Phagocytosis and killing of Mycobacterium avium complex by human neutrophils.

Organisms belonging to the Mycobacterium avium complex (MAC) cause life-threatening bacteremia in immunocompromised patients. Monocytes and macrophages are thought to be responsible for ingestion and killing of MAC. However, it has been suggested that neutrophils may play a role in the early immune response to MAC infection. Here, neutrophils in autologous plasma were incubated (at 0 and 37 degrees C) with M. avium labeled with Auramine O, a potent fluorochrome. Neutrophil phagocytosis was measured by flow cytometry. Neutrophils incubated at 37 degrees C showed an increase in fluorescence over time with a maximum at 15 min, whereas neutrophils on ice showed no time-dependent increase in FL1. At 15 min Fl 1 at 37 degrees C was twice as high as FL1 at 0 degrees C. Examination under the fluorescent microscope showed multiple intracellular fluorescent mycobacteria. Results in nine independent experiments showed time-dependent decrease of colony-forming units in neutrophil-associated live M. avium. Significant killing was observed within 30 min and was complete by 120 min. Observation by electron microscopy clearly confirmed the presence of intraphagosomal MAC, both intact and with evidence of degradation. These data demonstrate that MAC is rapidly phagocytized and killed by human neutrophils. The newly established flow cytometry method should be useful in further studies of neutrophil function and of the role of G-CSF and other cytokines in MAC disease.

Analysis of single and double-stained alveolar macrophages by flow cytometry.

The quantification of cell surface antigens on human alveolar macrophages using flow cytometry is complicated by strong autofluorescence which varies with cell size and granularity. We report here a new method for overcoming the analytical problems caused by autofluorescence. After positioning the unstained cells along the 0, 0; 10(4), 10(4) diagonal on all three fluorescence dot-plots (FL1 vs. FL2; FL2 vs. FL3; FL1 vs. FL3) of a single-laser flow cytometer (excitation wavelength at 488 nm) and adjusting compensation so that the reference FL3 channel profile is not changed by PE-staining, the cell population on the FL3 histogram is arbitrarily classified into subpopulations having similar autofluorescence intensity. These are subsequently back-gated onto the FL1 vs. FL2 dot-plot and separately analyzed. The percentage of stained cells in the whole population is then calculated on the basis of absolute numbers or as the weighted mean of all the subpopulations. This approach permits the analysis not only of single-stained but also double-stained human alveolar macrophages.

Expression of surface markers on alveolar macrophages from symptomatic patients with HIV infection as detected by flow cytometry.

Alveolar macrophages (AMs) harvested from 32 HIV-infected patients with respiratory problems (opportunistic pulmonary infections, n = 12; other lung disease, n = 20) and 13 healthy controls were stained with a panel of 15 monoclonal antibodies directed against surface antigens implicated in cell function. Antigen expression was quantified by flow cytometry and expressed as relative linear median fluorescence intensity (RLMFI). On AMs of patients, as compared with controls, there was a significant enhancement of HLA DP (12.1 +/- 1.5 vs 6.5 +/- 0.9, p = 0.01, M +/- SEM, RLMFI units), CD11b (3.4 +/- 0.5 vs 1.7 +/- 0.4, p = 0.014), CD11c (8.9 +/- 1.0 vs 4.8 +/- 0.8, p = 0.0046), CD14 (2.1 +/- 0.3 vs 1.0 +/- 0.2, p = 0.0009), and CD33 (1.7 +/- 0.1 vs 1.0 +/- 0.2, p = 0.0093). No significant differences could be established for HLA-DR (36.9 +/- 5.8 vs 30.9 +/- 7.5, NS), HLA-DQ (3.4 +/- 0.3 vs 3.1 +/- 0.6, NS), CD54 (1.9 +/- 0.3 vs 1.2 +/- 0.1, NS), CD13 (2.5 +/- 0.6 vs 1.5 +/- 0.3, NS), CD36 (1.4 +/- 0.2 vs 0.9 +/- 0.3, NS), CD71 (10.3 +/- 1.9 vs 8.9 +/- 1.8, NS), CD25 (0.8 +/- 0.0 vs 0.9 +/- 0.1, NS), 27E10 (1.1 +/- 0.1 vs 0.8 +/- 0.3, NS), RM3/1 (1.9 +/- 0.4 vs 1.5 +/- 0.4, NS), and CD4 (1.5 +/- 0.3 vs 1.0 +/- 0.0, NS). The expression of CD14 and CD11b, but not of HLA class II antigens and CD71, was increased in the smaller cell population compared with the larger, thus suggesting monocyte recruitment. The increased expression of HLA-DP, CD11c, CD14, and CD33 on the patients' AMs was independent of smoking habits. The degree of immunodeficiency as indicated by the absolute peripheral CD4 count, the character of HIV-related pulmonary disease, and the prophylactic use of pentamidine or zidovudine did not significantly modify the antigen expression pattern. It is concluded that HIV infection may lead, most probably indirectly, to enhanced expression of surface antigens by local upregulation and/or recruitment of monocytes from the peripheral circulation. The functional significance of enhanced marker expression requires further clarification.

Disseminated growth of Hodgkin's-derived cell lines L540 and L540cy in immune-deficient SCID mice.

Local tumor growth has been reported after subcutaneous and intraperitoneal injection of Hodgkin's disease (HD) derived cell lines into different immunodeficient mouse strains. An animal model with disseminated growth of tumor cells would be useful for studying the in vivo biology of HD cells as well as for preclinical testing of new therapeutic regimens. For this purpose the HD-derived cell lines L540, L540cy, L428, and KM-H2 were injected intravenously into SCID mice. In contrast to L428 and KM-H2, widespread neoplasia occurred after a period of four to six weeks following injection of L540 and the subline L540cy. Lymph nodes were found to be the preferred site of tumor growth. CD30 surface antigen expression on Hodgkin cells and the karyotype of the tumor cells were preserved in the animal host. Thus, to a large extent, the SCID mouse model mimics the dissemination pattern of Hodgkin's disease in man. To evaluate the role of adhesion molecule expression in the dissemination of HD-derived cell lines, CD44 and members of the immunoglobulin, integrin, selectin, and Fc receptor families were quantified by flow cytometry. CD30 expression was also measured. Although CD44 expression has been correlated with dissemination in non-Hodgkin's lymphoma (NHL), this was not the case in the Hodgkin's SCID mouse model. CD44 was not expressed on the disseminating cell lines L540 and L540cy but was expressed in the nondisseminating lines L428 and KM-H2.

Pentamidine aerosol increases the number of alveolar macrophages in HIV-infected patients.

In order to determine the possible effect of aerosolized pentamidine on the cellular composition of the bronchoalveolar lavage fluid in HIV-infected patients, differential counts of 22 consecutive patients who had been rebronchoscopied after 3-19 months were reviewed. Eleven patients were started on pentamidine prophylaxis subsequent to their first presentation. Eleven patients had never taken pentamidine or had discontinued the prophylactic regimen. Compared to first bronchoscopy, the bronchoalveolar lavage (BAL) from patients on regular prophylaxis revealed a significant increase in absolute alveolar macrophage (AM) counts at second presentation (20.8 +/- 11.2 to 50.3 +/- 39.4 x 10(5) cells/100 ml BAL; P less than 0.01). The AM counts of those without pentamidine remained essentially unchanged. Lymphocytes, including CD4 and CD8 subtypes, and neutrophils did not change over time in either group. The results of this retrospective analysis suggest that, in addition to its antimicrobial action, pentamidine may modulate local lung defence mechanisms, particularly by increasing the absolute number of AM.

Identification and quantification of human peripheral blood lymphocytes and monocytes following migration into nitrocellulose filters.

Previously, in order to evaluate the migration of specific types of lymphocytes (e.g., T or B cells, CD4 or CD8 subsets) using the Boyden chamber technique, the relevant cell populations have first been purified. We report here a method which permits identification of the lymphocytes following migration into the nitrocellulose filters. After fixation, the filters are exposed to specific anti-lymphocyte monoclonal antibodies and the reaction is visualized via a second gold-linked antibody. Monocyte-depleted lymphocytes isolated from human peripheral blood are routinely used for migration but mixed mononuclear cell preparations can also be used. This was shown by comparing lymphocyte migration in monocyte-containing and monocyte-depleted cell suspensions isolated from the same donor. The presence of monocytes did not influence the migration of normal resting T lymphocytes. With human peripheral blood as starting material the method is most suitable for the evaluation of T cell migration since, in most instances, T cells are the predominant lymphocyte population. When the B cell count is normal (5-10% total lymphocytes), quantification of B cell migration requires enrichment but not complete purification of this population. Migrating monocytes can also be identified. However, the antibody staining is less intense on the spread monocytes and, therefore, quantification must be performed by eye rather than with the Optomax image analyser.

L428 cells derived from Hodgkin's disease produce E rosette-inhibiting factor.

Diminished rosetting capacity of T cells is a well-known phenomenon in Hodgkin's disease, and inhibitors of E rosette formation have been reported to be present in the plasma of patients with Hodgkin's disease. The cell line L428, representing an in vitro counterpart of Hodgkin and Sternberg-Reed cells, could be shown to release a factor capable of suppressing the binding of sheep red blood cells (SRBC) to normal peripheral-blood T lymphocytes or to a T-cell line (L735). At maximally effective concentrations, RIF (rosette inhibiting factor) inhibited T lymphocyte rosetting by approximately 40% (mean from 184 healthy controls). The diminished E rosetting of T lymphocytes from Hodgkin's patients was not further suppressed by added RIF. This factor inhibited binding of SRBC to their target cells at 37 degrees C but not at 4 degrees C. The factor could be stored lyophilized at -20 degrees C and was stable at 56 degrees C (30 minutes). RIF was inactive below pH 6 and above pH 9 or after trypsin digestion. Purification by affinity, ion exchange, and molecular sieve chromatography showed activity peaks at 12.5 Kd, 25 Kd, 50 Kd, and 100 Kd.

Effects of recombinant tumor necrosis factor (rHuTNF alpha) on human neutrophils and monocytes: in vitro, ex vivo and in vivo.

In in vitro experiments, tumor necrosis factor (rHuTNF alpha) was found to inhibit spontaneous and directed migration of normal human neutrophils and monocytes. RHuTNF alpha showed no chemoattractant activity. In conjunction with a Phase-2 clinical trial, we also studied in vivo rHuTNF alpha effects on neutrophil and monocyte number and function. At the time of maximal plasma TNF levels (30 min), marked neutropenia and monocytopenia were observed. Isolated neutrophils were activated for superoxide production but were unable to migrate. Monocyte migration was inhibited at later times. Neutrophil migratory function recovered between 4 and 8 h but was again depressed at 24 h. The data demonstrate the complexity of the response to TNF, comprising direct and indirect effects which are concentration-, time- and place-dependent. They further suggest that the only neutrophils and monocytes available for participation in an anti-tumor activity of TNF in vivo are those present in the tumor at the outset.

Inhibition of human neutrophil migration by supernatants from Hodgkin's disease-derived cell lines.

The contribution of defective neutrophil function to the increased susceptibility to infection observed in patients with Hodgkin's disease is unclear. We describe cell-directed inhibition of normal human neutrophil migration by serum-free culture supernatants of the Hodgkin-derived cell line L428 KSA, tentatively termed Hodgkin-derived leucocyte factor (HDLF). This factor inhibits both random migration and migration toward different chemoattractants, appears to bind to the cell surface and is stable at 56 degrees C but destroyed at 100 degrees C. Hodgkin-derived leucocyte factor also stimulates basal neutrophil superoxide production but the cells remain fully responsive to n-formyl-methionylleucylphenylalanine. Gel filtration chromatography shows a single peak of migration-inhibitory and superoxide-stimulatory activity at approximately 70,000 g mol-1. Hodgkin-derived leucocyte factor migration inhibition persists in neutrophils from a patient with chronic granulomatous disease. Activity of HDLF is completely destroyed by trypsin but unaffected by the protease inhibitor phenyl-methylsulphonylfluoride. Hodgkin's factor appears to be different from previously described neutrophil migration inhibitory factors.

Limitations in the use of indium-111-oxine-labeled leucocytes for the diagnosis of occult infection in children.

Fifteen children underwent scintigraphy with indium 111 (111In)-labeled white blood cells (WBC) for the detection of a local suppuration. The procedure generally contributed to a correct diagnosis. False negative results were observed in 5 children, but in two of them positive foci were also present. The missed lesions were 2 liver abscesses, 1 lung abscess, foci of osteomyelitis and 1 pericarditis. Two cases of chronic granulomatous disease are presented in which increased leucocyte accumulation was not observed in proven instances of infection.

111Indium-oxine-labeled leukocytes in the diagnosis of localized infection in patients with neoplastic disease.

One hundred twenty-nine 111In-oxine-labeled leukocyte scintiscans have been performed in 117 patients with cancer in order to diagnose localized infectious disease. Of the 115 contributive scans, 40 were in patients with localizing signs, whereas in 75 fever of unknown origin constituted the indication for this examination. The overall specificity of the method was 95.4%, the overall sensitivity 86%, and the global accuracy 91.3%. In 10 cases with localizing signs, the 111In-oxine granulocyte scintigram allowed exclusion of the diagnosis of infection, whereas in 17 instances without localizing signs, a focal infectious process was demonstrated. Heterologous donor leukocytes were used successfully in five instances. With the exception of accumulation of label at the site of an osteolytic metastasis in one case, no uptake was observed in primary or secondary tumors. It is concluded that 111In-oxine-labeled leukocytes constitute a valuable tool in the diagnosis and localization of infection in patients with malignant disease.

A comparison of the effects of soluble stimuli on free cytoplasmic and membrane bound calcium in human neutrophils.

Calcium dynamics in human neutrophils have been studied using Quin 2 fluorescence as a measure of free cytoplasmic calcium and chlortetracycline fluorescence as an indicator of membrane-bound calcium. The results show that 1) FMLP-induced increased cytoplasmic calcium likely comes from at least two different pools. Calcium is released from one only after a high affinity receptor interaction and from the second also after a lower affinity interaction. The initial increment in cytosolic calcium does not appear to originate in the pool(s) reflected by CTC fluorescence. 2) Cytochalasin B strikingly alters the FMLP effect on membrane associated calcium, inducing a marked "recovery" phase which could be a reflection of fusion of granule membranes with the plasma membrane. 3) PMA, at concentrations inducing extensive specific granule release (less than or equal to 10 ng/ml) has no measurable direct effect on membrane-bound or cytosolic calcium. However, PMA inhibits a subsequent CTC fluorescence response to FMLP and following the ionophore, A23187, it induces a clear decrease in cytosolic calcium. These indirect effects may be explained in terms of PMA's activation of protein kinase C.

Specific staining of the thyroid follicular cell apical membrane by nitroblue tetrazolium.

Nitroblue tetrazolium (NBT) has been widely used to demonstrate redox systems, particularly in phagocytizing polymorphonuclear leukocytes. Based on the striking similarities between redox metabolism in leukocytes and in the thyroid, we studied NBT reduction by microscopic techniques and NBT effects in thyroid metabolism in dog-thyroid slices in vitro. We observed specific localization of NBT reduction product along the apical membrane of the follicular cell. Hence, NBT is a potentially useful market of thyroid-cell polarity.

Prostaglandin formation in bacteria : a reevaluation.

We report here that production and release of PGE2 do not occur in common bacteria. The apparent production in the presence of arachidonic acid, previously reported (1) may be explained by PGE2 contamination and autooxidation of the AA used. The presence of PGE2 like material in some but not all isolates of Propionibacterium acnes is confirmed.

Prostaglandin formation in bacteria.

We report here that intact Gram positive and Gram negative bacteria, in the presence of exogenous arachidonic acid, produce and release PGE2 and PGF2 alpha into the medium as measured by radioimmunoassay. The seven bacterial strains so far studied release 6.5-50.9 ng PGE2 and less than 0.02-0.51 ng PGF2 alpha per mg bacterial protein during a 1 hour incubation, quantities of the same order of magnitude as those observed in mammalian systems. PGE2 and PGF2 alpha formation in bacteria are inhibited by indomethacin.