Sheep red cell binding to human lymphocytes treated with neuraminidase; enhancement of T cell binding and identification of a subpopulation of B cells.

Neuraminidase treatment of normal human lymphocytes enhances their capacity to form SRBC rosettes; more red cells are bound and the rosettes are more stable. Under these conditions approximately 90% of peripheral blood lymphocytes form rosettes. In addition to the effects on T cells, another population of lymphocytes which possess surface immunoglobulin and have the Fc receptor acquire the rosette-forming property after neuraminidase. This subpopulation of 'B' cells represents approximately half of the lymphocytes with surface immunoglobulin but is not found among the leukemic lymphocytes of patients with chronic lymphocytic leukemia. Electron microscope observations indicate close approximation and intimate association of the red cell and lymphocyte membranes after neuraminidase.

Scanning electron microscopy of human lymphocyte-sheep erythrocyte rosettes.

Human lymphocytes of known B or T derivation were examined by scanning electron microscopy (SEM) before and after rosetting with SRBC. After collection of the cells onto silver membranes the samples were prepared for SEM by the critical point drying method. Sheep RBC frequently underwent sphero-echinocyte transformation and multiple projections extended from their surfaces. This was readily noticeable after storage of SRBC in the cold and washing in Hanks, but more prominent after rosetting. These erythrocyte surface alterations were less apparent when freshly withdrawn cells were used. Spontaneous sheep erythrocyte rosettes (E-R), a marker for human T lymphocytes, were prepared with normal peripheral blood lymphocytes (PBL), thymic cells, and cultured T cells. EAC-rosettes (EAC-R), used to identify B lymphocytes with complement receptors, were prepared with normal PBL and cultured B cells. The majority of rosetting T lymphocytes had generally smooth surfaces while about 20% had an intermediate number of microvilli and 15% were more villous and indistinguishable from villous B cells. Studies of rosetting thymocytes and cultured T cells however indicated that the surface of some T cells alters on rosetting, becoming more villous and thus account for the higher numbers of villous T cells seen in E-rosettes. Point to point contact sites between SRBC and T lymphocytes were more frequent than broad zones of attachment. The majority of rosetting B lymphocytes had multiple microvilli, about 25% had a moderate number of microvilli and less than 10% had smooth surfaces similar to those of most T cells. Areas of contact between EAC and B lymphocytes were frequently broad zones of attachment. The study confirms that in many cases B and T lymphocytes can be distinguished by their surface architecture as seen under the SEM; however, about 20% of rosetting B and T cells have similar surfaces with intermediate numbers of surface microvilli and cannot be distinguished by SEM without parallel immunologic identification.

Studies on human peripheral blood lymphocytes in vitro. I. Biological and physicochemical properties of the pokeweed mitogen.

The saline extract from the roots of Phytolacca americana (pokeweed) possesses three biological properties; hemagglutinin, leukagglutinin, and mitogen. Fractionation and further purification on calcium phosphate column chromatography revealed that the biologically active substance was eluted in the front moving fraction with 0.05 M phosphate buffer pH 7.5. Analytical separation on polyacrylamide gels in disc electrophoresis yielded a single homogeneous band with an R(f) value of 0.43 containing all three biological activities. This fraction had an ultraviolet absorption spectrum similar to PHA, was stable to both periodate and mercaptoethanol treatment and gave a single band in double diffusion and immunoelectrophoretic analysis against the antibody prepared to the crude PWM saline extract. Absorption studies with red cells or stroma revealed that the hemagglutinin could be selectively removed without significantly altering the mitogen, whereas absorption with leukocytes resulted in loss of both the mitogenic and leukagglutinating activities.

Studies on human peripheral blood lymphocytes in vitro. II. Morphological and biochemical studies on the transformation of lymphocytes by pokeweed mitogen.

A study of the kinetics of RNA and DNA synthesis in PWM-stimulated lymphocytes revealed that RNA synthesis preceded the onset of DNA synthesis by approximately 24 hr and that DNA synthesis and transformation was maximal between 66 to 78 hr. Histochemical and radioautographic studies on PWM stimulated cultures indicated that at 72 hr 50 to 60% of the cell population had been transformed by PWM, and that a distinct cell type bearing cytologic resemblance to the early plasma cell had emerged. The RNA sedimentation profile for newly synthesized RNA in PWM-stimulated cells showed that a large peak of 45 to 50 S material was formed after 24 and 40 hr. PWM thus produces a distinctive transformation of human peripheral blood lymphocytes.

Infection of monocyte-derived macrophages with human immunodeficiency virus type 1 (HIV-1). Monocyte-tropic and lymphocyte-tropic strains of HIV-1 show distinctive patterns of replication in a panel of cell types.

To characterize the host range of different strains of HIV-1, we have used four types of cells, primary monocyte-derived macrophages (MDM), primary PBL, a promonocyte cell line (U937), and a CD4+ T cell line (SUP-T1). These cells were infected with three prototype strains of HIV-1, a putative lymphocyte-tropic strain (IIIB), and two putative monocyte-tropic strains (SF162 and DV). Infections were monitored by assays for infectious virus, for cell-free and cell-associated viral antigen (p24), and for the proportion of cells infected by immunohistochemical staining. It was concluded that: (a) the use of four different cell types provides a useful biological matrix for distinguishing the tropism of different strains of HIV-1; this matrix yields more information than the infection of any single cell type. (b) A monocyte-tropic strain of HIV-1, such as strain SF162, shows a reciprocal host range when compared with a lymphocyte-tropic strain such as IIIB; strain SF162 replicates well in primary MDM but not in U937 or SUP-T1 cells, while strain IIIB replicates well in both U937 and SUP-T1 cells but not in MDM. (c) Both lymphocyte-tropic and monocyte-tropic strains of HIV-1 replicate well in PBL. (d) The promonocyte cell line, U937, and the T cell line, SUP-T1, differ markedly from primary cells, such as MDM and PBL, in their ability to support the replication of different strains of HIV-1; these cell lines cannot be used as surrogates for primary cells in host range studies of HIV-1 strains.

Studies on lysosomes. XI. Characterization of a hydrolase-rich fraction from human lymphocytes.

Pure suspensions of human lymphocytes were separated from peripheral blood by means of nylon wool, homogenized in 0.34 M sucrose-0.01 M EDTA solution, and fractionated by differential centrifugation. The bulk of acid hydrolase activity was found to be concentrated in a 20,000 g x 20 min granular fraction, whereas nuclear, debris, and supernatant fractions contained lesser concentrations of hydrolases. Acid hydrolase activity present in the granular fraction showed appropriate "latency" as judged by its dose-dependent release into the 20,000 g x 20 min supernatant after exposure to membrane-disruptive agents such as streptolysin S, filipin, and lysolecithin. Heparin proved to be necessary in the suspending medium so that reproducible homogenization and cell fractionation could be obtained. Even excessive contamination of lymphocyte suspensions with platelets did not appreciably alter the acid hydrolase activity of lymphocyte homogenates or the distribution of enzymes in subcellular fractions. Discontinuous density-gradient centrifugation of a 500 g x 10 min supernatant, containing both acid hydrolase-rich organelles and mitochondria, resulted in partial resolution of hydrolase-rich organelles from mitochondria. Fine structural studies of the intact lymphocytes showed the presence of acid phosphatase-positive, membrane-bounded organelles. Electron microscopy of the "large granule" (20,000 g x 20 min) fraction of such lymphocytes demonstrated 80-90% mitochondria, 5-10% platelets, and 5-10% membrane-bounded acid phosphatase-positive structures. The data indicate the presence in human peripheral blood lymphocytes of acid hydrolase-rich granules which possess many of the biochemical and structural characteristics of lysosomes in other tissues.

Thiol suppression of human immunodeficiency virus type 1 replication in primary cord blood monocyte-derived macrophages in vitro.

We investigated the effects of glutathione (GSH), the major naturally occurring thiol, and a pharmacologic thiol precursor of GSH, N-acetyl cysteine (NAC), on the expression of human immunodeficiency type 1 (HIV-1) in primary cord blood and adult donor monocyte-derived macrophages (MDM). HIV-1 infection of cord blood and adult MDM was accomplished after incubating 10-15-d-old cultures for 4 h with a monocyte-tropic strain of HIV-1 (Bal). After 1 wk in culture cell supernatants were tested for reverse transcriptase (RT) activity. MDM were exposed to 5, 10 and 20 mM concentrations of both GSH and NAC before infection, during infection, and after infection was established. GSH and NAC suppressed the replication of HIV-1 in both primary cord blood and adult donor MDM in a concentration dependent fashion. These suppressive effects were more pronounced in cord-derived cells than in adult-derived cells. In cells treated with GSH or NAC before infection, there was no significant rise in RT activity as compared with controls. Similarly, when cells were treated with GSH and NAC and simultaneously infected, there was also no significant rise in RT activity after 1 wk in culture. In cells treated after infection was established, RT values were suppressed 80-90% that of untreated controls. This effect persisted for 1-2 wk after exposure to GSH and NAC. Untreated controls demonstrated syncytium formation and lost characteristics of spreading and elongation 2 wk after HIV-1 infection, whereas most of the treated cells remained free of syncytium and retained cytoplasmic spreading, adherence, and elongation. These data are consistent with other studies of thiol suppression of HIV-1 replication and demonstrate a similar observation for primary cultured cord MDM. These results may offer new approaches toward cellular protection after infection with HIV-1.

Modulation of neutrophil function by lysozyme. Potential negative feedback system of inflammation.

Host responses to infectious organisms should be modulated so that tissue-damaging products of inflammatory cells do not produce excessive destruction of normal tissue. Lysozyme, which is continuously secreted by monocytes, which, in turn, migrate relatively late to inflammatory areas, was found to significantly dampen several responses of neutrophils to inflammatory stimulants. Thus, human lysozyme obtained and purified from the urine of patients with monocytic leukemia (but not its structurally similar and comparably cationic analogue, eggwhite lysozyme) depresses chemotaxis of normal neutrophils to activated complement, bacterial supernate, and N-formylmethionyl-phenylalanine. In addition, human (but not eggwhite) lysozyme depresses oxidative metabolism (hexose monophosphate shunt activity) and superoxide generation of neutrophils. The specificity of the suppressive effects was indicated by inhibition studies with rabbit antihuman lysozyme antibody, and with the trisaccharide of N-acetylglucosamine, a specific inhibitor of lysozyme. The results suggest that lysozyme, a product of inflammatory cells themselves, may function in a negative feedback system to modulate the inflammatory response.

Ca2+ responses to interleukin 1 and tumor necrosis factor in cultured human skin fibroblasts. Possible implications for Reye syndrome.

Elevated concentrations of cytokines were found in the plasma of patients acutely ill with Reye syndrome (RS) but not in control subjects or recovered RS patients. To determine whether this disorder involves a genetically determined abnormal response to cytokines, the effects of tumor necrosis factor (TNF) and IL-1 on intracellular free Ca2+ were compared in cultured skin fibroblasts from control subjects and patients with RS. IL-1 and TNF caused rapid, transient, and concentration-dependent increases in cytosolic free Ca2+. The peak cytosolic free Ca2+ was greater and occurred at higher concentrations of IL-1 and TNF in patient cells than in cells from age-matched controls. In control cells, the Ca2+ transient diminished sharply with increasing amounts of IL-1 or TNF above the maximum stimulatory concentration. In contrast, in patient fibroblast this bell-shaped curve of concentration dependency was much less apparent. Bradykinin-stimulated Ca2+ transients were similar in the two groups and did not exhibit the bell-shaped concentration dependency. Thus, plasma cytokine levels are elevated in RS patients and the Ca2+ response to cytokines is increased in cells derived from these patients. We propose that the increased response reflects a genetic defect in cytokine receptor-modulated signal transduction.

The key role of peptidoglycan in the opsonization of Staphylococcus aureus.

In an effort to determine the staphylococcal cell surface component(s) of importance in opsonization, cell walls (peptidoglycan and teichoic acid) and peptidoglycan were isolated from Staphylococcus aureus strain H grown in [3H]glycine-containing broth. After incubation of the cell walls and peptidoglycan with various opsonic sources, uptake by human polymorphonuclear leukocytes was measured. The opsonic requirements for phagocytosis of cell walls and peptidoglycan were found to be similar to those of intact bacteria. Removal of teichoic acid from the cell wall did not affect opsonization. Likewise, a teichoic acid-deficient mutant strain of S. aureus H was opsonized in a manner similar to that of the parent strain. Immunoglobulin G functioned as the major heat-stable opsonic factor and both the classical and alternative pathways participated in opsonization. Kinetic studies revealed that opsonization of peptidoglycan, as well as C3-C9 consumption by peptidoglycan, proceeded at a slower rate via the alternative pathway (C2-deficient serum) than when the classical pathway was present (normal serum). The ability of peptidoglycan to activate C3-C9 was significantly reduced when normal and C2-deficient sera were preabsorbed with peptidoglycan at 2 degrees C suggesting that antibodies to peptidoglycan may be involved in activation of both the classical and alternative complement pathways. Thus, peptidoglycan appears to be the key cell wall component involved in staphylococcal opsonization, and it is suggested that host response to peptidoglycan, a major cell wall component of most gram-positive bacteria, may be related to the development of "natural immunity" to this group of microorganisms.

Leukocytosis and natural killer cell function parallel neurobehavioral fatigue induced by 64 hours of sleep deprivation.

The hypothesis that sleep deprivation depresses immune function was tested in 20 adults, selected on the basis of their normal blood chemistry, monitored in a laboratory for 7 d, and kept awake for 64 h. At 2200 h each day measurements were taken of total leukocytes (WBC), monocytes, granulocytes, lymphocytes, eosinophils, erythrocytes (RBC), B and T lymphocyte subsets, activated T cells, and natural killer (NK) subpopulations (CD56/CD8 dual-positive cells, CD16-positive cells, CD57-positive cells). Functional tests included NK cytotoxicity, lymphocyte stimulation with mitogens, and DNA analysis of cell cycle. Sleep loss was associated with leukocytosis and increased NK cell activity. At the maximum sleep deprivation, increases were observed in counts of WBC, granulocytes, monocytes, NK activity, and the proportion of lymphocytes in the S phase of the cell cycle. Changes in monocyte counts correlated with changes in other immune parameters. Counts of CD4, CD16, CD56, and CD57 lymphocytes declined after one night without sleep, whereas CD56 and CD57 counts increased after two nights. No changes were observed in other lymphocyte counts, in proliferative responses to mitogens, or in plasma levels of cortisol or adrenocorticotropin hormone. The physiologic leukocytosis and NK activity increases during deprivation were eliminated by recovery sleep in a manner parallel to neurobehavioral function, suggesting that the immune alterations may be associated with biological pressure for sleep.

Impaired metabolic function and signaling defects in phagocytic cells in glycogen storage disease type 1b.

Patients with glycogen storage disease (GSD) type 1b (1b), in contrast to patients with GSD type 1a (1a), are susceptible to recurrent bacterial infections suggesting an impairment in their immune system. In this study, phagocytic cell (neutrophil and monocyte) respiratory burst activity, as measured by superoxide anion generation, oxygen consumption, and hexose monophosphate shunt activity, was markedly reduced in both neutrophils and monocytes from GSD 1b patients as compared with either GSD 1a patients or healthy adult control cells. Degranulation, unlike respiratory burst activity, was not significantly different in neutrophils from GSD 1b patients as compared with controls. Both neutrophils and monocytes from GSD 1b patients showed decreased ability to elevate cytosolic calcium in response to the chemotactic peptide f-Met-Leu-Phe. In addition, calcium mobilization in response to ionomycin was also attenuated suggesting decreased calcium stores. Thus, reduced phagocytic cell function in GSD 1b is associated with diminished calcium mobilization and defective calcium stores. Defective calcium signaling is associated with a selective defect in respiratory burst activity but not degranulation.

Neutrophil chemotaxis and enzyme release competitive inhibition by a diazoacetamide pepsin inhibitor.

Treatment of human neutrophils with a reagent (diazoacetylnorleucine methyl ester plus copper ion) which covalently labels the active site of the acid proteases, pepsin and cathepsin D, inhibits neutrophil chemotaxis and enzyme release stimulated by the chemoattractants pepstatin and formylmethiony peptides. In contrast, chemotaxis and enzyme release in response to zymosan activated serum are not affected. Furthermore, diazoacetylnorleucine methy ester plus copper competes with [3H]formylmethionyl leucylphenylalanine for binding to neutrophils. Since pepstatin shares binding sites with formylmethionyl leucylphenylalanine, the present data suggest that diazoacetylnorleucine methyl ester plus copper reacts with the neutrophil receptor for pepstatin and formylmethionyl peptides, and thus may be useful in further characterization of this structure.

Enhancement in vitro of the low interferon-gamma production of leukocytes from human newborn infants.

Interferon-gamma (IFN-gamma), a lymphokine produced by lymphocytes with the help of monocytes, is essential for host resistance to intracellular pathogens. Leukocytes from normal term newborn infants cannot produce IFN-gamma in vitro in response to stimulation by antigen or mitogens in vitro or in vivo. We investigated the production of IFN-gamma in vitro using endotoxin from Salmonella typhimurium as a stimulus. In contrast to those from adults, mononuclear cells derived from the cord blood of newborn infants did not produce IFN-gamma in response to this endotoxin. We investigated the contribution of the functional immaturity of cord blood monocytes to this relative inability to produce IFN-gamma. Aging of the monocytes for 2 weeks in vitro or treatment of freshly isolated cord blood monocytes with conditioned medium (from cultures of mononuclear cells from healthy adults) greatly enhanced IFN-gamma production stimulated by endotoxin. Furthermore, recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), or IFN-gamma was able to substitute in part for the conditioned medium from adult cells. Thus correction of the functional immaturity of monocytes derived from newborn infants can result in enhanced production of IFN-gamma in vitro.

Phorbol myristate acetate induced oxidation of 2',7'-dichlorofluorescin by neutrophils from patients with chronic granulomatous disease.

The oxidative metabolic burst of stimulated human polymorphonuclear leukocytes (PMNs) has been evaluated by the measurement of oxygen consumption, chemiluminescence, and oxygen radicals (O2-, H2O2, OH-) derived from activation of the hexose monophosphate shunt (HMPS). PMNs from patients with chronic granulomatous disease (CGD) are shown to lack functional NADPH oxidase and undetectable oxygen radical generation. However, using single cell analysis by flow cytometry and 2',7'-dichlorofluorescin (DCFH) oxidation by H2O2, significant DCFH oxidation by the PMA stimulated CGD PMNs was observed. Furthermore, 1mM potassium cyanide enhanced DCFH oxidation by control and CGD PMNs. DCFH oxidation by cells from an obligate heterozygous mother of an X-linked CGD patient was intermediate. These observations suggest that a PMA induced oxidase enzyme is present in CGD cells.

Use of colloidal silica (Sepracell-MN) for enrichment of dendritic cells from human peripheral blood: comparison with other methods.

Three methods are described for enrichment of dendritic cells from human peripheral blood. In method 1, mononuclear cells were incubated in plastic tissue culture flasks for two h. Nonadherent cells were removed. Adherent cells were washed to remove floating cells and incubated for 14 h at 37 degrees C in 5% CO2. Carbonyl iron was added, and the flasks were incubated for another 2 h. Nonadherent cells were subjected to centrifugation over metrizamide gradient. Phagocytic cells containing ingested carbonyl iron, small lymphocytes, and free carbonyl iron particles passed through the metrizamide, while the interface cell population was enriched for dendritic cells. The purity and yield of enriched dendritic cells were 52.8% and 0.05%, respectively. In method 2, adherent mononuclear cells were cultured overnight, and the released cells (released adherent cells) were centrifuged over metrizamide to separate low-density cells. Monocytes from this low-density cell population were removed by panning over human gamma globulin-coated plastic Petri dishes. In this method the average purity and yield of DC were 63.8% and 0.1%, respectively. In method 3, released adherent cells were treated with anti-CD5 and anti-CD14 monoclonal antibodies plus baby rabbit complement for 15 min, washed, and centrifuged with colloidal silica (Sepracell-MN). Centrifugation with Sepracell-MN was repeated three times. Low-density cells were panned twice over human gamma globulin-coated plastic Petri dishes. Nonadherent cells were highly enriched for DC. Contamination of T cells, B cells, and NK cells was undetectable by flow cytofluorometry. Contamination of monocytes was less than 2%. This method provided greater than 85.0% purity and 0.4% yield. This method (method 3) combines adherence, complement-dependent lysis, centrifugation with colloidal silica, and panning and provides the best yield and purity; it is therefore recommended for optimal purification of DC.

Decreased fibronectin biosynthesis by human cord blood mononuclear phagocytes in vitro.

Cultured cord blood monocytes synthesize significantly less fibronectin than cultured monocyte-macrophages from adult peripheral blood. Biosynthesis of the second component of complement in vitro is similar for both cell systems. The monocyte-macrophages from neonates and adults have similar functional and morphologic properties in long-term cultures. The decreased monocyte-macrophage biosynthesis of fibronectin observed in vitro may be in part related to the reduced plasma fibronectin concentrations and reticuloendothelial system hypofunction which occur in newborn infants.

Enhanced phospholipase A2 and C activities of peripheral blood polymorphonuclear leukocytes from patients with rheumatoid arthritis.

Increased concentrations of eicosanoids are found in synovial fluids (SF) from patients with rheumatoid arthritis (RA). SF polymorphonuclear leukocytes (PMN), which derive from peripheral blood, usually account for approximately 90% of the cells in RA SF. Since eicosanoid precursor fatty acids (FA) are liberated by phospholipases from membrane phospholipids, we examined phospholipase A2 and C activities in peripheral blood PMN from patients with RA. Peripheral blood PMN from patients with RA (RA-PMN) exhibit greater phospholipase A2 activities against phosphatidylcholine (PC) and phosphatidylethanolamine (PE), and greater phospholipase C activities against PC, PE, and phosphatidylinositol (PI) than PMN obtained from normal volunteers (N-PMN).

Isolation and flow cytometric characterization of newborn mouse brain-derived microglia maintained in vitro.

Microglia have been identified in the white matter of developing and adult mouse brain using different murine macrophage markers. While several techniques for the isolation of murine microglia have been described, the small cell yields and partial purification have limited the progress of these studies. We now describe the isolation of murine microglia using a modification of McCarthy and de Vellis method. Brain tissues from 1-2 day old newborn mice were mechanically and chemically dissociated and maintained in in vitro culture for 3 weeks. In primary dissociated brain cultures, microglia are observed after 10 days migrating from small colonies. After 16-20 days, brain-derived microglia were isolated with high cell yields by continuous shaking of the cultures for 16 hr. In contrast to resident murine peritoneal macrophages, microglia express less Class II (Ia) antigen and a small percentage express L3T4 (CD4) antigen by flow cytometry.

Centrifugal enhancement of human immunodeficiency virus type 1 infection and human cytomegalovirus gene expression in human primary monocyte/macrophages in vitro.

In an effort to facilitate the efficiency of human immunodeficiency virus type 1 (HIV-1) and/or human cytomegalovirus (HCMV) infection in primary monocyte/macrophages in vitro, the effect of low-speed centrifugation was studied. The infectivity of three strains (Bal, Ada-M, and IIIB) of HIV-1 tested was significantly enhanced by centrifugal inoculation at a force of 1500g for 60 min. Reverse transcriptase activity and HIV-1 p24 antigen in primary monocyte/macrophages infected by a centrifugal inoculation technique were detectable 3-7 days earlier and were more than 10-fold greater in magnitude (at an early stage of the infection) than those of control cells infected by the conventional inoculation technique. Examination of the cells by indirect immunofluorescence revealed higher expression of HIV-1 p24 protein in the monocyte/macrophages infected by the centrifugal inoculation technique. These differences were directly related to centrifugal inoculation and were evident up to 3 weeks after infection. Enhancement was not observed when centrifugation was carried out before or after HIV-1 infection. Centrifugal inoculation of HCMV also enhanced its immediate-early and early gene expression up to 30- to 50-fold, although neither late nuclear antigens and glycoproteins of HCMV nor infectious virus was detected in HCMV-infected monocyte/macrophage cultures. These results show that centrifugal inoculation is a useful technique for improving the efficiency of HCMV and HIV-1 infection in vitro.