At G ≥ 66 s −1, doublet lifetimes increased, with increasing G reaching values twice those for the L-selectin-blocked controls. This correlated with capture efficiencies in excess of 20%, and, at G ≥ 110 s −1, led to the rapid formation of large aggregates, and this in the absence of exogenous chemotactic stimuli. Moreover, the aggregates almost completely broke up when the shear rate was reduced below 66 s −1. Partial inhibition of aggregate formation was achieved by blocking β 2-integrin receptors with antibody. By direct observation of the shear-induced interactions between neutrophils, these data reveal that steady application of a threshold level of shear rate is sufficient to support homotypic neutrophil aggregation.

Nonlinear Flow Affects Hydrodynamic Forces and Neutrophil Adhesion Rates in Cone–Plate Viscometers

We present a theoretical and experimental analysis of the effects of nonlinear flow in a cone–plate viscometer. The analysis predicts that flow in the viscometer is a function of two parameters, the Reynolds number and the cone angle. Nonlinear flow occurs at high shear rates and causes spatial variations in wall shear stress, collision frequency, interparticle forces and attachment times within the viscometer. We examined the effect of these features on cellular adhesion kinetics. Based on recent data (Taylor, A. D., S. Neelamegham, J. D. Hellums, et al. 1996. Biophys. J. 71:3488–3500), we modeled neutrophil homotypic aggregation as a process that is integrin-limited at low shear and selectin-limited at high shear. Our calculations suggest that selectin and integrin on-rates lie in the order of 10 −2–10 −4/s. They also indicate that secondary flow causes positional variations in adhesion efficiency in the viscometer, and that the overall efficiency is dependent not only on the shear rate, but also the sample volume and the cone angle. Experiments performed with isolated neutrophils confirmed these predictions. In these experiments, enhancing secondary flow by increasing the sample volume from 100 to 1000 μl at 1500/s for a 2° cone caused up to an ∼45% drop in adhesion efficiency. Our results suggest that secondary flow may significantly influence cellular aggregation, platelet activation, and endothelial cell mechanotransduction measurements made in the viscometer over the range of conditions applied in typical biological studies.

LFA-1/ICAM-3 mediates neutrophil homotypic aggregation under fluid shear stress.

We found that human neutrophils undergo homotypic aggregation by loading the physiological range of fluid shear stress (12-30 dynes/cm2). Under the fluid shear stress, an increase of intracellular Ca2+ concentration of neutrophils was observed. This increase of intracellular Ca2+ concentration was caused by Ca2+ influx, and the blockage of the flux by NiCl2 suppressed the neutrophil homotypic aggregation. Furthermore, this neutrophil aggregation under fluid shear stress was completely inhibited by pretreatment with antibody against LFA-1 or ICAM-3. These results suggested that NiCl2-sensitive Ca2+ channel played an important role in LFA-1/ICAM-3-mediated neutrophil homotypic aggregation under fluid shear stress.

Phosphatidylinositol 3-Kinase Mediates Chemoattractant-Stimulated, CD11b/CD18-Dependent Cell-Cell Adhesion of Human Neutrophils: Evidence for an ERK-Independent Pathway

We examined the role of phosphatidylinositol 3-kinase (PI 3-K) in FMLP-stimulated cell-cell adhesion of human neutrophils. The specific PI 3-K inhibitors wortmannin and LY294002 inhibited neutrophil homotypic aggregation stimulated by chemoattractants such as FMLP (50% inhibitory concentration (IC50) approximately 11 nM and 13 microM, respectively) but not PMA. Wortmannin also inhibited FMLP-stimulated adhesion of neutrophils to human endothelial cell monolayers, suggesting a common signaling pathway for homotypic and heterotypic adhesion. Neither CD11b/CD18 expression nor expression of an activation-specific epitope of CD11b/CD18 was affected by wortmannin in FMLP-stimulated cells. Moreover, wortmannin also inhibited the aggregation of egranulate neutrophil cytoplasts that lack the capacity for CD11b/CD18 up-regulation. Although wortmannin inhibited neutrophil lysosomal enzyme release, it had no effect on FMLP-stimulated up-regulation of CD35 in intact neutrophils, suggesting discrepant signaling pathways for specific granule degranulation and secretory vesicle release. Aggregation of human neutrophils is associated with activation of the mitogen-activated protein kinases Erk1 and -2, and Erk is activated in response to PI 3-K in some cell types. However, wortmannin inhibited FMLP stimulation of neutrophil Erk only at concentrations (IC50 > or = 1 microM) inconsistent with an effect on PI 3-K. Our data indicate that PI 3-K mediates neutrophil adhesion by a mechanism independent of CD11b/CD18 up-regulation, suggesting that PI 3-K acts either parallel to, or downstream of, Erk.

The multistep process of homotypic neutrophil aggregation: a review of the molecules and effects of hydrodynamics.

Homotypic adhesion of neutrophils stimulated with chemoattractant is analogous to capture on vascular endothelium in that both processes are supported by L-selectin and beta 2-integrin adhesion receptors. Under hydrodynamic shear, cell adhesion requires that receptors bind sufficient ligand over the duration of intercellular contact to withstand the hydrodynamic stresses. Using cone and plate viscometry to apply a uniform linear shear field to suspensions of neutrophils and flow cytometry to quantitate the size distribution of aggregates formed over the time course of formyl peptide stimulation, we conducted a detailed examination of the affect of shear rate and shear stress on the kinetics of cell aggregation. The efficiency of aggregate formation was fit from a mathematical model based on Smoluchowski's two-body collision theory. Over a range of venular shear rates (400-800 s-1), approximately 90% of the single cells are recruited into aggregates ranging from doublets to grouping larger than sextuplets. Adhesion efficiency fit to the kinetics of aggregation increased with shear rate from approximately 20% at 100 s-1 to a maximum level of approximately 80% at 400 s-1. This increase to peak adhesion efficiency was dependent on L-selectin and beta 2-integrin, and was resistant to shear stress up to approximately 7 dyn/cm2. When L-selectin was blocked with antibody, beta 2-integrin (CD11a,b) supported adhesion at low shear rates (< 400 s-1). Aggregates formed over the rapid phase of aggregation remain intact and resistant to shear up to 120 s. At the end of this plateau phase of stability, aggregates spontaneously dissociate back to singlets. The rate of cell disaggregation is linearly proportional to the applied shear rate. The binding kinetics of selectin and integrin appear to be optimized to function within discrete ranges of shear rate and stress, providing an intrinsic mechanism for the transition from neutrophil tethering to firm but reversible adhesion.

Modeling the reversible kinetics of neutrophil aggregation under hydrodynamic shear

Neutrophil emigration into inflamed tissue is mediated by beta 2-integrin and L-selectin adhesion receptors. Homotypic neutrophil aggregation is also dependent on these molecules, and it provides a model system in which to study adhesion dynamics. In the current study we formulated a mathematical model for cellular aggregation in a linear shear field based on Smoluchowski's two-body collision theory. Neutrophil suspensions activated with chemotactic stimulus and sheared in a cone-plate viscometer rapidly aggregate. Over a range of shear rates (400-800 s-1), approximately 90% of the single cells were recruited into aggregates ranging from doublets to groupings larger than sextuplets. The adhesion efficiency fit to these kinetics reached maximum levels of > 70%. Formed aggregates remained intact and resistant to shear up to 120 s, at which time they spontaneously dissociated back to singlets. The rate of cell disaggregation was linearly proportional to the applied shear rate, and it was approximately 60% lower for doublets as compared to larger aggregates. By accounting for the time-dependent changes in adhesion efficiency, disaggregation rate, and the effects of aggregate geometry, we succeeded in predicting the reversible kinetics of aggregation over a wide range of shear rates and cell concentrations. The combination of viscometry with flow cytometry and mathematical analysis as presented here represents a novel approach to differentiating between the effects of hydrodynamics and the intrinsic biological processes that control cell adhesion.

The anti-FcγRIII MAb 3G8 induces neutrophil activation via a cooperative action of FcγRIIIb and FcγRIIa

Human neutrophils express two types of Fcγ receptors, the transmembrane FcγRIIa and the glycan-phosphatidylinositol-anchored FcγRIIIb, that show synergism in provoking a cellular response. To analyse further the requirements for this synergism to occur we used the monoclonal antibody 3G8, directed against FcγRIII This antibody is able to induce neutrophil activation, as measured by an increase in the intracellular free Ca 2+ concentration and homotypic neutrophil aggregation, but only when the Fe part of the antibody is able to interact with FcγRIIa. We observed that binding of the Fab parts of 3G8 mAb to two FcγRIIIb molecules and binding of the Fc part to one FcγRIIa molecule is required, because a bispecific antibody, 2B1, in which only one 3G8 Fab is present, did not induce neutrophil activation. Moreover, engagement of one FcγRIIa molecule and two FcγRIIIb molecules on the same cell is instrumental to achieve activation of the mAb 3G8. The activation of neutrophils by the 3G8 antibody represents a further example of synergistic activation of neutrophils via Fcγ receptors.

Fluid shear stress induces actin polymerization in human neutrophils

We have previously reported that a physiological range of shear stress induces neutrophil homotypic aggregation mediated by lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-3 (ICAM-3) interactions. To further characterize the homotypic aggregation, actin polymerization was investigated in neutrophils stimulated by shear stress in comparison with formyl-methionyl-leucyl-phenylalanine (fMLP). In fMLP-stimulated neutrophils, actin polymerization was localized in the pseudopods, and this reaction was not mediated by a cytosolic level of Ca2+. In contrast to fMLP stimulation, the actin polymerization induced by shear stress in a cone-plate viscometer was localized in cell-cell contact regions, and this polymerization required the increase of intracellular Ca2+. This shear stress-induced actin polymerization was not observed when neutrophils were pretreated with anti-LFA-1 or anti-ICAM-3 antibody. In conclusion, LFA-1 and ICAM-3 interaction mediated by the increase of [Ca2+]i generated the intercellular signal in order to accumulate F-actin in the cell-cell contact regions. © 1996 Wiley-Liss, Inc.

The effect of bovine seminal plasma on the function and integrity of bovine neutrophils

Spermatozoa, by virtue of their high content of polyunsaturated fatty acids and limited antioxidant activity, are highly sensitive to lipid peroxidation. Sources of reactive oxygen species in the female genital tract include neutrophils recruited in response to insemination or those contained within the ejaculate, or even spermatozoa themselves. This study was undertaken to investigate the effect of seminal plasma of bulls on bovine neutrophils. Seminal plasma was removed from ejaculates of 6 healthy bulls by centrifugation. Neutrophils were isolated from jugular venous blood samples obtained from lactating Holstein cows. Seminal plasma was found to decrease measured Superoxide anion production by activated neutrophils (P = 0.008) in a concentration-dependent manner over the range tested (0 to 16%). This effect may reflect Superoxide scavenging by the seminal plasma or direct inhibition of the neutrophils. It has the potential physiological advantage of protecting spermatozoa from oxidative damage. Seminal plasma was found to reduce homotypic neutrophil aggregation dramatically (P < 0.0001). At a seminal plasma concentration of 0.2% neutrophil aggregation was virtually abolished. Homotypic neutrophil aggregation depends on the CD 11b/CD 18 (Mac-1) receptor, which also functions as the C3bi receptor (known as complement receptor-3 or CR3). If this receptor is down-regulated, the ability of neutrophils to phagocytose complement-opsonized microorganisms would be compromised. Finally, seminal plasma was found to increase neutrophil permeability to trypan blue in a concentration and time dependent fashion (P < 0.0001), indicating a direct cytotoxic effect. Taken together, these findings indicate a profound inhibition of neutrophil function by bovine seminal plasma. While these mechanisms are likely to protect spermatozoa from oxidative damage or phagocytosis, they also have the potential to diminish defense against pathogenic microorganisms.

LFA-1/ICAM-3 mediates neutrophil homotypic aggregation under fluid shear stress

We found that human neutrophils undergo homotypic aggregation by loading the physiological range of fluid shear stress (12–30 dynes/cm2). Under the fluid shear stress, an increase of intracellular Ca2+ concentration of neutrophils was observed. This increase of intracellular Ca2+ concentration was caused by Ca2+ influx, and the blockage of the flux by NiCl2 suppressed the neutrophil homotypic aggregation. Furthermore, this neutrophil aggregation under fluid shear stress was completely inhibited by pretreatment with antibody against LFA-1 or ICAM-3. These results suggested that NiCl2-sensitive Ca2+ channel played an important role in LFA-1/ICAM-3-mediated neutrophil homotypic aggregation under fluid shear stress. © 1996 Wiley-Liss, Inc.

LFA‐1/ICAM‐3 mediates neutrophil homotypic aggregation under fluid shear stress

We found that human neutrophils undergo homotypic aggregation by loading the physiological range of fluid shear stress (12–30 dynes/cm2). Under the fluid shear stress, an increase of intracellular Ca2+ concentration of neutrophils was observed. This increase of intracellular Ca2+ concentration was caused by Ca2+ influx, and the blockage of the flux by NiCl2 suppressed the neutrophil homotypic aggregation. Furthermore, this neutrophil aggregation under fluid shear stress was completely inhibited by pretreatment with antibody against LFA-1 or ICAM-3. These results suggested that NiCl2-sensitive Ca2+ channel played an important role in LFA-1/ICAM-3-mediated neutrophil homotypic aggregation under fluid shear stress. © 1996 Wiley-Liss, Inc.