Adhesion Of Polymorphonuclear Granulocytes Research Articles

Differential Adhesion of Polymorphous Neutrophilic Granulocytes to Macro- and Microvascular Endothelial Cells under Flow Conditions

Objective: As one of the important active barriers in the human organism, endothelial cells (EC) play a central role in the biological reaction to a variety of stimuli, e.g. during the induction and regulation of inflammation, as well as in the reaction to transplantation and biomaterial implantation. In the study of endothelial function, the most widely used in vitro model is that of human umbilical vein EC (HUVEC), i.e. an EC type of embryonic and macrovascular origin. However, many of the important pathological processes occur at microvascular level, thus questioning the validity of the HUVEC model. Moreover, the morphological and functional heterogeneity of the endothelium in the various organs, e.g. kidney, liver and lung, must be taken into consideration. The purpose of the present study was to use a dynamic cell culture system to compare the reactions of HUVEC and human pulmonary microvascular EC (HPMEC) to pro-inflammatory stimulation. Methods: HUVEC and HPMEC in monolayer culture were stimulated by tumor necrosis factor-α (TNFα) in a parallel-plate flow chamber. Short- (4 h) and long-term (12 h) stimulation were compared. As a functional parameter, the adhesion of human peripheral blood polymorphonuclear granulocytes (PMN) to EC was quantitated both under venous and arterial flow conditions. Results: Short-term (4 h) TNFα stimulation and venous flow conditions elicited a 32% higher PMN adhesion to HPMEC compared with HUVEC, whereas under arterial flow conditions no statistically significant differences were found. Following longer-term (12 h) TNFα stimulation, PMN adhesion to HPMEC was 65% higher than to HUVEC under venous flow. Under arterial flow no differences were detected. Conclusion: The present results provide new data on the heterogeneity of the endothelium and affect a central element in microvascular pathology, namely granulocyte-endothelial interactions. Moreover, this paper emphasizes the necessity to evaluate the in vitro models of the endothelium with respect to the extrapolation to the situation in vivo.

Polymicrobial sepsis induces organ changes due to granulocyte adhesion in a murine two hit model of trauma

Polytrauma patients, who develop organ dysfunction, have often undergone multiple subsequent insults ("hits"). The sequence of organs that show a dysfunction mostly is lung, liver, kidney and heart. The aim of the present study was to investigate whether a second hit after trauma induces organ changes. Furthermore, it was of interest to identify possible pathogenic mediators such as polymorphonuclear granulocytes (PMN) and cytokines. For this purpose, a two hit model of systemic damage in mice was developed. Sepsis was induced by caecal ligation and puncture (CLP), which was preceded 48 hours by a femur fracture, the most common fracture of long bones in trauma patients. This fracture was combined with a haemorrhagic shock. In both mouse groups studied, a standardized femur fracture was produced using a blunt guillotine device with a weight of 500 g. This was followed by a haemorrhagic shock with substitution of ringer's lactate after 1 hour. In the study group, CLP was induced by puncturing the caecum using a 21G needle. As a control, sham animals underwent a laparotomy without CLP. Both groups were sacrificed after 48 or 96 hours. Clinical parameters were investigated on a daily basis to evaluate the animals' status. Lung, liver and kidney morphology was studied by light microscopy. PMN adhesion was determined by counting the number of adherent PMN per 100 microm of endothelium. Serum levels of TNF-alpha were measured after 48 and 96 hours. In the group submitted to laparotomy, all animals survived. The induction of polymicrobial sepsis by CLP resulted in an 85% (34/40) mortality within 96 hours after surgery (p < 0.05). The induction of a polymicrobial sepsis resulted in a significantly steady worsening of the clinical situation compared to the sham animals (p < 0.05). Lung morphology demonstrated significant changes at the end of the experimental period after 96 h in the two hit group. The alveolar septa were thickened and in all lungs haemorrhagic foci were observed. The number of PMN adhering to the pulmonary endothelium significantly increased at 96 hours. Some of the liver specimens in the two hit group showed focal hydropic degeneration and PMN infiltration. No kidney pathology was observed. This result coincided with an increase in TNF-alpha serum levels. A new rodent model mimicking the situation in the polytraumatized patient was developed. Although the animals showed minimal organ manifestation, a high percentage died probably due to cytokinemia. Furthermore, the increased TNF-alpha levels may lead to increased adhesion of PMN in the lung venules. This adhesion developed four days after the second hit. This might be the initial step for the development of extensive lung lesions in later phases. This model represents the SIRS more than MODS. This is a model for devolopment of posttraumatic disease due to cytokinemia and less for chronic multiple organ dysfunction and failure.

Cell Adhesion and Migration Properties of β2-Integrin Negative Polymorphonuclear Granulocytes on Defined Extracellular Matrix Molecules: RELEVANCE FOR LEUKOCYTE EXTRAVASATION

Regulated adhesion of leukocytes to the extracellular matrix is essential for transmigration of blood vessels and subsequent migration into the stroma of inflamed tissues. Although beta(2)-integrins play an indisputable role in adhesion of polymorphonuclear granulocytes (PMN) to endothelium, we show here that beta(1)- and beta(3)-integrins but not beta(2)-integrin are essential for the adhesion to and migration on extracellular matrix molecules of the endothelial cell basement membrane and subjacent interstitial matrix. Mouse wild type and beta(2)-integrin null PMN and the progranulocytic cell line 32DC13 were employed in in vitro adhesion and migration assays using extracellular matrix molecules expressed at sites of extravasation in vivo, in particular the endothelial cell laminins 8 and 10. Wild type and beta(2)-integrin null PMN showed the same pattern of ECM binding, indicating that beta(2)-integrins do not mediate specific adhesion of PMN to the extracellular matrix molecules tested; binding was observed to the interstitial matrix molecules, fibronectin and vitronectin, via integrins alpha(5)beta(1) and alpha(v)beta(3), respectively; to laminin 10 via alpha(6)beta(1); but not to laminins 1, 2, and 8, collagen type I and IV, perlecan, or tenascin-C. PMN binding to laminins 1, 2, and 8 could not be induced despite surface expression of functionally active integrin alpha(6)beta(1), a major laminin receptor, demonstrating that expression of alpha(6)beta(1) alone is insufficient for ligand binding and suggesting the involvement of accessory factors. Nevertheless, laminins 1, 8, and 10 supported PMN migration, indicating that differential cellular signaling via laminins is independent of the extent of adhesion. The data demonstrate that adhesive and nonadhesive interactions with components of the endothelial cell basement membrane and subjacent interstitium play decisive roles in controlling PMN movement into sites of inflammation and illustrate that beta(2)-integrins are not essential for such interactions.

Indirect enhancement of neutrophil activity and adhesion to cultured human umbilical vein endothelial cells by isoprostanes (iPF 2α-III and iPE 2-III)

Isoprostanes are metabolites of arachidonic acid found in blood under various conditions of oxidative stress. Because arachidonic acid derivatives are major mediators of inflammation, we investigated the potential inflammatory effects of iPF 2α-III (previously 8-isoPGF 2α) and iPE 2-III (8-isoPGE 2) on human polymorphonuclear granulocytes (PMN), as well as on human umbilical vein endothelial cells (HUVECs). The early activation marker CD11b on PMN and the adhesion molecules ICAM-1, E-selectin, and P-selectin on HUVECs were quantified by flow cytometry. Levels of the cytokines interleukin (IL)-6 and IL-8 were measured in the culture supernatant by enzyme-linked immunosorbent assay. Furthermore, adhesion of PMN to HUVECs was assessed. Neither isoprostane showed any direct stimulatory effects on PMN or HUVECs at concentrations of 0.1 or 1 μM: there was no acute elevation in expression of CD11b or P-selectin and no change of ICAM-1 or E-selectin after 4 or 24 h of incubation, respectively. The levels of interleukin IL-6 and IL-8 were also unaltered. However, PMN adhesion was significantly enhanced both after 4 and 24 h of incubation of HUVECs with iPF 2α-III, and CD11b expression on PMN was elevated by contact of these cells with the supernatant of pre-exposed HUVECs. Neither of these actions were inhibited by an endothelin receptor antagonist (bosentan) or a combined thromboxane A2/isoprostane-receptor antagonist (SQ29548). Thus, although not having a direct pro-inflammatory potential, isoprostanes might indirectly accentuate PMN stimulation. This seems to occur via a receptor-independent mechanism, perhaps the production of an active metabolite of isoprostanes by endothelial cells.

Involvement of ecto-ATPase and extracellular ATP in polymorphonuclear granulocyte-endothelial interactions

The adhesion of human polymorphonuclear granulocytes (PMN) with confluent human endothelial cells (line EAhy926) and with solid substrate coated by collagen and fibronectin (Fn) was studied by phase contrast microscopy and by the measurement of myeloperoxidase activity. The ecto-ATPase inhibitors suramin and Reactive Blue 2 (RB2) more than doubled the adhesion of PMN to endothelial cells. The cells hydrolyzed added ATP and this reaction was inhibited by suramin and RB2. The degree of ATP hydrolysis during PMN adherence depended on solid substrata and decreased in the order: non-stimulated endothelial cells, TNF-stimulated endothelial cells, collagen-coated surface, Fn-coated surface. In the same order adherence increased. The endogenous level of extracellular ATP in the PMN-endothelial coculture was around 25 nM. We conclude that PMN-endothelial adhesion is counteracted by an ecto-ATPase or by ATP receptors with ATPase activity. Such interactions may play a role in PMN rolling and diapedesis as well as in the pathophysiology of PMN activation by an anergic endothelium.

Mouse polymorphonuclear granulocyte binding to extracellular matrix molecules involves beta 1 integrins.

The mechanism of adhesion of purified mouse polymorphonuclear granulocytes (PMN) to extracellular matrix proteins characteristic of basement membranes and the interstitium has been investigated and compared with the adhesion of a mouse progranulocytic cell line, 32DC13, and a mouse monocytic cell line, WEHI 78/24. All three cell types bound specifically to fibronectin and vitronectin to different degrees under different cellular activation states. 32DC13 bound to fibronectin and vitronectin strongly, and this binding increased upon cellular activation with phorbol 12-myristate-13-acetate (PMA) but not with formyl-Met-Leu-Phe. Only 32DC13 showed significant binding to laminin-1. By contrast, WEHI 78/24 and PMN bound only fibronectin and vitronectin; this binding was weak and was altered only marginally upon activation with PMA. In the case of WEHI 78/24, a slight increase in adhesion both to fibronectin and to vitronectin was observed after cellular activation with PMA, while PMN adhesion to both substrates was slightly reduced. The mechanism of binding to fibronectin and vitronectin was similar in the three cell types. The integrin alpha5 beta1 mediated fibronectin adhesion, demonstrating for the first time the existence of a functionally active beta1 integrin on mouse PMN. Vitronectin binding was mediated by alpha(v) beta3, as demonstrated by the ability of alpha(v)-specific cyclic L-Arg-L-Gly-L-Asp-D-Phe-L-Val (RGDfV) peptide (EMD66203), and anti-beta3 antibody to inhibit cell adhesion. 32DC13 adhesion to laminin-1 was via the alpha6 beta1 integrin. None of the three cell types tested bound to the basement membrane proteins collagen type IV and perlecan, or to the interstitial stromal constituents tenascin, collagen types I, V and VI. Interestingly, perlecan and collagen type IV were found to repel all three cell types. The relative inability of PMN, WEHI 78/24, and 32DC13 to bind to extracellular matrix proteins characteristic of basement membranes and their ability to bind inflammatory markers of the interstitium is discussed with respect to leukocyte extravasation processes.

The Leukocyte Integrin Mac-1 (CD11b/CD18) Contributes to Binding of Human Granulocytes to Collagen

Adhesion of polymorphonuclear granulocytes (PMN) to extracellular matrix proteins has been shown to be important for their migration in vitro and is thought to participate in PMN recruitment to sites of inflammation. Isolated human PMN stimulated with PMA were found to adhere best to microtiter wells coated with the novel ECM glycoprotein undulin (27 ± 3% of PMNs added), followed by fibrinogen (25 ± 2%), collagen type VI (18 ± 2%), fibronectin (16 ± 2%), and laminin (15 ± 3%). PMN adhesion to other collagens ranged between 3 and 11%. Monoclonal antibodies recognizing CD18 and CD11b subunits of Mac-1 inhibited adhesion of PMN to collagens by an order of magnitude more effectively than to all noncollagenous substrates. F(ab′) 2 fragments of the anti-CD18 antibody were also able to block adhesion to collagens. Anti-LFA-1 (CD11a) and anti-CD44 antibodies did not significantly reduce adhesion. PMN adhesion was also inhibited by soluble collagens type II and VI (ID 50 approximately 75 μg/ml). Binding of soluble radiolabeled collagens type II and VI to PMNs was specific and saturable with apparent dissociation constants of 2.2 and 1.9 n M, respectively, and specific binding of collagens type II and VI was almost completely inhibited by anti-CD18, but not by control antibodies. These data indicate that Mac-1 function is required for binding of human PMN to collagens.

Polymorphonuclear granulocyte expression of CD11a/CD18, CD11b/CD18 and L-selectin in normal individuals

In this study direct immunofluorescence and flow cytometry with calibration using quantitative bead standards were used to enumerate the cell surface receptors CD11a/CD18, CD11b/CD18 and L-selectin. Holding blood at room temperature and fixation of samples prior to staining induced changes in expression, while immediate staining of polymorphonuclear granulocytes (PMN) in whole blood followed by fixation produced accurate values. The ranges of PMN adhesion molecule expression in 10 normal individuals were CD11a/CD18: 14794–28725, CD11b/CD18: 5300–11939 and L-selectin: 35662–61654 receptors per cell. Differences within individuals over 4 h were also observed. Adhesion molecule expression is used as an index of the adhesive function and state of activation of the cell. The data presented here shows that there is inherent variability in the expression of the PMN adhesion molecules between and within individuals, thus direct comparisons of PMN adhesion molecule expression between patients and “normals” must be interpreted with caution.

Polymorphonuclear granulocyte expression of CD11a/CD18, CD11b/CD18 and L-selectin in normal individuals.

In this study direct immunofluorescence and flow cytometry with calibration using quantitative bead standards were used to enumerate the cell surface receptors CD11a/CD18, CD11b/CD18 and L-selectin. Holding blood at room temperature and fixation of samples prior to staining induced changes in expression, while immediate staining of polymorphonuclear granulocytes (PMN) in whole blood followed by fixation produced accurate values. The ranges of PMN adhesion molecule expression in 10 normal individuals were CD11a/CD18: 14794-28725, CD11b/CD18: 5300-11939 and L-selectin: 35662-61654 receptors per cell. Differences within individuals over 4 h were also observed. Adhesion molecule expression is used as an index of the adhesive function and state of activation of the cell. The data presented here shows that there is inherent variability in the expression of the PMN adhesion molecules between and within individuals, thus direct comparisons of PMN adhesion molecule expression between patients and "normals" must be interpreted with caution.

Differential adhesion of granulocytes to five distinct phenotypes of cultured microvascular endothelial cells

Adhesion of isolated human polymorphonuclear granulocytes (PMNs) to five different phenotypes of cultured microvascular endothelial cells derived from bovine corpora lutea was investigated by measuring the myeloperoxidase content of cell lysates. Untreated and interleukin 1 (IL-1) -pretreated confluent monolayers were overlaid with unstimulated and phorbol ester (PMA)-stimulated PMNs in the absence and presence of the monoclonal antibody IB4 recognizing and functionally blocking β 2 (CD18) of the leukocyte integrins. Unstimulated PMN adhesion was highest on type 4, followed by type 3 and 5 endothelial cells. This adhesion was not inhibited by treatment with IB4. IL-1 pretreatment of endothelial cells resulted in a significant increase of PMN adhesion on types 1, 2, and 4, most of which was also β 2 integrin-independent. PMA-stimulation of PMNs increased adhesion to maximal values on cell types 1 and 5, which was largely blocked by IB4. Type 2 endothelial cells supported significantly less PMA-stimulated PMN adhesion than all other types. In the presence of IB4, adhesion of PMNs to untreated and IL-1-pretreated type 3 and 4 endothelial cells was significantly reduced by PMA. This reduction of β 2 integrin-independent adhesion by PMA stimulation is compatible with possible shedding of the lectin-like leukocyte adhesion molecule, l-selectin, from PMNs. Differential PMN adhesion may reflect distinctive expression of endothelial adhesion molecules in different phenotypes of microvascular endothelial cells. Endothelial specialization within the microcirculation may have important functional consequences for the inflammatory response in vivo.

The peripheral lymph node homing receptor, LECAM-1, is involved in CD18-independent adhesion of human neutrophils to endothelium

The binding of polymorphonuclear granulocytes (PMN) to activated vascular endothelium is a crucial step in the recruitment of PMN to an inflammatory site. Studies employing cytokine-activated endothelium in culture have shown that PMN binding involves the CD18 family of leukocyte integrins, but also CD18-independent adhesion mechanism(s) on PMN that have not been defined. We unify here two previously disparate approaches to study cell adhesion events between endothelial cells and leukocytes. We show that antibodies to human LECAM-1, the peripheral lymph node homing receptor that is also expressed on PMN, partially inhibit the adhesion of human PMN not only to HEV in frozen sections of lymph node tissue, but also to cytokine-activated human umbilical vein endothelium in vitro. Inhibition with anti-LECAM-1 antibodies and anti-CD18 antibodies is additive. Furthermore, the anti-LECAM-1 antibodies inhibit the adhesion of CD18-deficient PMN to cytokine activated human endothelial cells. These findings indicate that LECAM-1 and CD18-mediated binding mechanisms are independent, and act coordinately or sequentially to mediate PMN attachment to cytokine activated endothelium.

In vitro determination of the force required for detachment of human polymorphonuclear granulocytes (PMN) from cultured endothelium and immobilized albumin

Detachment of adherent human PMN from cultured endothelial cells (human umbilical vein, HUVEC) and from immobilized albumin (bovine serum albumin, BSA) was investigated as a function of shear stress. Non-activated PMN did not adhere to either of the substrates «2%). 20 min after activation with PMA (phorbol myristate acetate, 10 nM), 48 ± 5% (mean ± SEM, n=15) of the total number of PMN added had adhered to albumin, and 18 ± 4% (n=9) to HUVEC. The dishes with pre-adhered PMN were subjected to shear stress in the range occurring in venules in vivo, using a rotating plate-and-cone (cone angle 2.50 ) apparatus made of transparent plastic to allow observation of the cells during the assay. On HUVEC, the majority of adhered PMN (66 ± 3%, n=9) was removed by a shear stress of 0.12 Pa (1.2 dyn/cm2), while the same shear stress removed only 15 ± 5% (n=9) of the PMN adhered to BSA. Further increase of shear stress to 2.3 Pa (23 dyn/cm2) removed 39 ± 4% (n=14) of the PMN adhered to BSA. Using spherical and semispherical approximations for the shape of the adhered PMN, the adhesive forces between PMA-activated PMN and HUVEC were calculated to be on the order of 0.05 nN per PMN, while most PMN on BSA adhered with a force of more than 4 nN per PMN. The force needed to detach PMN from HUVEC is more than one order of magnitude lower than value reported for PMN adhesion to venular endothelium in vivo. The present results suggest that the endothelium may playa regulatory role in PMN adhesion and limit the attachment force to allow emigration.

Shear-dependent inhibition of granulocyte adhesion to cultured endothelium by dextran sulfate

Adhesion of polymorphonuclear granulocytes (PMNs) in microvessels occurs in the presence of shear forces exerted by the blood flow. To model this in vitro, phorbol myristate acetate (PMA)-activated PMN were exposed to shear stress on cultured human umbilical vein endothelial cells (HUVECs) and on plastic dishes coated with bovine serum albumin (BSA). PMN adhesion to HUVECs averaged 36% of the total PMNs added and was reduced to 21% by shear stress of approximately 1.5 dynes.cm-2. On BSA, adhesion was reduced from 59% to 35%. Dextran sulfate (molecular weight 500,000) inhibited PMN adhesion in a dose-dependent manner when shear stress was applied. At a concentration of 1 mg.ml-1, inhibition was 72% on HUVECs and 76% on BSA. Half-maximal inhibition was reached at approximately 1 microgram.mL-1 dextran sulfate, corresponding to 2 nmol/L. Without shear stress, dextran sulfate had no effect on HUVECs and only a moderate effect on BSA. The murine monoclonal antibody (MoAb) 60.3, recognizing an epitope on the leukocyte adhesion glycoprotein CD18, inhibited PMN adhesion equally well with and without shear. A low dose of MoAb 60.3 enhanced the effect of dextran sulfate without shear stress. Flow cytometry (FACS) did not show inhibition of MoAb 60.3 binding to PMNs by dextran sulfate. These results indicate that a dextran sulfate-inhibitable adhesion process is important for PMN adhesion in the presence of shear stress.

Shear-Dependent Inhibition of Granulocyte Adhesion to Cultured Endothelium by Dextran Sulfate

Adhesion of polymorphonuclear granulocytes (PMNs) in microvessels occurs in the presence of shear forces exerted by the blood flow. To model this in vitro, phorbol myristate acetate (PMA)-activated PMN were exposed to shear stress on cultured human umbilical vein endothelial cells (HUVECs) and on plastic dishes coated with bovine serum albumin (BSA). PMN adhesion to HUVECs averaged 36% of the total PMNs added and was reduced to 21% by shear stress of approximately 1.5 dynes.cm-2. On BSA, adhesion was reduced from 59% to 35%. Dextran sulfate (molecular weight 500,000) inhibited PMN adhesion in a dose-dependent manner when shear stress was applied. At a concentration of 1 mg.ml-1, inhibition was 72% on HUVECs and 76% on BSA. Half-maximal inhibition was reached at approximately 1 microgram.mL-1 dextran sulfate, corresponding to 2 nmol/L. Without shear stress, dextran sulfate had no effect on HUVECs and only a moderate effect on BSA. The murine monoclonal antibody (MoAb) 60.3, recognizing an epitope on the leukocyte adhesion glycoprotein CD18, inhibited PMN adhesion equally well with and without shear. A low dose of MoAb 60.3 enhanced the effect of dextran sulfate without shear stress. Flow cytometry (FACS) did not show inhibition of MoAb 60.3 binding to PMNs by dextran sulfate. These results indicate that a dextran sulfate-inhibitable adhesion process is important for PMN adhesion in the presence of shear stress.

Effects of Nontransferrin‐bound Iron on the Aggregation and Adhesion of Polymorphonuclear Granulocytes (PMN)

Annals of the New York Academy of SciencesVolume 526, Issue 1 p. 363-364 Effects of Nontransferrin-bound Iron on the Aggregation and Adhesion of Polymorphonuclear Granulocytes (PMN) I. M. Hoepelman, I. M. Hoepelman Department of Medicine University Hospital Utrecht, The NetherlandsSearch for more papers by this authorJ. Verhoef, J. Verhoef Department of Microbiology University Hospital Utrecht, The NetherlandsSearch for more papers by this authorJ. J. M. Marx, J. J. M. Marx Department of Medicine University Hospital Utrecht, The Netherlands Department of Hematology University Hospital Utrecht, The NetherlandsSearch for more papers by this author I. M. Hoepelman, I. M. Hoepelman Department of Medicine University Hospital Utrecht, The NetherlandsSearch for more papers by this authorJ. Verhoef, J. Verhoef Department of Microbiology University Hospital Utrecht, The NetherlandsSearch for more papers by this authorJ. J. M. Marx, J. J. M. Marx Department of Medicine University Hospital Utrecht, The Netherlands Department of Hematology University Hospital Utrecht, The NetherlandsSearch for more papers by this author First published: June 1988 https://doi.org/10.1111/j.1749-6632.1988.tb55529.xCitations: 1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article.Citing Literature Volume526, Issue1Hemochromatosis: Proceedings of the First International ConferenceJune 1988Pages 363-364 RelatedInformation