Receptors for the chemoattractants C5a and IL-8 are clustered on the surface of human neutrophils.

We have used high-resolution field emission scanning electron microscopy with backscatter electron imaging to detect immunogold-labeled C5a and interleukin-8 (IL-8) receptors on human blood neutrophils. The receptors were labeled with receptor-specific antibodies in combination with secondary antibody conjugated to immunogold. When neutrophils were isolated in a "nonactivated" state, both of these receptor populations were expressed primarily in clusters on nonprojecting domains of the cell membrane. When these cells were double labeled for C5a and IL-8 receptors, intermixing of these receptor species in a common cluster was not found. When neutrophils were isolated in an "activated" state, by mixing the blood with N-formylmethionyl-leucyl-phenylalanine, the cells were seen to be elongated and ruffled at their anterior pole, but the C5a receptors did not disperse or redistribute on the surface of the peptide-activated cells. Analysis of the distribution of human C5a receptors expressed by transfected mouse L-cell fibroblasts showed the C5a receptors to be clustered, but expressed on nonprojecting and projecting domains of the cell surface. These observations provide new information on the topographical expression of leukocyte receptors involved in directing cell migration.

Preservation of spatial organization and antigenicity of leukocyte surface molecules by aldehyde fixation: flow cytometry and high-resolution FESEM studies of CD62L, CD11b, and Thy-1.

We used transmission and scanning electron microscopy in conjunction with immunogold labeling to study cell surface molecules for evidence of distribution-function relationships. Ascription of functional significance to surface distribution therefore requires preservation of cell morphology and maintenance of molecular expression and distribution through the multiple steps of cell preparation. These requirements prompted us to compare two methods for preparing leukocytes for analysis of surface molecule distribution: one method involved using low temperature to "stabilize" cell morphology and surface molecular organization through immunolabeling; the other involved fixation of the cells with dilute glutaraldehyde before their isolation and labeling. Binding of primary antibodies to several surface molecules, measured by flow cytometry, was comparable for cells prepared by the two methods. Cell morphology and molecular distributions, assessed by high-resolution field emission SEM, were likewise comparable. These results support the conclusion that cell morphologies and CAM distributions previously reported were not affected by exposure of the cells to low temperature through isolation and immunolabeling. Our additional observation that Thy-1 is expressed on both non-projecting and projecting membrane domains of mouse lymph node lymphocytes and rat thymocytes represents a third and new pattern of surface molecule distribution.

A central role for microvillous receptor presentation in leukocyte adhesion under flow.

Leukocyte adhesion to endothelium requires specialized mechanisms for contact initiation under flow. L-selectin (CD62L), an efficient initiator of adhesion, is clustered on the tips of leukocyte microvilli. To test whether microvillous presentation is critical for contact formation ("tethering"), we transfected lymphoid cells with chimeras of L-selectin and CD44, an adhesion molecule that is excluded from microvilli. CD44 transmembrane and intracellular (TM-IC) domains targeted the L-selectin ectodomain to the planar body, whereas L-selectin TM-IC segments conferred CD44 ectodomain clustering on microvilli. Wild-type and chimeric transfectants bound similarly to anti-ectodomain MAbs in static assays, but MAb binding under flow was much more efficient in the context of microvillous presentation. Similarly, wild-type and chimeric L-selectin possessed equivalent lectin activity, but microvillous presentation dramatically enhanced contact initiation on a native ligand. These findings demonstrate a critical role for receptor topography in leukocyte adhesion and suggest a novel regulatory mechanism of leukocyte trafficking.

Spatial distribution of L-selectin (CD62L) on human lymphocytes and transfected murine L1-2 cells.

We have examined the topographical distribution of L-selectin on surface membrane domains of human lymphocytes and murine L1-2 cells transfected to express human L-selectin. L-selectin was immunolocalized using murine monoclonal DREG 200 Fab antibody and a 12 nm colloidal gold-conjugated secondary antibody. Cell surface morphology and surface distribution of gold-labelled L-selectin were visualized using backscatter electron images obtained by high-resolution, field emission scanning electron microscopy. The topographical morphologies of lymphocytes of both types were complex. The surface of human lymphocytes was composed of both microvilli and ruffles; that of the murine cells was composed of long microvilli and few, if any, ruffles. L-selectin on human lymphocytes was observed primarily as focal clusters on the apical surfaces of ruffles and microvilli. Similarly, on the transfected murine cells, L-selectin was detected predominantly on the apical surface of microvilli. We conclude that L-selectin has a common spatial distribution and clustered organization on all leukocytes examined to-date, and that these features of receptor expression likely facilitate rolling of circulating leukocytes on the endothelial surface.

alpha 4 integrins mediate lymphocyte attachment and rolling under physiologic flow.

Of the several families of adhesion receptors involved in leukocyte-endothelial cell interactions, only the selectins have been shown to initiate leukocyte interaction under physiologic shear; indeed, beta 2 (CD18) intergrins responsible for neutrophil arrest are unable to engage without prior selectin-mediated rolling. In contrast, alpha 4 (CD49d) integrins are shown here to initiate lymphocyte contract ("tethering") in vitro under shear and in the absence of a selectin contribution. The alpha 4 integrin ligands MAdCAM-1 and VCAM-1 support loose reversible interactions including rolling, as well as rapid sticking and arrest that is favored following integrin activation. Moreover, alpha 4 beta 7 mediates L-selectin (CD62L)-independent attachment of blood-borne lymphocytes to lamina propria venules in situ. Scanning electron microscopy of alpha 4 beta 7hi lymphoid cells reveals that, like L-selectin, alpha 4 beta 7 is highly concentrated on microvillous sites of initial cellular contact, whereas the beta 2 integrin LFA-1 is excluded from villi. Thus, alpha 4 but not beta 2 integrins can initiate leukocyte adhesion under flow, a capacity that may be in part a function of topographic presentation on microvilli.

Thermal injury, the inflammatory process, and wound dressing reduce human neutrophil chemotaxis to four attractants.

The purpose of this study was to assess the influence of thermal injury and the inflammatory process on chemotactic responses of neutrophils to four attractants (N-formyl-methionyl-leucyl-phenylalanine, the complement fragment C5a, interleukin-8, and leukotriene B4) under agarose, expression of Mac-1 (CD11b/CD18) adherence receptors on the cell surface, and polymerization of actin in the cell cytoplasm. Circulating neutrophils were isolated from peripheral blood, and exudate neutrophils from fluid collecting under two different wound dressings applied to abrasion sites of healthy subjects and to skin graft donor sites of patients with burns. Burn injury reduced the chemotactic responses of circulating neutrophils to all four attractants, suggesting a "global" defect in chemotactic function. Patient-exudate neutrophils collected under Tegaderm exhibited further decrements in all chemotactic responses, and patient-exudate neutrophils collected under Biobrane were nonmotile. The exudate neutrophils collected under Biobrane expressed high levels of Mac-1 receptors and irreversibly polymerized actin, which may contribute to the nonmotility of these exudate cells.

Detection and spatial distribution of the beta 2 integrin (Mac-1) and L-selectin (LECAM-1) adherence receptors on human neutrophils by high-resolution field emission SEM.

We have developed a method utilizing high-resolution field emission SEM and backscatter electron imaging of immunogold for detection of cell adhesion receptors on the surface of unfixed human neutrophils, using indirect immunogold localization of specific murine monoclonal antibodies (MAb) to the cell adhesion receptors L-selectin (LECAM-1) and the beta 2 integrin (Mac-1). We have observed that these two receptor populations occupy different membrane domains on the surface of unactivated human neutrophils. LECAM-1 was observed to occur in clusters on the tips of microvilli or membrane ruffles and was seldom detected on the membrane of the cell body. On the other hand, Mac-1 was found mainly on the membrane of the cell body in unactivated neutrophils, either singly or in small clusters, and was only rarely encountered on microvilli or ruffles. In contrast, the distribution of Mac-1 on activated, spreading neutrophils was markedly increased (up-regulated) and occurred in clusters on both the membrane of the cell body and also of surface projections, i.e., microvilli and ruffles. The unique distributions of LECAM-1 and Mac-1 on the surface of unactivated human neutrophils, as observed by high-resolution LVSEM, confirm the spatial relationships of these receptor types as predicted by models for the attachment of circulating neutrophils to vascular endothelium and their emigration to sites of inflammation.

Actin polymerization contributes to neutrophil chemotactic dysfunction following thermal injury.

The agent(s) and mechanism(s) responsible for suppression of neutrophil chemotaxis in association with major thermal injury have not been identified. We have proposed that the reduced random motility characterizing patients' cells may contribute to their generalized chemotactic dysfunction. Here we report that actin polymerization may be responsible for the loss of neutrophil motility associated with major thermal injury. Using a fluorescent ligand specific for polymerized or filamentous actin (NBD-phallacidin) in conjunction with flow cytometry, we have discovered that peripheral blood and exudate neutrophils from patients with major thermal injury contain increased levels of actin in a stably polymerized form. Because cyclic polymerization and depolymerization of actin is essential to cell motility, we suggest that actin polymerization may contribute in a major way to the attenuation of neutrophil random and chemotactic functions induced by major thermal injury.

Down-regulation of homing receptors: a mechanism for impaired recruitment of human phagocytes in sepsis.

Receptors known as DREG adhesion molecules on human neutrophils and monocytes provide for homing of these phagocytic leukocytes to sites of inflammation. They mediate the initial adhesive interaction of the leukocytes to vascular endothelial cells and are then shed from the cell surface in response to chemotactic factors and inflammatory mediators. Systemic accumulation of these agents following major injury or sepsis may therefore promote shedding of DREG receptors from circulating leukocytes and impair their recruitment to sites of inflammation. To test this hypothesis, we have analyzed the expression of DREG receptors on neutrophils and monocytes from 25 patients admitted to the Surgical Intensive Care Unit. Receptor expression was measured by flow cytometry of cells stained with murine monoclonal DREG-56 anti-DREG antibody. For 14 nonseptic patients, mean monocyte positivity for DREG was reduced from 64% to 40%. For 11 septic patients, mean neutrophil and monocyte positivity for DREG was reduced from 94% to 82% and 64% to 34%, respectively. These results suggest that monocytes are more affected than neutrophils in vivo by conditions expected to stimulate shedding of DREG and that sepsis promotes shedding of these adherence receptors. Accumulation of DREG-negative monocytes in association with sepsis may be sufficient to impair their recruitment to inflammatory sites and limit their contribution to host defense against infection and tissue repair.

Mechanisms of loss of human neutrophil chemotaxis following thermal injury.

The increased susceptibility to infection of patients with thermal injury is related to loss of host defense, which is reflected, in part, by the temporal loss of chemotactic function of leukocytic phagocytes. Our studies of patient neutrophils to define the mechanism of this phenomenon involved evaluation of both random and chemotactic migratory functions of patient neutrophils, measurement of receptors for chemotactic ligands, and measurement of receptors mediating substrate adherence of the cells. Measurements of migratory functions were made using the under-agarose technique and measurements of receptor expression were accomplished by flow cytometry using fluorescein-labeled ligand or receptor-specific antibody. We conclude that loss of chemotaxis in response to C5a/C5adesArg is the results of down-regulation of receptors for C5a and of reduced motility, and that loss of chemotaxis in response to the tripeptide FMLP is the result of reduced motility alone. Measurements of changes in the expression of "adherence" (iC3b) receptors revealed that up-regulation occurs early and can be sustained for weeks after injury. These results are taken to suggest that either hyper- or hypo-adherence could explain the loss of random migratory function observed for patient cells. Evidence of auto-oxidative alteration of cytoskeletal elements, to produce loss of random migratory function, also is reviewed. Considering the evidence for activation of the complement cascade after thermal injury C5a and C5adesArg are likely primary factors in effecting the down-regulation of C5a receptors, stimulation of secretion to mobilize iC3b receptors, and stimulation of respiration to auto-oxidize cell components. Such evidence of injury-mediated complement activation included data derived from application of a novel immunoassay for iC3b.

Influence of minor thermal injury on expression of complement receptor CR3 on human neutrophils.

Thermal injury is well known to inhibit functions of the circulating neutrophil related to its role in host defense against infection, but the mechanism(s) of this phenomenon are not fully understood. To gain further clues to these mechanisms, the authors have studied patients with thermal injury in terms of altered expression of neutrophil cell membrane receptors for the opsonic complement-derived ligand C3bi--complement receptor Type 3, or CR3. CR3 expression was selected for study because an increase in the number of receptors on the cell surface can be stimulated by products of complement activation known to accumulate after thermal injury and because of the role of CR3 in phagocytic and adherence functions of the neutrophil. Expression of CR3 was monitored semiquantitatively by flow cytometry with the use of a murine monoclonal antibody (OKM1) specific for an antigen (CD11) associated with this receptor. Patients evaluated were limited in this study to those with minor degrees of thermal injury (second-degree burn involving less than 20% of total body surface area) so that possible confounding effects of major injury and its complications could be eliminated. It was observed that patient neutrophil CR3 becomes significantly up-regulated during the first week, as early as 1 day after injury. The maximum level of expression of CR3 averaged greater than 150% (range, 70-314%) of the respective minimum level observed for each patient. The minimum levels of expression of CR3 on patient neutrophils, reached 11-37 days after injury for 7 of 8 patients, were comparable to the level of expression of CR3 on unstimulated control neutrophils. Such temporal up-regulation of patient neutrophil CR3 suggests the early generation of stimuli of CR3 mobilization in response to thermal injury. Increased numbers of CR3 on patient neutrophils may augment microbicidal function and enhance or inhibit delivery of cells to the burn site.