Regulated expression and function of CD11c/CD18 integrin on human B lymphocytes. Relation between attachment to fibrinogen and triggering of proliferation through CD11c/CD18.

CD11c/CD18 (p150,95) is a beta 2 integrin expressed by myeloid, natural killer and certain lymphoid cells such as some cytotoxic T cell clones and B cell malignancies. We have studied the expression and function of CD11c on resting and activated B lymphocytes. Flow cytometry, immunoprecipitation, and mRNA analyses showed that cell activation with phorbol esters or with a variety of stimuli such as Staphylococcus aureus or anti-mu antibodies in combination with cytokines induced de novo CD11c/CD18 cell surface expression on most B cells while CD11b expression was not affected. Functional analysis of CD11c/CD18 on B cells revealed that it plays a dual role. First, CD11c/CD18 is implicated in B cell proliferation, as demonstrated by the ability of several anti-CD11c monoclonal antibodies to trigger comitogenic signals; and second, the newly expressed CD11c/CD18 mediates B cell binding to fibrinogen. Our data conclusively demonstrate the role of CD11c/CD18 on both B cell activation and adhesion processes.

Transfection of cells from patients with leukocyte adhesion deficiency with an integrin beta subunit (CD18) restores lymphocyte function-associated antigen-1 expression and function.

Leukocyte adhesion deficiency (LAD) is an inherited immunodeficiency disease that is characterized by the deficient expression of the leukocyte adhesion glycoproteins lymphocyte function-associated antigen-1 (LFA-1), Mac-1, and p150,95. This loss of expression is attributed to heterogeneous defects in the common beta subunit shared by these glycoproteins. Here we demonstrate that expression of the LFA-1 alpha beta heterodimer in EBV-transformed B lymphoblastoid cells from LAD patients can be recovered after transfection with the beta subunit cDNA contained in an EBV-based vector. Four patients with differing severities of LAD comprising three distinct classes of mutations were studied. Flow cytometry analysis of stably transfected patient cells revealed near normal levels of expression of both the alpha and beta chains of LFA-1, and immunoprecipitation studies confirmed that fully processed alpha and beta chains were being expressed at the cell surface. In addition, Northern analysis of mRNA expression also demonstrated that the transfected LAD patient cells were expressing high quantities of exogenous beta subunit mRNA. Functional studies such as homotypic adhesion and adhesion to a purified counterreceptor for LFA-1, intracellular adhesion molecule-1, demonstrated that LFA-1 function had been restored in the stably transfected LAD patient cell lines. These studies unequivocally show that the defect in cells from patients with LAD is in the leukocyte integrin beta subunit.

Neutrophil adherence to isolated adult canine myocytes. Evidence for a CD18-dependent mechanism.

Cardiac myocytes were isolated from adult dogs and incubated with isolated canine neutrophils (PMN). Intercellular adhesion was low and unchanged by stimulation of the PMN with zymosan activated serum or platelet activating factor (PAF) at concentrations that significantly enhance PMN adhesion to protein-coated glass and canine endothelial cell monolayers. Intercellular adhesion was significantly increased only when both myocytes and PMN were stimulated (e.g., myocytes incubated with IL-1, tumor necrosis factor, or phorbol myristate acetate, and PMN were chemotactically stimulated). Inhibitors of protein synthesis diminished the IL-1 beta-induced effect by greater than 80%. The IL-1 beta, PAF-stimulated PMN-myocyte adhesion was associated with substantial H2O2 production. Under conditions with low PMN-myocyte adhesion (i.e., IL-1 beta alone, PAF alone, or no stimulus) H2O2 production was generally less than 5% of that occurring with high adhesion. An anti-CD18 monoclonal antibody (R15.7) inhibited stimulated PMN-myocyte adhesion by greater than 95% and reduced H2O2 production by greater than 90%. Control isotype-matched, binding, and nonbinding antibodies were without effect on adherence or H2O2 production. The results indicate that cytokine stimulation of adult myocytes induces expression of a ligand involved in CD18-dependent adherence of canine neutrophils.

Identification of activated T cell receptor gamma delta lymphocytes in the liver of tumor-bearing hosts.

T cell receptor (TcR)gamma delta cells are known to be a minor population of T lymphocytes in the blood (less than 10%) and other peripheral lymphoid organs in healthy donors. We demonstrated here that a large proportion of TcR gamma delta cells, i.e., up to 30% of mononuclear cells (MNC) were detectable in the liver, but not other lymphoid organs of cancer patients. More importantly, the majority of such TcR gamma delta cells (greater than 70%) were shown to be lymphoblastic by electron microscopy. An activation marker of T lymphocytes, Leu-19 (CD56) was also highly expressed on the hepatic TcR gamma delta cells. The possibility of hepatic TcR gamma delta cells being activated was further examined in mice. C3H/He mice injected with syngeneic tumor cells were demonstrated to have an increased number of liver MNC; such MNC showed an ability to proliferate in vitro. These mice eventually had a considerable proportion of TcR gamma delta cells in the liver, showing activation markers, the Ia and LFA-1 antigens. These results suggest that the liver may be an important organ for activation and probably expansion of TcR gamma delta cells especially in tumor bearing hosts.

Recombinant human granulocyte-macrophage colony-stimulating factor in patients with advanced malignancy: a phase Ib trial.

We evaluated the toxic, hematopoietic, and immunomodulatory effects of recombinant human granulocyte-macrophage colony-stimulating factor (rHuGM-CSF). The rHuGM-CSF was administered at doses up to 50 micrograms/kg by daily 2-hour intravenous infusions to 11 patients with advanced malignancy. It induced dose-related increases in cells of the myeloid series, but it had no significant effect on reticulocyte or platelet counts. Bone marrow cellularity increased with higher doses of rHuGM-CSF, but there was a dose-related decrease in the number of colony-forming units--granulocyte-monocyte--and colony-forming units--granulocyte-erythrocyte-monocyte-megakaryocyte--per 10(5) bone marrow cells. The rHuGM-CSF caused transient increased expression of CD11b and CD16 on granulocytes but increased expression of HLA-DR and decreased expression of the high-affinity Fc receptor on monocytes and no change in monocyte production of H2O2. Thus, rHuGM-CSF has potent effects on granulocyte, eosinophil, and monocyte numbers in the peripheral blood and bone marrow. In addition, it enhances the expression of monocyte and granulocyte activation-associated surface markers.

Surface characteristics, morphology, and ultrastructure of human adherent lymphokine-activated killer cells.

Human adherent lymphokine-activated killer (A-LAK) cells represent a population of highly cytotoxic, interleukin-2 (IL-2)-activated peripheral blood lymphocytes that have large granular lymphocyte (LGL) morphology and display natural killer (NK) cell-associated surface markers (CD3-CD56+). The ultrastructure of A-LAK cells also is consistent with that of highly activated NK cells. After their initial isolation, continued culture of A-LAK cells in the presence of IL-2 resulted in cyclic shifts between adherent and non-adherent phases with about 90% of the cells floating and non-adherent. All of these A-LAK cells were spheroidal with numerous villi and pseudopodia. In the adherent phase, A-LAK cells were motile, moving along the solid surface at a speed of about 1 micron per minute, and polar, with a ruffled leading edge at one end of the cell and a terminal tuft of villi at the opposite end. Transmission electron microscopy of these cells also demonstrated a high degree of internal polarity, with the nucleus at the leading edge of the cell and richly granulated cytoplasm at the terminal end. Extensive cytoskeletal structures, multivesicular bodies, and an active Golgi complex characterized these cells. A-LAK cells in the adherent phase were found to express numerous point contacts (podosomes) and larger adherence structures containing polymerized actin, which appear to be important for interactions of these cells with the substrate. Increased expression of adhesion molecules in association with surface structures mediating adherence is also responsible for effective binding of A-LAK cells to solid surfaces.

Multiple receptors on human monocytes are involved in adhesion to cultured human endothelial cells.

Monocytes exhibit significant basal (unstimulated) adherence to human umbilical vein endothelium (HUVE), which is only partially inhibited by an anti-CD18 monoclonal antibody (mAb) (60.3). We examined factors modulating the residual, CD18-independent monocyte binding to HUVE by pretreating monocytes with mAb 60.3 to eliminate CD18-dependent binding. Basal adherence was reduced from 32% +/- 2% to 14% +/- 2% with mAb 60.3 (means +/- SE of eight experiments; P less than 0.01). mAb 60.3-treated monocytes were incubated with tumor necrosis factor-gamma (TNF-alpha), interleukin-1 (IL-1), lipopolysaccharide (LPS), N-formylmethionyl-leucyl-phenylalamine (FMLP), or phorbol myristate acetate (PMA). Only PMA affected CD18-independent binding. Pretreatment with PMA alone reduced adherence to 21% +/- 2% (mean +/- SE of eight experiments; P less than 0.01). In conjunction with mAb 60.3, PMA virtually eliminated monocyte adherence to HUVE (7% +/- 1%, mean +/- SE of eight experiments; P less than 0.01). We also examined CD18-independent monocyte binding to endothelial-leukocyte adhesion molecules (E-LAMs) induced by pretreatment of HUVE with LPS. Monoclonal antibody 60.3-treated monocytes increased adherence from 14% +/- 2% with unstimulated HUVE to 37% +/- 2% with LPS-stimulated HUVE (mean +/- SE of four experiments; P less than 0.01). Monocytes pretreated with both mAb 60.3 and PMA increased adherence from 5% +/- 1% with the unstimulated HUVE to 18% +/- 1% with the LPS-stimulated HUVE (mean +/- SE of four experiments; P less than 0.01). This result implies the presence of a CD18-independent and PMA-insensitive receptor on human monocytes for an E-LAM induced by LPS. In summary, we have identified two CD18-independent mechanisms of monocyte adherence to HUVE; a PMA-sensitive mechanism mediating basal adherence and a PMA-insensitive mechanism involved in binding to E-LAMs.

Characteristics of the chemotactic activity of heparin cofactor II proteolysis products.

The physiological function of the serpin (serine proteinase inhibitor) heparin cofactor II (HCII) is not well understood. A role for HCII as an inhibitor of thrombin in the presence of dermatan sulfate and heparin has been proposed. Neutrophils (PMN) are the major cellular component of acute inflammation. HCII can be proteolytically inactivated by cathepsin G (CG) and elastase (LE), which are released by stimulated PMN. We have recently shown that reaction products of HCII with CG and LE are potent chemotaxins for PMN. Monocytes (monos) appear later in the course of inflammation than do PMN. They differentiate into macrophages in the tissues and participate in healing of damaged tissue and initiating immune responses. We found that the proteolysis products of HCII were chemotactic for monocytes in a fashion similar to their effects on PMN. At 10(-8) to 10(-9) M, the chemotactic activity of HCII proteolysis products was comparable to that of 10(-8) M N-formyl-Met-Leu-Phe (fMLP). The chemotactic activity of HCII-proteinase reaction products is mediated by a different mechanism than that of alpha 1 proteinase inhibitor (alpha 1 PI)-LE complexes or fMLP. Our data suggest that chemotactic activity generated by proteolysis of HCII is not due to the conformational change induced by cleavage of the exposed loop near the reactive site nor by release of the reactive site peptide. We also compared the effects of HCII reaction products and fMLP on expression of Mac-1 and p150,95 adhesive proteins. Mac-1 has been implicated in mono adhesion and chemotaxis and as a potential initiator of coagulation. The surface expression of Mac-1 was not increased above control levels by incubation of leukocytes with HCII digests, even though fMLP did increase surface Mac-1. Proteolysis products of HCII could play a role in the initial influx of PMN into a thrombus, and in the transition from acute to chronic inflammation, or to granulation and healing.

Quantitation of the host cell infiltration kinetics of the nonimmunogenic colon 26 tumor by multiparameter flow cytometry.

In this study, we examined the kinetics of host cell infiltration into the nonimmunogenic Colon 26 tumor. We found that 1 x 10(4) cells were required to produce tumors in 100% of mice. The vivo doubling time was 42.5 h, and a barely palpable tumor contained 8 x 10(6) cells. No evidence of concomitant immunity was found. The number of host cells infiltrating the in vivo tumors increased at the same rate as the number of tumor cells, but averaged only 22% of total cells. Cycling T lymphocytes were present in the host cell infiltrate of this tumor. In addition, approximately 50% of in vivo Colon 26 cells were Thy-1.2 positive. The observed characteristic of low immunogenicity makes it a useful murine model for studying human malignant tumors.

A rapid turbidimetric assay of phagocytosis and serum opsonizing capacity.

An in vitro assay has been developed to measure the opsonizing capacity of serum and the extent of bacterial uptake by phagocytes. Various micro-organisms were preopsonized for 10 min with a serum concentration previously determined to be optimal for the respective types of micro-organism. Subsequently, neutrophils from a healthy donor were added to the preopsonized bacteria in a cuvette of a spectrophotometer. The decrease in turbidity at 400 nm, resulting from the uptake of the micro-organisms by the neutrophils, was measured for 20-30 min and the area under the curves was taken as a measure of the opsonizing capacity of the serum or the phagocytic capacity of the neutrophils. The results correlated well with standard opsonophagocytic assays. By excluding Ca2+ from the buffer of the assay, phagocytosis was distinguished from the combined response of phagocytosis and aggregation. In the presence of Ca2+ ions, both phagocytosis and aggregation contributed to the decrease in turbidity. In the absence of Ca2+, phagocytosis was normal, but aggregation was completely inhibited. Phagocytosis in the absence of Ca2+ was also observed using microscopic and radiometric methods of evaluation. Neutrophils from a patient with a deficiency of leukocyte adhesion molecules, ingested as many bacteria as did normal neutrophils without Ca2+. Experiments with NaF, to inhibit phagocytosis, indicated that the change in turbidity measured in the absence of Ca2+ was mainly caused by phagocytosis, not by attachment of bacteria to the neutrophils. The opsonizing capacity of sera, as determined in our assay, depended both on antibodies and on an intact complement system and the inter-assay variance was less than 5%. We found a close correlation between turbidity changes measured in the presence or absence of Ca2+, suggesting that both phagocytosis and aggregation are opsonin-dependent. This assay is applicable to a variety of opsonizing fluids and micro-organisms, and can be used for assessing the phagocytic capacity of patients' neutrophils as well as for assessing the opsonizing capacity of patients' sera.

Examination of macrophage cell surface antigen regulation by rIFN-gamma and IFN-alpha/beta utilizing digital imaging by a novel laser detection system. Anchored cell analysis station (ACAS) 470.

The use of a computer-controlled scanning laser instrument, the ACAS 470, for the analysis of cell-surface antigens on adherent macrophages is described. The association of specific cell surface antigens with macrophage differentiation and the modulation of these markers by environmental signals has long been recognized. However, direct analysis of individual macrophages for cell-surface antigen expression by conventional methods has been fraught with difficulties. Primary macrophage cultures form highly adherent monolayers in vitro. To be analyzed by conventional flow cytometric methods (e.g., FACS analysis of non-adherent lymphoid populations), the adherent macrophages must be detached by enzymatic or mechanical methods which can result in damage to the cell membranes and/or strip off cell surface antigens. Other conventional techniques, e.g., antibody plus complement-mediated cytotoxicity or immunofluorescent microscopy, are semiquantitative at best. Surface antigen analysis using ELISA techniques provides a total population measure of changes in cell surface antigen expression, but fails to provide the number of antigen-positive cells or the density of antigen per cell. The ACAS 470 obviates all of these technical problems and allows for an analysis of individual adherent cells for the density and topography of antigen per cell, as well as expression of an antigen within a population. Using the ACAS 470, we have examined the expression of Ia antigens (class II major histocompatibility antigens) and the Mac-1 antigen (C3bi receptor) on thioglycollate-elicited murine macrophages, basally and after treatment with interferons. In this study, the density and distribution of these antigens per cell, as well as their expression within a population, are reported.

Brain microglia constitutively express beta-2 integrins.

Localization of beta-2 integrins in normal and Alzheimer disease temporal cortex was studied immunohistochemically. Resting microglia were found to express constitutively CD11a (LFA-1), CD11b (Mac-1, CR3), CD11c (P150, 95; CR4), and CD18 (beta-2). They were also found to express constitutively leukocyte common antigen and the immunoglobulin receptor Fc gamma RI. The intensity of expression of each of these antigens was enhanced on reactive microglia in Alzheimer disease tissue. HLA-DR was detected on only a few microglia in control tissue, but was intensely expressed on large numbers of reactive microglia in Alzheimer tissue. These data are consistent with a leukocyte origin and a phagocytic role for microglia. They provide further evidence of an inflammatory response of brain tissue in Alzheimer disease. The microglia were found to make up 9-12% of the total glial population in gray matter and 7.5-9% in white matter.

Expression of adhesion molecules on lymphocytes/monocytes and hepatocytes in human liver grafts.

The regulation of expression of adhesion molecules in human liver grafts in the course of rejection and inflammatory reactions was studied. The tissue distribution of adhesion receptor molecules and ligand molecules in graft biopsies taken during complications was compared to that in normal liver and reference biopsies taken at the time of transplantation.

The regulation of CD11b integrin levels on human blood leukocytes and leukotriene B4-stimulated skin by a specific leukotriene B4 receptor antagonist (LY293111).

CD11b is part of the beta2-integrin Mac-1 and plays an important role in neutrophil adhesion. Leukotriene B4 (LTB4) is an active upregulator of neutrophil CD11b-expression, acts as a potent chemoattractant to neutrophils and is also known to upmodulate epidermal proliferation. We performed a placebo-controlled study on LY293111, an oral LTB4 receptor antagonist. Twenty healthy male volunteers were randomised over three treatment groups that received placebo, 48 mg, or 200 mg drug twice daily for 10 days. Before and after treatment, flow cytometrical CD11b assessment was performed on in vitro LTB4-stimulated peripheral blood neutrophils. Additionally, skin biopsies were taken at 24 and 72 h after epicutaneous LTB4 application, before and after treatment. The effects on skin were assessed immunohistochemically using various markers. All observed effects were dose related. CD11b upregulation on blood neutrophils was significantly suppressed in both treatment groups compared to placebo. In skin, a significant suppression of inflammation and hyperproliferation occurred. Pronounced inhibition was observed on neutrophil migration into the epidermis and the inflammatory infiltrate was decreased. A similar but weaker response was seen in the dermis. The number of cycling cells as well as suprabasal keratin-16 expression were decreased in both treatment groups. LY293111 proved to be a potent inhibitor of LTB4-induced cutaneous inflammation and hyperproliferation. The potent antiinflammatory effect in vivo and the fact that in the present study the compound showed no clinically significant side effects make it an interesting drug in the future treatment of inflammatory conditions predominated by neutrophils.

Expression of leukocyte adhesion molecules in human endometrium.

In the present investigation the distribution of molecules that are involved in the leukocyte binding was studied in human endometrium. The expression of intercellular adhesion molecule-1 (ICAM-1), lymphocyte function-associated antigen (LFA-1), and HLA-DR molecules was studied in 13 proliferative and 10 secretory endometria as well as cultures of endometrial glands and stroma by avidin-biotin complex (ABC) procedure using monoclonal antibodies. The ICAM-1 and HLA-DR molecules were both strongly expressed in the lymphoid and endothelial cells. ICAM-1 expression was uniform in the epithelium, whereas the HLA-DR molecules were preferentially expressed in the epithelial cells in the basalis. Expression of both epithelial HLA-DR and ICAM-1 molecules was enhanced adjacent to lymphoid aggregates. ICAM-1 molecule was uniformly expressed in the stromal cells in the basalis and the functionalis, whereas the HLA-DR molecules were expressed exclusively in the stromal cells surrounding the lymphoid cells. ICAM-1 expression in the epithelial and stromal cells was confirmed in the isolated intact stromal cells and glands by immunohistochemistry. Although stromal and epithelial cells propagated in vitro expressed ICAM-1, they were rarely HLA-DR positive. The expression of LFA-1 was confined to the lymphoid cells. The high level of expression of ICAM-1, LFA-1, and HLA-DR molecules in human endometrial constituents may contribute to the presence, aggregation, and preferential distribution of lymphoid cells in human endometrium.

A new method for isolation of salivary neutrophils and determination of their functional activity.

The purposes of this study were to develop a new method for isolating salivary neutrophils (SPMNs), and to determine their functional activity. Studies of neutrophils in the oral cavity have been largely limited to crevicular PMNs, because of difficulties in obtaining viable SPMNs free of epithelial cells. A method to obtain SPMNs is presented. Donors performed rapid sequential rinsings by placing in their mouths 15 mL of Hanks' balanced salt solution [free of calcium or magnesium ions (HBSS-CMF)], which contained 0.1% gelatin, then swishing the solution for 30 s, and expectorating into a polypropylene receptacle containing 400 mL 4 degrees C HBSS-CMF. This sequence was repeated for 20 min. The collected solution was stirred for 10 min, the cells were washed, and the re-suspended pellet was passed sequentially through a 20-microns and a 10-microns nylon mesh. The cells consisted of 97.7 +/- 1.7% SPMNs, only 2.3% epithelial cells, and were almost free of oral debris. The SPMNs were studied for CD11b expression, H2O2 production, and F-actin polymerization. SPMNs had significantly higher resting values for CD11b, H2O2 production, and F-actin polymerization compared with blood neutrophils (BPMNs). SPMNs responded to stimulation by chemotactic peptide or phorbol ester in a dose-dependent fashion, with levels of CD11b, H2O2, and F-actin comparable with BPMNs at optimal stimulant concentrations. The elevated resting levels of CD11b, H2O2, and F-actin for SPMNs were probably caused by exposure to gingival and oral bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)

Accessory molecules expressed on the peripheral blood or synovial fluid T lymphocytes from patients with Sjögren's syndrome or rheumatoid arthritis.

We previously demonstrated that the cells expressed by activation antigens were increased in several T cell subsets from the peripheral blood (PB) and joint fluid (JF) of patients with Sjögren's syndrome (SS) or rheumatoid arthritis (RA). In the present report, we further determined by three-color flow cytometry the homing receptors (Leu8) for peripheral lymph nodes expressed on naive (CD45RA+) or memory (CD45RA-) CD4+ cells and on the two subsets of CD8+ cells (CD11b+ and CD11b-) in the PB and JF from SS and RA patients. In addition, the activation antigens (HLA-DR) and two adhesion molecules, including the alpha-chain of the leukocyte function associated antigen-1 (LFA 1 alpha: CD11a) and its ligand (intercellular adhesion molecule-1: ICAM-1: CD54) expressed on T cells, were compared. We found that CD45RA-CD4+ cells were markedly increased in JF, while CD45RA+CD4+ cells were almost absent. Leu8+ cells were decreased in both CD45RA-CD4+ and CD8+CD11b-cells (cytotoxic T cells) in the JF, and were also decreased in the CD8+CD11b-subset in the patients' PB. Furthermore, activated (DR+) T cells were markedly increased in JF, and the cells expressed more adhesion molecules in both the PB and JF from patients, compared with the DR-T cells. The DR+ T cells therefore are considered to be memory T cells, which are more efficient for cell-to-cell interactions. These observations also suggest that the Leu8- and DR+ T cells with increased adhesion molecules might preferentially migrate into inflammatory tissues, and that naive T cells are being further converted to to memory T cells by in vivo stimulation within the tissues.

Human leukocyte antigen (HLA) frequency among patients with nasopharyngeal carcinoma in Taiwan.

In order to assess the association between human leukocyte antigens (HLA) and nasopharyngeal carcinoma (NPC), a total of 10 familial and 10 sporadic NPC patients and 171 unrelated healthy controls were studied. HLA typing was performed using commercial trays which defined 30 specificities of HLA-A, B and C loci and 10 specificities of HLA-D locus according to the method of Tiwari and Terasaki. HLA-A2, B16 and DR1 were found to be higher among patients with NPC than unrelated healthy controls with an odds ratio (OR) and a 95% confidence interval of 5.91 (2.1-16.6), 6.00 (2.0-18.0) and 6.89 (1.3-37.5), respectively. Further analysis showed that A2(+) B16(+) haplotype was significantly associated with a much higher risk of NPC (OR = 15.5) as compared with A2(-) B16(-) haplotype. No difference in frequency distributions of HLA-A, B, C and D antigens was observed between familial and sporadic NPC patients.

Morphologic assessment of leukocyte-endothelial cell interactions in mesenteric venules subjected to ischemia and reperfusion.

Intravital microscopic studies of the mesenteric microcirculation have demonstrated that leukocyte adherence and emigration in postcapillary venules are a characteristic feature of tissues exposed to ischemia-reperfusion. The objectives of this study were to determine whether: (1) neutrophils are the predominant leukocytes that adhere and emigrate in postischemic mesenteric venules, and (2) leukocyte adherence and/or emigration are a prerequisite for reperfusion-induced increases in venular permeability. Leukocyte kinetics in cat mesenteric venules (25-35 microns diameter) were evaluated using both intravital microscopy and quantitative morphometry. The intestine and mesentery were exposed to 60 min of ischemia, followed by 60 min reperfusion. Some animals were pretreated with a monoclonal antibody (MoAb IB4) against the leukocyte adhesion glycoprotein, CD11/CD18. Vessels observed by intravital microscopy and adjacent venules of similar diameter were excised and processed for light (LM) and electron microscopy (EM). Horseradish peroxidase (HRP), administered intravenously, was used to assess vascular permeability by EM. By LM, the control (nonischemic) mesentery is sparsely populated by plasma cells, mast cells, and leukocytes; 30-50% of the resident population is neutrophils. Ischemia-reperfusion led to a significant increase in the number of extravascular cells, with neutrophils accounting for greater than 80% of the total cell population. Control and ischemic venules demonstrated no leakage of HRP into the interstitium. However, venules exposed to ischemia and reperfusion demonstrated HRP leakage between endothelial cells and into the surrounding interstitium; neutrophils were adherent to the luminal surface of the endothelium, transmigrating the vessel wall, and in the surrounding interstitium. Animals pretreated with MoAb IB4 presented the same cell profile as nonischemic controls, with no adherent or transmigrating neutrophils. However, some HRP leakage was noted following reperfusion in venules treated with MoAb IB4. The results of this study indicate that: (1) neutrophils are the predominate leukocytes that adhere and emigrate in postischemic venules, and (2) inhibition of leukocyte adhesion does not completely prevent the venular dysfunction associated with ischemia-reperfusion.

In vivo and in vitro HeNe laser effects on phagocyte functions.

The goal of this work was to evaluate the effect of helium-neon (HeNe) laser irradiation on immunocompetent cells. We used the in vivo skin window method and in vitro granulocyte function tests. The study of cellular migration showed a marked decrease in vitro and in vivo in a dose-independent manner. Superoxide release was not modified by laser irradiation. The granulocyte's aggregation, when using PHA and PMA, presented a reduction that was statistically very significant, not as a subordinate dose. An increase of the release of ATP was demonstrated only at 4 joules and precedes granulocyte aggregation. When using Ca2+ ionophore A23187 as stimulus, laser irradiation at 1, 2 or 4J did not show any modification of granulocyte aggregation. The monoclonal antibody 60.1, which identifies a membrane antigen fundamental for aggregation and chemotaxis, is expressed in normal amounts on granulocyte membranes both before and after irradiation with a HeNe laser. In fact, Laser irradiation preferentially attacks the area of the cellular centrosome that determines a modification of cellular morphology. The electron microscope and immunofluorescence study with a monoclonal antibody have pointed out a disorganization of the microtubules. The alteration of some of the granulocyte functions is correlated to the damage in the centrioles. The granulocyte mitochondrial system and surface membrane remain intact, and this explains the normal production and release of free radicals. Further experiments are necessary to evaluate the clinical application of lasers in various diseases with immunophagocytic pathogenesis.