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These studies reveal that the enumeration of peripheral blood mononuclear cells by fluorescence microscopy and automated flow microfluorometry show a high degree of correlation whether the cells came from normals, individuals with common variable immunodeficiency, chronic lymphocytic leukemia of B cell origin or chronic lymphocytic leukemia of T cell origin. There was excellent agreement between these two methods when counting positive cells stained by the pan-T monoclonal antibodies OKT3 and Leu-1, the helper T reagents OKT4 and Leu-3a, and the suppressor T antibodies OKT8 and Leu-2a. The values obtained for B cells using a pan-B (HB-2) cell antibody analyzed by fluorescence microscopy and automated flow microfluorometry gave a correlation coefficient of 0.86. The percentage of cells identified by antibodies with reactivities toward peripheral blood monocytes (MMA or Leu-M1), the HLA-DR determinant, and HNK-1 (Leu-7) positive cells gave correlation coefficients of 0.90, 0.90, and 0.80 respectively when compared by the 2 methods mentioned above. These data suggest that comparable values for lymphocyte subpopulations in human blood samples can be obtained using the most convenient and available technology.

A biotin-avidin horseradish peroxidase system is described for the localisation of membrane-bound monoclonal antibodies by low power transmission electron microscopy. An indirect bridge technique was used, with a biotin-labelled goat anti-mouse immunoglobulin antibody and horseradish peroxidase-conjugated avidin D. The technique provided preparations of high morphological and ultrastructural quality which facilitated accurate cellular identification. Positive immunostaining was specific and precisely defined.

A simple, sensitive, rapid and reproducible method is described for the measurement of HLA-DR expression on monocytes. The assay is based on an enzyme-linked immunostaining of the cells in suspension form followed by colour development. The non-specific binding of anti-HLA-DR to Fc receptors was taken into account by incubation of cells with an irrelevant monoclonal antibody (antirubella-G7H9). The binding of the anti-rubella monoclonal with the cells was subtracted from the binding of anti-HLA-DR to cells. Using three different cell lines (K562, CTLL and Daudi), the specificity of the assay proved to be acceptable and the assay remained sensitive even when only 10(4) cells/well were used. The assay has a number of potential applications.

Cytokines released from monocytes upon stimulation by lipopolysaccharide cause a number of cells to undergo proliferative and synthetic changes. At least one of these cytokines affects hepatocytes in vivo causing increased synthesis of a series of acute-phase proteins. We have established an in vitro micro-assay for hepatocyte stimulating factor (HSF) using primary cultures of normal rat hepatocytes. Measurement of increased synthesis of alpha 2-macroglobulin and decreased synthesis of albumin caused by exogenously added factor constitute a sensitive parameter for quantifying HSF. For comparing various cytokines preparations, we have defined a unit of HSF activity in terms of a stimulation index. We have used this assay to follow some preliminary attempts to isolate the factors responsible for stimulation of synthesis of acute-phase reactant by the liver.

We have used IL-1 alpha and IL-1 beta specific RNA standards in an RNA dot blot assay to rapidly and quantitatively assess the differential expression of the two human interleukin-1 mRNAs. This approach enabled us to minimize inaccuracies due to differences in specific activities of the probes, transcript size, and transfer and/or hybridization efficiencies of the two transcripts. We were thus able to accurately determine the steady state levels and molar ratio of the two species of IL-1 mRNA in human peripheral blood monocytes (PBM) and alveolar macrophages (AM). PBM, upon stimulation by lipopolysaccharide (LPS), expressed approximately ten-fold more IL-1 beta mRNA than IL-1 alpha. AM, however, expressed only two-fold more IL-1 beta then IL-1 alpha. PBM aged in vitro for 7 days prior to LPS stimulation, expressed at least 20-fold more IL-1 beta than IL-1 alpha RNA and at levels significantly lower than either PBM or AM.

A simple method was devised for counting small numbers (10(4)-10(6)) of adherent mononuclear phagocytes, including populations containing multinucleated giant cells, which often arise during cultivation of human blood monocytes. Coverslips with adherent cells were transferred into small volumes (50-200 microliters) of 0.1 M citric acid, pH 2.2, containing 0.05% naphthol blue black and 1.0% of either Triton X-100 or Cetavlon. Triton X-100 was adequate for use with monocytes and macrophages from early cultures. However, Cetavlon was preferable for use with older cultures of adherent human mononuclear cells in order to prevent aggregation of the nuclei from giant cells. When multinucleated cells were present, separately stained coverslips were inspected to determine the mean number of nuclei per cell. This value, together with the number of nuclei per coverslip, permitted calculation of the number of cells per coverslip. The latter value is not readily derived from measurements of protein or DNA content in populations containing multinucleated giant cells. This counting method was simpler and more sensitive than several previously reported methods for enumerating adherent macrophages.

By limiting dilution of WEHI 164 mouse fibrosarcoma cells we have isolated a cell line, WEHI 164 clone 13, which is extremely sensitive to cytotoxic factor (CF) derived from human monocytes. By using WEHI 164 clone 13 in a MTT tetrazolium cytotoxicity assay it was found that CF supernatants from activated monocytes had to be diluted 10(5)-10(6) times to reach the dose which produced 50% dead cells (LD50). By comparing the LD50 of different target cells, WEHI 164 clone 13 cells were found to be approximately 10(3) times more sensitive for CF-induced cytotoxicity as compared to WEHI 164 parental cells and approximately 10(2) times more sensitive as compared to actinomycin D-treated L929 cells. Treatment of the WEHI 164 clone 13 cells with actinomycin D did not increase their sensitivity for CF-induced cytotoxicity. Recombinant tumor necrosis factor (rTNF) also mediated high cytotoxicity towards WEHI 164 clone 13 cells, with an LD50 of 2 X 10(-3) ng/ml. Neutralizing CF antiserum completely inhibited the toxic activity of rTNF. WEHI 164 clone 13 cells were highly sensitive to monocyte-mediated cytotoxicity in that 1-2 monocytes were able to kill at least 5000 of these target cells. Neutralizing TNF antiserum completely inhibited monocyte-mediated cytotoxicity. These results indicate that the high level of cytotoxicity mediated by CF supernatants and monocytes on WEHI 164 clone 13 cells is due to TNF as the effector molecule.

Among the effector molecules induced in monocytes by the cellular immune response is tissue factor (TF), the initiating receptor/cofactor of the extrinsic coagulation protease cascade that is also frequently observed on human tumor cells. Other cellular activators have also been described on monocytes and tumor cells. Analyses of the cellular immune procoagulant response would be aided by a simple and efficient form of quantitation. An assay for cellular procoagulant activity (PCA) induction and expression was developed utilizing the chromogenic thrombin substrate tosyl-Gly-Pro-Arg-p-nitroanilide acetate. The constitutive or induced PCA of a variety of cells was analyzed. Peripheral blood mononuclear cells, peritoneal exudate cells, 13762 Mat B (III) mammary carcinoma cells, 1591-RE fibrosarcoma cells, the macrophage cell line WEHI-265, a detector of PCA inducing lymphokines, or mixtures of these cells were incubated with or without stimuli, e.g., endotoxin, in 96-well microplates. After incubation the cells were assayed for PCA by addition of the chromogenic substrate for thrombin using fibrinogen depleted plasma as a source of the coagulation proteins factors VII, X, V and prothrombin. The absorbance at 405 nm was determined. Spontaneous cleavage of the chromogenic substrate restricted the assay to total analysis times of less than 14 min. The 13762 Mat B (III) rat tumor which constitutively expressed tissue factor-like procoagulant activity induced measurable substrate hydrolysis with as few as 100 cells/well. It was observed that the chromogenic substrate assay was approximately twice as sensitive as conventional clotting assays for procoagulant activity. Endotoxin stimulated human peripheral blood mononuclear cells and mouse peritoneal exudate cells were readily analyzed. The procoagulant activity of approximately 280 LPS-stimulated human monocytes generated sufficient thrombin to provide a significant measurable signal within 10 min. Also supernatants from mixed lymphocyte cultures as well as from immune lymphocyte responses to syngeneic tumor cell cultures induced procoagulant activity in the macrophage like cell line WEHI-265 as determined with the assay for thrombin generation. The hydrolysis of the substrate was attributed to thrombin formation since the induced cleavage was abolished by hirudin, the highly specific active site inhibitor of thrombin. This chromogenic thrombin assay can be used for measuring induction of viable cell expression or total cellular procoagulant activity rapidly and efficiently in large replicate numbers suitable for a variety of analyses of cellular immune responses including clonal analyses of gene induction.

Bovine blood mononuclear cells were isolated by density gradient centrifugation on Ficoll-Paque. Phagocytic mononuclear cells were characterized functionally by ingestion of fluorescent latex beads. After incubation with beads the cells were treated with Triton X-100 and propidium iodide (PI) to stain DNA. Cells were analyzed with a FACS-III instrument connected to a Nuclear Data-6660 multiparameter computer system. The computer was used to evaluate the 2 parameter histograms in order to enumerate the percentage of cells with different numbers of associated beads. With this system we also obtained information about cell concentration and number of beads per cell. Results from flow cytometry and manual counting by fluorescence microscopy were compared and good correlation (r = 0.91) was obtained. During the first hours of incubation latex beads adhered to cell surfaces as demonstrated by FCM histograms and fluorescence microscopy. Blood mononuclear phagocytes have to be incubated for several hours before significant phagocytotic activity can be detected.

An improved immunoenzyme assay for measuring cell-surface beta 2-microglobulin (beta 2 m) is presented in which quantitation is achieved by reference to soluble beta 2 m-induced inhibition of horseradish peroxidase-labelled rabbit antibody. Some consideration is given to kinetics of binding and dissociation, and their implications for interpretation are discussed.

An assay for determining the DNA content of mononuclear phagocytes is described. The assay is efficient and very sensitive, measuring as little as 1 microgram of DNA. Content of DNA is a linear function of the number of mononuclear phagocytes in the sample. One million murine macrophages contain 10.1 +/- 0.36 microgram of DNA. Consequently, samples of as few as 100,000 macrophages can be accurately quantified to be related to other biochemical analyses.