Plasmacytoid monocytes in Jessner's lymphocytic infiltration of the skin.

Plasmacytoid monocytes are normal cell constituents of the human lymph node and have been found to form perivascular clusters in a case of lymphocytic infiltration of the skin. This study was undertaken to analyze the occurrence of plasmacytoid monocyte clusters in biopsy specimens from 54 patients with lymphocytic infiltration of the skin using light microscopy and immunohistochemistry. Variably sized clusters of plasmacytoid monocytes were observed in close association with dermal venules in 16 of 54 biopsy specimens and were composed of medium-sized cells, admixed with pyknotic cells and, occasionally, with tangible body macrophages. Immunohistochemistry on paraffin and on frozen sections facilitated the recognition of plasmacytoid monocytes and showed an immunophenotype similar to that observed previously on reactive lymph nodes. It is concluded that, in analogy with reactive lymph nodes, plasmacytoid monocytes represent a common constituent of the skin-associated lymphoid tissue. The striking perivascular distribution and the immunophenotypical characteristics of these monocyte-derived cells suggest they may have a role in the process of lymphocyte recruitment into the skin.

Quantitation and biological activities of native tumour necrosis factor from LPS-stimulated human monocytes.

Human mononuclear cells (MNC) were stimulated in culture with LPS to produce tumour necrosis factor (TNF). The natural tumour necrosis factor (nTNF) was quantitated by ELISA and immunoblotting using rabbit antibodies to human recombinant TNF (rTNF) and the biotin-avidin-peroxidase system. Biologically active nTNF was determined by its cytotoxic activity for actinomycin-D treated mouse fibroblasts and by its lethal effect in D-galactosamine sensitized endotoxin-resistant mice. Two different LPS preparations (Salmonella abortus equi and Pseudomonas aeruginosa) induced the formation of comparable amounts of nTNF. MNC from different donors, however, showed large variations in their ability to produce nTNF. The amount of nTNF induced in response to LPS could be enhanced by priming the MNC with interferon. The amounts of nTNF determined by ELISA generally correlated well with the activity of the nTNF in the two biological assays. On a weight basis, the lethal activity of nTNF in D-galactosamine treated mice was very similar to that of human rTNF. Immunoblotting revealed a single band of nTNF with the same molecular weight (17 kD) as human rTNF. The lethality induced by nTNF was inhibited by rabbit anti-human rTNF antibodies.

Anti-inflammatory lipocortin 1 production by peripheral blood leucocytes in response to hydrocortisone.

The presence and amount of the anti-inflammatory protein lipocortin 1 was determined in plasma and peripheral blood leucocytes by a highly specific, enzyme-linked immunosorbent assay. Within 120 min of a single intravenous dose of 100 mg hydrocortisone, the intracellular concentrations of lipocortin 1 in peripheral monocytes in 7 of 8 healthy men increased by a median of 225% (range 129-507%) compared with pretreatment levels, and mononuclear cell-surface lipocortin increased by a median of 224% (range 76-483%). Placebo injections had no effect. There was no increase at any time in free plasma or polymorph-associated lipocortin. In 3 of 4 subjects, induction of lipocortin was also observed when whole unseparated blood was incubated in vitro after steroid administration, but cells which had first been isolated and purified were refractory to such induction. Thus rapid changes in the concentration of an active anti-inflammatory protein can occur in man after normal therapeutic doses of hydrocortisone.

Relationship of one form of human histamine-releasing factor to connective tissue activating peptide-III.

We have previously reported purification of three forms of histamine-releasing factors (HRFs) from mixtures of streptokinase-streptodornase stimulated human mononuclear cells and platelets with apparent molecular masses of 10-12, 15-17, and 40-41 kD (1989. J. Clin. Invest. 83:1204-1210). We have also prepared mouse MAbs against the 10-12-kD HRF (1989. J. Allergy Clin. Immunol. 83:281). Affinity-purified 10-12-kD HRF appears as a broad band upon polyacrylamide gel electrophoresis in the presence of SDS. We determined the NH2-terminal amino acid sequence of the top and bottom halves of this broad band. Sequence analysis revealed striking homology between this HRF and connective tissue activating peptide-III (CTAP-III), a platelet-derived 8-10-kD protein known to cause mitogenesis and extracellular matrix formation in fibroblast cultures. 19 of 21 NH2-terminal residues in the top half of the HRF band were identical to the NH2-terminal sequence of CTAP-III. 20 of 21 NH2-terminal residues in the bottom half were identical to the NH2-terminal sequence of neutrophil-activating peptide-2, which is derived from CTAP-III by proteolytic cleavage between residues 15 and 16. Purified CTAP-III also released histamine from basophils. Rabbit antiserum raised against either native or recombinant CTAP-III recognized affinity-purified HRF in immunodot blot assays, and MAb against HRF recognized CTAP-III in both dot blot and microtiter plate based immunoassays. These data demonstrate the first structural, functional, and immunologic relationship between one form of human HRF and a previously described cell product.

Variation in the composition of gingival inflammatory cell infiltrates.

Biopsy specimens were taken at gingivectomy from 18 adult patients undergoing treatment for chronic marginal periodontitis. They were embedded so that the cut surface of the gingiva was parallel to the top of the block to obtain a comprehensive view in a transversal plane of the inflammatory cell infiltrate near the bottom of the pocket. Sections were stained with HES or with toluidine blue for histological description, and acid alpha-naphthyl acetate esterase (ANAE) was used to differentially stain T lymphocytes, plasma cells and monocytes/macrophages. Sections stained with HES showed that the density and size of the cell infiltrates varied along the circumference of a tooth over very short distances and on various surfaces on neighbouring teeth. Differential counts of cells stained for ANAE demonstrated great variation in the composition of the cell infiltrates, particularly along the pocket epithelium. The predominating ANAE positive cell type in this area was T lymphocytes, while in the central connective tissue, plasma cells predominated. There was no systematic covariation between the localization of the gingiva (i.e. mesial, facial, etc.) and the composition of the cell infiltrates. The local variation in the composition of the cellular infiltrate most likely reflects local variability in the noxious substances (i.e. plaque composition) within the periodontal pocket, and in the resulting local inflammatory response.

A novel monoclonal antibody induces the differentiation of monocyte leukemic cells.

A cell surface antigen (possibly a receptor) on human myeloid cell lines, that may play a crucial role in the differentiation of promonocytic cells, was detected by a murine monoclonal antibody (MAb 710F). When the myeloid cell lines, HL-60, THP-1 and U937, were cultured with the MAb 710F following pretreatment with phorbol 12-myristate 13-acetate (PMA), they displayed both cellular spreading and a strong adherence to the culture dish substratum. On transforming from their original round shape, the induced cells displayed the well-developed microvilli, spindles, or raffles that are characteristic of macrophages or dendrocytes. Similar morphological changes were not induced by treatment with either PMA or MAb 710F alone. By contrast, the lymphoid cell lines did not respond to these reagents. These results suggested that a signal for cellular adhesion and cytoskeletal reconstitution was triggered by the binding of MAb 710F to an antigen on the PMA-primed cells. Thus, the antigen 710F, which is preferentially expressed on peripheral blood monocytes, may represent a cell surface receptor closely associated with differentiation. The antigen/receptor 710F was shown to be a N-glycosylated protein with Mr. of 35-70k. This MAb may prove useful as a tool for both studies on the differentiation of myeloid lineage cells as well as on monocyte/macrophage adhesion function.

Changes in overall plasma norepinephrine turnover and lymphomonocyte beta-adrenoceptor number during combined caloric and sodium restriction in normotensive obese subjects.

The aim of the study was to evaluate in eight normotensive obese patients the influence of low sodium intake (9 mEq/day) on the sympathetic activity modifications induced by caloric restriction (2511 kJ/day). As compared to the isocaloric salt balanced diet, 7 days of normosodic underfeeding induced a decrease in the overall norepinephrine turnover (clearance and appearance rate) and 24 hours urinary output, whereas the combined caloric and salt restriction significantly increased the norepinephrine appearance rate and even more the norepinephrine clearance but, on the other hand, decreased the beta-adrenergic receptor number on the lymphomonocyte surface, suggesting a reduced peripheral sensitivity to catecholamines. Therefore, the utility of the combined sodium and caloric restriction in the treatment of the normotensive obese patients remains still questionable.

Identification of the monocyte chemotactic protein from human osteosarcoma cells and monocytes: detection of a novel N-terminally processed form.

The chemotactic activity for monocytes in culture supernatants from double-stranded RNA-stimulated human MG-63 osteosarcoma cells and from LPS-stimulated human monocytes was purified to homogeneity and characterized by amino acid sequence analysis. The chemotactic protein derived from the fibroblastoid osteosarcoma cells had a blocked N-terminus but sequencing of tryptic fragments showed that it was identical with a recently identified monocyte chemoattractant designated MCP-1 or MCAF isolated from glioma or myelomonocytic cells, respectively. Preparations of monocyte -derived chemotactic activity appeared to contain not only the blocked protein, but also a novel N-terminally processed form of this molecule, lacking 5 amino acid residues.

Cellular distribution, regulation, and biochemical nature of an Fc alpha receptor in humans.

In these studies, we characterize an Fc receptor (FcR) for IgA that is present on human granulocytes, monocyte/macrophages, and their corresponding cell lines. Receptor expression appears to be constitutive but can be selectively upregulated on monocyte cell lines by stimulation with a phorbol ester and polymeric IgA. Both the induction requirements and ligand specificity of the IgA receptor differ from the IgG receptors, Fc gamma R I, II, and III, that are also expressed on monocytes and granulocytes. IgA binding to the cell surface receptor is mediated via the Fc alpha region. The Fc alpha R is a heterogenously charged, approximately 60-kD molecule with an isoelectric point of 4.5-5.6 that binds monomeric or polymeric IgA1 and IgA2 molecules. This transmembrane glycoprotein appears to be composed of 32- and 36-kD protein cores with multiple N-linked carbohydrate moieties. We conclude that this Fc alpha R represents a novel member of the FcR family that may have a distinctive role in host defense.

Effect of thyrotropin-releasing hormone on immune functions of peripheral blood mononuclear cells.

The tripeptide thyrotropin-releasing hormone (TRH) works as a hypothalamic hormone, but is found also outside the brain in intrinsic nerve fibers of the gastrointestinal tract. There is evidence that TRH modulates the activity of immunocompetent cells, although there are only very few data on TRH-mediated immune effector functions. Since we could recently show that TRH inhibits monocyte activities we were also interested in other possible TRH modulated immune functions. Peripheral blood mononuclear cells (PBMC) from ten healthy subjects were cultured for 7 days and pulsed with 0.125 and 0.250 microgram/ml Pokeweed mitogen (PWM). 10(-12) to 10(-6) M TRH was added simultaneously with PWM. Lymphocyte proliferation [(3H]thymidine incorporation), interferon-gamma (IFN-gamma) activity (RIA) and immunoglobulin activities (IgG, IgM, IgA; ELISA) were determined in the supernatants. We could demonstrate a TRH-dependent decrease in PWM-pulsed IgG activity with significant (alpha = 0.05) values at 10(-8) and 10(-10) M (-29 +/- 6%/-16 +/- 3% for PWM 0.125 microgram/ml and -17 +/- 9%/-11 +/- 9% for PWM 0.250 microgram/ml). This inhibitory effect could be abolished by an anti-TRH antiserum. There was no TRH effect on IgM and IgA activities, IFN-gamma activity and lymphocyte proliferation compared with the PWM stimulated values alone. The described TRH effect on the polyclonal IgG response by PBMC gives further evidence for a functional link between the immune system and the endocrine system, although its underlying mechanism is not yet clear.

Procoagulant activity of mononuclear phagocytes from different anatomical sites in patients with gynecological malignancies.

Mononuclear phagocytes exhibit complex interactions with cancer cells and might contribute to fibrin formation associated with malignancy through the production of procoagulant activity (PCA). We have studied the PCA of peritoneal macrophages in 8 patients with advanced (stages III or IV) ovarian cancer and of macrophages from regional lymph nodes in 14 patients with limited (stages I or II) uterine cancer; peritoneal and lymph-node macrophages from patients with benign gynecological tumors were used as reference cell populations. In all patients, PCA of blood monocytes was also studied. Peritoneal and lymph-node macrophages obtained from patients with ovarian and uterine cancer, respectively, expressed far higher levels of basal PCA than the corresponding cell populations from patients with benign tumors (p less than 0.001). PCA of blood mononuclear cells from patients with ovarian, but not with uterine cancer, was significantly higher (p less than 0.001) than that of control cells. High levels of D-dimer, a specific product derived from plasmin-induced degradation of stabilized fibrin, were found in all ascitic fluids and in all plasma samples but one from patients with ovarian cancer. In contrast, all controls and all uterine cancer patients but one had normal plasma D-dimer. Our findings suggest that local activation of host macrophages for PCA production might contribute to fibrin formation within the tumoral mass. In advanced cancer, blood monocytes may also be activated to produce PCA and thus contribute to activation of intravascular coagulation and, possibly, to thrombo-embolic complications frequently associated with disseminated malignancy.

Millimolar concentrations of ascorbic acid in purified human mononuclear leukocytes. Depletion and reaccumulation.

Ascorbic acid (vitamin C) was found in isolated human mononuclear leukocytes and their purified components in millimolar concentration. Intracellular ascorbic acid was depleted greater than 96% during cell culture and was rapidly reaccumulated after addition of physiologic concentrations of ascorbic acid to the extracellular medium. Purified cells maintained concentration gradients of ascorbic acid as large as 100-fold across the plasma membrane. The ability to vary intracellular ascorbic acid concentrations over such a wide range makes it possible for the first time in these cells to study ascorbic acid function in direct relationship to intracellular concentration.

Development, characterization, and subcellular location of DNAse activity in HL-60 cells and monocytes.

The digestion of DNA from intact bacteria by human phagocytic cells was measured by the release of solubilized radiolabeled DNA. Two subclones from the human promyelocytic HL-60 cell line were unable to digest bacterial DNA unless they were previously induced to mature by incubation for several days with 1.25% dimethylsulfoxide (DMSO). The maximal capacity of DMSO-induced HL-60 cells to digest DNA was similar to that of monocytes purified from peripheral blood (PB) and much greater than that of neutrophils. The increasing capacity to digest DNA during maturation was associated with the development of acid DNAse activity, measured in a cell-free system, and slightly preceded development of 12-O-tetradecanoyl phorbol 13-acetate-stimulated respiratory burst activity. The acid DNAse had a pH optimum of 5.0 and did not require the presence of calcium or 2-mercaptoethanol (2-ME). A third subclone of HL-60 cells was able to digest DNA from intact bacteria without previous maturation, however, and this was associated with the presence of an alkaline DNAse which had a pH optimum between 7.0 and 8.0 and showed a dependence on calcium and 2-ME for maximal activity. The subcellular location of acid DNAse in DMSO-induced HL-60 cells was similar to that of monocytes in having a bimodal distribution on fractionated sucrose density gradients. The dense peak (mean density 1.195 g/mL) was located in the same region of the gradient as primary granule enzymes but the light peak (mean density 1.137 g/mL) did not codistribute with either plasma membrane, endoplasmic reticulum, or mitochondria, suggesting accumulation in a different organelle.

Effects of transforming growth factor-beta on long-term human cord blood monocyte cultures.

Transforming growth factor-beta (TGF-beta) modulates growth and differentiation in many cell types and is abundant in bone matrix. We recently showed that human cord blood monocytes cultured in the presence of 1,25(OH)2D3 acquire some features of osteoclast precursors. Since TGF-beta has been shown to influence bone resorption in organ culture, we have studied the effect of TGF-beta (1-1,000 pg/ml) on cord blood monocyte cultures. These cells were cultured on plastic substrate during 3 weeks in the presence of 20% horse serum and 10(-9) M 1,25(OH)2D3. TGF-beta, from a concentration of 10 pg/ml in the culture medium, decreased in a dose dependent manner the formation of multinucleated cells. At a concentration of TGF-beta of 1 ng/ml, the multinucleated cells were reduced to 2.1% +/- 0.3%, compared to 19.3% +/- 1.5% in control cultures. TGF-beta inhibited in a dose-dependent manner the proliferation of cord blood monocytes as assessed by 3H-thymidine incorporation at 7 and 14 days of culture. The fusion index was also decreased by 3 weeks of treatment with TGF-beta. Indomethacin did not reverse the inhibitory effects of TGF-beta. The expression of the osteoclastic phenotype was assessed using two different antibodies: 23C6, a monoclonal antibody directed against the vitronectin receptor, which is highly expressed by osteoclasts but not by adult monocytes, and an antibody to HLA-DR, which is not present on osteoclast. TGF-beta decreased the expression of HLA-DR and increased in a dose-dependent manner the proportion of 23C6-labeled cells; these results suggest that TGF-beta could modulate a differentiation effect to the osteoclastic phenotype. However, when cord blood monocytes were cultured on devitalized rat calvariae prelabeled with 45Ca, TGF-beta did not induce any 45Ca release from bone cultured with monocytes, suggesting that full osteoclastic differentiation was not achieved. These results emphasize the complex role of TGF-beta in the local regulation of bone cell differentiation and in bone remodeling.

c-myc proto-oncogene expression in peripheral blood mononuclear cells from patients with primary Sjögren's syndrome.

We examined peripheral blood mononuclear cells (PBMC) from 16 patients with Sjögren's syndrome (SS) and 7 normal control subjects for the expression of the c-myc proto-oncogene. Patients with SS were found to have a significantly increased expression of c-myc messenger RNA compared with normal individuals. No abnormal forms of c-myc RNA were detected in the SS patients. DNA analysis did not show deletion, rearrangement, or amplification in the c-myc proto-oncogene. The methylation status of the c-myc gene in patients with SS was found to be comparable with that of the control subjects. Nuclear run-off assays showed increased transcription of the c-myc gene in some patients but normal transcription in others, suggesting that posttranscriptional mechanisms are also involved in the increased c-myc messenger RNA observed in these patients. Two patients with primary SS and B cell lymphomas were found to have normal c-myc expression in their PBMC. These results demonstrate the presence of activated PBMC in patients with primary SS and delineate some of the mechanisms that are involved at the molecular level. We speculate that increased c-myc expression may represent an early permissive event in the progression toward neoplasia in these patients.

Glycosphingolipid patterns of peripheral blood lymphocytes, monocytes, and granulocytes are cell specific.

We analyzed the amounts and types of glycosphingolipids (GSLs) from peripheral blood lymphocytes, monocytes, and granulocytes isolated by counter-current elutriation. The three cell types contained different amounts of neutral and acidic GSLs. The highest amount of neutral GSLs (109 micrograms/10(8) cells) was found in granulocytes, with considerably less found in monocytes (11 micrograms/10(8) cells) and lymphocytes (4 micrograms/10(8) cells). The neutral GSLs were composed of four types of lipids, GL1 through GL4 (mono-, di-, tri-, and tetraosylceramide). The highest percentage of GL1 was detected in lymphocytes and the lowest percentage in granulocytes, with the reverse order observed for GL2. GL3 and GL4, which were minor components of the neutral GSLs, were highly cell specific, with lymphocytes containing GL3 and GL4 of the globo series, granulocytes containing GL3 and GL4 of the lacto or neolacto series, and monocytes containing GL3 and GL4 of both types. The acidic GSL, sialosyl hexaosylceramide (lacto-series), was abundant in granulocytes but not in monocytes or lymphocytes. Another ganglioside, GM3, although present in all three cell types, was most abundant in monocytes and lymphocytes, whereas sialosyl paragloboside was higher in granulocytes than in lymphocytes and monocytes. These results indicate that peripheral blood lymphocytes, monocytes, and granulocytes have distinct "GSL fingerprints."

A selective defect in tissue factor mRNA expression in monocytes from AIDS patients.

Expression of tumor necrosis factor (TNF alpha), tissue factor (TF), and interleukin 1-beta (IL-1 beta) mRNA was evaluated in monocytes isolated from patients infected with human immunodeficiency virus (HIV). There was a significant depression (66%) of the induced level of TF mRNA expression in response to lipopolysaccharide. Conversely, the response of TNF alpha and IL-1 beta, following LPS induction, was "normal." TF mRNA reduction was also observed to a lesser degree in AIDS-related complex patients (20%) but not in asymptomatic seropositives. TF is necessary for initiation of the coagulation protease cascade, leading to thrombin production and fibrin deposition, which play a role in inflammatory responses. Its selective reduction may be a factor in the diminished resistance to secondary infections observed in AIDS. Further, since the TF defect increases as patients progress toward AIDS, it may serve as a marker for disease progression.

32P-postlabelling analysis of DNA adducts in monocytes of smokers and passive smokers.

In a controlled study, ten male volunteers were subjected to different smoking and passive smoking conditions. After 60 h of strictly controlled nonsmoking, five smokers were exposed to mainstream smoke only, while five nonsmokers were exposed to the gas phase of environmental tobacco smoke (ETS). In a second experiment smokers were mainstream and ETS exposed, while nonsmokers were exposed to complete ETS. Blood was drawn before and after smoking and DNA adducts were analysed from blood monocytes by the 32P-postlabelling assay, using the nuclease P1 enhancement method. We detected DNA adducts in monocytes of all probands. These adducts unrelated to smoking showed interindividual differences but only minor intraindividual changes in four samples of the same donor. After smoking interindividually variable additional adducts were visible in active smokers only. These smoking-related adducts had disappeared after 40 h of nonsmoking and reappeared again in three out of five smokers after the second smoking period. We conclude that smoking causes an interindividually variable pattern of DNA adducts in active smokers. These adducts disappear in less than 2 d, owing to the fast turnover of monocytes in the intravascular system. The effects described could not be observed in heavily exposed passive smokers.