Showing papers in "Journal of Leukocyte Biology in 1989"

Differentiation, maturation, and proliferation of macrophages in the mouse yolk sac: a light-microscopic, enzyme-cytochemical, immunohistochemical, and ultrastructural study.

Abstract: Primitive macrophages first appear in the blood islands of the mouse yolk sac on the ninth day of gestation. After the tenth day of fetal life, these cells differentiate Into fetal macrophages and become mature, with the development of Intracellular organelles. They appear in the mesenchymal layer and further immigrate into the extraembryonic coelom. The fetal macrophages do not show any cytochemical peroxidase or 5’-nucleotldase activity, and they possess a marked proliferative capacity. Promonocytes or monocytes that have an incomplete ultrastructure emerge in the blood islands of the yolk sac a day after the occurrence of the fetal macrophages. These events suggest that fetal macrophages differentiate from primItive macrophages before the development of promonocytes or monocytes in the mouse yolk sac; they actively proliferate and are colonized into the embryonic tissues. These results also Indicate that the ontogeny of the monocyte/macrophage is different in the early embryo compared with its later developmental stages.

Macrophage subset repopulation in the spleen: differential kinetics after liposome-mediated elimination.

Abstract: Different macrophage subsets can be discriminated in the well defined compartments of the mouse spleen by specialized functions and the presence of specific surface determinants. Red pulp macrophages, marginal zone macrophages, and marginal metallophilic macrophages are eliminated simultaneously within 24 hr by a single injection with liposome-entrapped dichloromethylene diphosphonate (DMDP). After such elimination, these subsets show a striking difference in their kinetics of reappearance: Red pulp macrophages are back in normal numbers after 1 week, the marginal metallophilic macrophages take 2 weeks to regain fully their position at the border of the marginal zone and periarteriolar lymphocyte sheath, but it takes over 1 month for complete reappearance of the marginal zone macrophages. Marginal zone lymphocytes, also affected by treatment with the liposome-entrapped drug, reappeared in the marginal zone within 2 weeks, indicating that marginal zone macrophages are not required for their localization and/or retention there. Approximately 2 weeks after treatment, all cells in the spleen have returned to normal numbers with the exception of marginal zone macrophages, which can be found only sporadically at that time. The results indicate that these macrophage subpopulations must have different precursor requirements. The differential reappearance of the macrophages creates the possibility of studying lineage analysis and will help to unravel the precise function of the marginal zone macrophages and marginal metallophilic macrophages in particular.

Interferon-induced indoleamine 2,3-dioxygenase activity in human mononuclear phagocytes.

Abstract: Interferon (IFN)-induced tryptophan degradation, catalyzed by indoleamine 2,3dloxygenase (IDO), has been shown to mediate antimicrobial activity in epithelial cells. IDO activity has also been augmented in peripheral blood mononuclear cells (PBMC) treated with IFN or interleukin-2 (lL-2). The effector cells in this population have now been further characterized. PBMC5 were isolated from normal donors, separated Into monocyteandlymphocyte populatlona by placticadharenee, treatedwith IFN or lL-�, and cultivated in medium supplemented with [3H]tryptophan. Culture supernatants were collected after a 48-h incubation and fractionated by high-performance liquid chromatography; radioactivIty was determined in fractions corresponding to tryptophan and its metaboiltes. IFN-y and IFN-(3 induced IDO activity only in monocytes (plastic-adherent, nonspecific esterase-positive PBMCs). The Induction of IDO actIvity by 11-2 requIred both monocytes and lymphocytes. Interaction was required between these populations for Induction of IDO by 11-2, due to production of IFN-’j by I lymphocytes, with subsequent IFN-y-mediated induction of IDO in monocytes. A number of myeioid cell lines as well as monocyte-derlved macrophages were also tested for their ability to be induced to degrade tryptophan in response to IFN treatment Monocyte-derived macrophages were found to retain their capacity to be Induced by IFN-’y and IFN-13 to degrade tryptophan after differentiation, and to possess seven times more IDO activity per cell than IFN-lnduced monocytes. However, the presence of lipopolysaccharide (LPS) in the culture medIum was required for the maximum Induction of IDO activity by IFN-�. Furthermore, higher concentrations of LPS were sufficient to induce IDO activity in macrophages in the absence of exogenous IFN.

MH-S, a murine alveolar macrophage cell line: morphological, cytochemical, and functional characteristics.

Abstract: A continuous cell line of murine alveolar macrophages (AM), designated MH-S, has been established following transformation of cells obtained by bronchoalveolar lavage from Balb/cJ mice with simian virus 40 (SV40). Thirty days after infection of the AM cultures, foci of rapidly proliferating cells were recovered and these have been propagated continuously for more than 36 mo. Following its initial isolation in Fischer's medium supplemented with L-cell-conditioned medium and horse and fetal bovine serum, the cell line is now routinely grown in RPMI-1640 medium containing 10% fetal bovine serum in the absence of conditioned medium. MH-S cells were adherent, lacked contact inhibition, and were trypsin-sensitive. They expressed intracellular T-antigen and incorporated 3H-thymidine (DNA synthesis) with a doubling time of approximately 48 h but doubled in number in 96 h. MH-S exhibited typical macrophage morphology, was greater than 98% esterase-positive, negative for peroxidase, and expressed cell surface Ia and Mac-1 antigens. The cells were Fc receptor-positive as demonstrated by rosette formation with, and phagocytosis of, antibody-coated sheep erythrocytes. Constitutive IL-1 secretion was significantly increased following stimulation of the cells with lipopolysaccharide. Like freshly isolated AM, MH-S cells suppressed the in vitro plaque-forming cell (PFC) response in a dose-dependent manner when cultured with splenic lymphocytes. This cell line should facilitate studies where homogeneous populations of AM are desirable, especially those involved in determining the immunological functions of AM and their potential role in lung pathology.

Regulation of Monokine Gene Expression: Prostaglandin E2 Suppresses Tumor Necrosis Factor but Not Interleukin‐1α or β‐mRNA and Cell‐Associated Bioactivity

Abstract: Prostaglandin E2 (PGE2)-mediated suppression of macrophage interleukin-1 alpha,beta and tumor necrosis factor-alpha synthesis was examined at the cellular and molecular levels. Treatment of lipopolysaccharide (LPS)-stimulated adjuvant-elicited murine macrophages with 5 x 10(-7) M PGE2 caused a 70% reduction in cell-associated TNF but had no suppressive effect on cell-associated interleukin-1 (IL-1) activity. Consistent with this result, Northern blot and nuclear transcription analyses demonstrated suppression of TNF mRNA but PGE2 had no effect on IL-1 alpha and IL-1 beta mRNA accumulation, as compared to LPS controls. Immunoperoxidase staining for cell-associated TNF alpha, IL-1 alpha, and IL-1 beta demonstrated that PGE2 suppressed TNF, but not IL-1 alpha or -beta expression, supporting the bioassay data. These results imply that PGE2-mediated regulation of IL-1 alpha,beta and TNF alpha is quite distinct. Synthesis of TNF appears to be regulated at least at the level of transcription, whereas that for IL-1 alpha and -beta is regulated post-transcriptionally.

Heterogeneity of macrophages in the rat evidenced by variability in determinants: two new anti-rat macrophage antibodies against a heterodimer of 160 and 95 kd (CD11/CD18).

Abstract: A set of three monoclonal antibodies (MoAbs), ED1, ED2, and ED3, has been shown to recognize in situ different subsets of macrophages in the rat. This macrophage diversity can be correlated with differences in stage of differentiation of cells belonging to one lineage. The present study quantifies this antigen distribution in the macrophage fractions of several lymphoid organs provided by Percoll centrifugation. Four new MoAbs (ED4, ED7, ED8, and ED9) raised against macrophages are included in this study. The tissue distribution of each of the four new MoAbs is determined by immuno- and enzymehistochemistry on cryostat sections.The MoAbs recognize distinct subpopulations of macrophages. The new MoAbs ED4, ED7, ED8, and ED9 recognize granulocytes and other unrelated cell types, as well as cells of the mononuclear phagocyte system. ED7 and ED8 recognize a surface heterodimer of M, 160,000 and 95,000.

Comparative study of cytotoxicity, tumor necrosis factor, and prostaglandin release after stimulation of rat Kupffer cells, murine Kupffer cells, and murine inflammatory liver macrophages.

Abstract: Resident murine liver macrophages had no natural cytotoxicity for the TNF-resistant target cell line P815. Activation of these cells was only obtained by a combination of IFNy and LPS. Inflammatory murlne macrophages were in a primed stage and could be actIvated by LPS alone in the absence of lFNy. Rat resident macrophages resembled functionally the Inflammatory macrophages of the mouse liver rather than the resident macrophages. They displayed natural cytotoxicity against all targets tested and were further activated by LPS in the absence of lFNy. Similar results were obtained with respect to macrophage-depleted nonadherent NPC: Mouse NPC had a low level of NK activity against Yac-1 cells. Treatment with pyran copoiymer resulted in a strong increase of cytotoxicity against Yac-1; furthermore, a TNF-dependent killing of Wehi 164 and TNF-lndependent cytotoxicity against P815 cells were now acquired. In the rat NPC prepared from unstimuiated animals expressed high levels of natural cytotoxicity against all targets. No major differences could be observed between inflammatory MO and Kupffer cells of rat and mouse liver wIth regard to TNF production and TNF-dependent killing of Wehi 164 tumor cells. The same was true for the spectrum of secreted prostanoids. Upon activation of all cell populations a marked shift toward the production of PGE2 occurred. Experiments Involving the cyclooxygenase Inhibitor indomethacin showed enhanced TNF-dependent tumor cell killing by nonactivated MO in the absence of prostanoid production.

Differential production of tumor necrosis factor, macrophage colony stimulating factor, and interleukin 1 by human alveolar macrophages

Abstract: Human alveolar macrophages (AMO) have been investigated for their ability to produce three monokines, tumor necrosis factor-alpha (TNF), macrophage colony stimulating factor (CSF-1), and interleukin 1-beta (IL-1). No TNF activity was found in supernatants of unstimulated AMO cultured for 20 h, although TNF mRNA was detected in the cells by Northern blot analysis. Stimulation of the cells with lipopolysaccharide (LPS) induced production and release of high levels of TNF into the culture supernatant. Increased levels of TNF mRNA were detectable at 90 min after LPS stimulation by dot blot analysis, reaching maximum expression between 4 and 8 h and declining thereafter. TNF activity peaked at approximately 8 h in the AMO supernatants. After 24 h TNF production had ended. Compared to autologous monocytes the AMO produced 5.7 times more TNF on a per cell basis (activity accumulated in 20 h supernatants). Uncultured AMO expressed CSF-1 mRNA which was translated into active protein recovered in supernatants upon culture in the absence of stimulus. The addition of LPS to AMO slightly reduced both mRNA levels and amount of factor in the supernatants. In contrast to the AMO, monocyte production of CSF-1 was enhanced by LPS. CSF-1 production by AMO continued for at least 48 h of culture. Spontaneous production of low amounts of IL-1 was found in one-third of the AMO samples, while low levels of IL-1 mRNA were present in all tested preparations. LPS stimulation induced increase in IL-1 mRNA within 90 min; mRNA levels peaked between 12 and 20 h and stayed high for at least 42 h. However, while all LPS-stimulated AMO expressed high levels of IL-1 mRNA biologically active IL-1 was again detected only in a fraction of the AMO supernatants. These results show that the production of monokines CSF-1, TNF, and IL-1 is differentially regulated by LPS in alveolar macrophages and has different responses as compared to monocytes. The longevity of the messages for each of the factors is possible indicators of the relative contribution of these factors to the response to endotoxin-induced injury and repair processes in the lung.

Human neutrophils activated by interferon-γ and tumour necrosis factor-α kill Entamoeba histolytica trophozoites in vitro

Abstract: The interaction between cytokine-activated human neutrophils and Entamoeba histolytica trophozoites was studied as well as the mechanism(s) involved. Treatment of neutrophils with rIFN-gamma alone allowed them to kill 30% of E. histolytica trophozoites; however, rIFN-gamma and rTNF-alpha pretreatments in combination increased neutrophil killing to 70%. In the absence of direct contact between neutrophils and amebae, rIFN-gamma-treated neutrophils were shown to kill 70% of amebae, and rIFN-gamma- and rTNF-alpha-treated neutrophils killed 97% of amebae. Neutrophil enhancement of amebicidal activity following cytokine treatments was correlated with increased neutrophil resistance to amebic contact-dependent killing and was shown to be 73% H2O2 dependent.

Natural killer cell function in patients with acquired immunodeficiency syndrome and related diseases.

Abstract: This review describes current knowledge of changes in natural killer (NK) cell function in acquired immunodeficiency syndrome (AIDS)-related disorders, vis-a-vis associated abnormalities in NK cytolytic function, NK cell subset distribution, NK cytopathology, and lymphokine regulation. NK cells, which are closely associated with large granular lymphocytes, are spontaneously cytotoxic to tumor and virally infected targets. As such, they may play a role in natural resistance to human immunodeficiency virus type 1 (HIV-1)-associated disorders and other opportunistic infections. Yet, peripheral blood NK activity is frequently reduced in patients with HIV-1-induced disease. NK cells are heterogeneous both with respect to their expression of serologically defined membrane antigens and functional activity. In AIDS-related syndromes, there appears to be a diminution of the NK pool (CD16+ cells) involved in cytolytic function, while there is an elevation of the NK pool that coexpresses NK (Leu 7+) and T (CD8+) cell markers, which show little or no involvement in cytolytic function. The impairment of in vitro NK function is not associated with a reduced frequency of lytic conjugates of effectors and target cells nor with the recycling capacity of these effector cells but rather is associated with defects in the NK cell lytic machinery following formation of such conjugates. NK cells in AIDS patients show an impairment in effector cell microtubule rearrangement following target cell interaction. The causes of NK cell dysfunction in AIDS-related disorders remain unknown. NK cells do not appear to express the CD4 epitope of the HIV receptor, nor have they been demonstrated to be susceptible to infection by HIV-1. There appears to be a preponderance of immature NK cells and a lymphokine imbalance in patients with HIV-1 associated disease. Interleukin-2 can partially restore diminished in vitro NK function. Elucidation of the involvement of the NK compartment in natural resistance to HIV-1 merits further investigation.

Interleukin-1 and interleukin-6 stimulate acute-phase protein production in primary mouse hepatocytes.

Abstract: Primary mouse hepatocytes were treated with the acute-phase mediators interleukin-1, interleukin-6, and glucocorticoids, singularly and in combination, in order to delineate the spectrum of proteins induced by the stimulatory factors. As found for rat and human liver cells, mouse hepatocytes responded to the cytokines by increasing production of acute-phase proteins, which in mice include haptoglobin, alpha 1-acid glycoprotein, complement C-3, serum amyloid A, and hemopexin. Serum amyloid A was unusual in that only the acidic peptide form responded to treatment with IL-1 and IL-6; the more basic form remained unchanged. In addition, an unidentified secretory protein was induced only by mixtures containing IL-6. The present study shows that a combination of IL-1, IL-6, and glucocorticoids is required for regulation of acute-phase plasma protein production in mouse liver cells.

Selective depletion of rat neutrophils by in vivo administration of a monoclonal antibody.

Abstract: A monoclonal antibody (RP-3) that depletes rat neutrophils selectively in vivo was developed by hybridization of mouse myeloma cells (P3-X63.Ag8.653) and spleen cells of BALB/c mice sensitized with peritoneal neutrophils of WKA/Hok rats. RP-3 reacted with rat neutrophils but not with lymphocytes, macrophages, natural killer cells, basophils, eosinophils, or tissues of various organs. The mitogenic responsiveness to concanavalin A (ConA), phytohemagglutinin (PHA), and lipopolysaccharide (LPS) of rats given RP-3 was not significantly different from that of normal rats. Administration of RP-3 into the peritoneal cavity of rats that had been kept under specific pathogen-free (SPF) or clean conditions induced selective depletion of circulating neutrophils to under 100/mm3 (0.5% WBC). The numbers of monocytes, lymphocytes, and platelets were not changed. Administration of 2 ml of RP-3 reduced blood neutrophils to under 100/mm3 for approximately 24 h, and administration of 1 ml caused depletion for approximately 12 h.

Enhancement of the expression of activation markers on human peripheral blood mononuclear cells by in vitro culture with retinoids and carotenoids.

Abstract: Retinoids (retinol, retinal, retinoic acid, retinyl beta-glucuronide, and 13-cis retinoic acid) and carotenoids (beta-carotene and canthaxanthin) were evaluated for their immunomodulatory effects on human peripheral blood T-lymphocyte subpopulations and natural killer (NK) cells. Peripheral blood mononuclear cells (PBMC) from healthy young volunteers were isolated and incubated for 72 hours at various levels of retinoids and carotenoids including a physiological concentration (10(-8) M). Expression of surface antigens for total T cells, T-helper and T-suppressor cells, and activation markers (transferrin receptor, HLA-Dr antigen, and interleukin 2 receptor) were analyzed with an EPICS V flow cytometer. Retinoic acid and 13-cis retinoic acid (13-cRA) produced significant increases in the percentage of cells with markers for total T-helper cells, with a minimal effect on percentage of lymphocytes with markers for NK cells. However, beta-carotene (BC), canthaxanthin (CTX), and retinyl beta-glucuronide (RBG) dramatically increased the percentage of PBMC with markers for NK cells and produced a smaller increase in lymphocytes with surface antigens identifying them as T-helper cells. Furthermore, retinol and retinal did not show significant change either in the percentage of lymphocytes with markers for T-helper cells or in the helper/suppressor ratio. An increase in the expression of HLA-Dr antigen and transferrin receptors was greater when cells were incubated with 13-cRA than with either BC, CTX, or RBG, while carotenoids produced a greater increase in the expression of IL-2 receptors than 13-cRA. Our study indicates that both retinoids and carotenoids might be activating different subpopulations of immune cells.

Development, Differentiation, and Maturation of Fetal Mouse Yolk Sac Macrophages in Cultures

Abstract: The development and differentiation of macrophages in the fetal mouse yolk sac were studied morphologically in four different culture experiments. In the culture of mouse embryos with yolk sac, the development of fetal macrophages was demonstrated to precede that of promonocytes and monocytes in the yolk sac. In vitro differentiation of the fetal macrophages was consistent with the results of our previous in vivo observation indicating that fetal macrophages were differentiated from primitive macrophages, but not from the monocytic cell series. Differentiation of primitive macrophages into fetal macrophages, before the development of promonocytes and monocytes, was reproduced in the culture of cell suspensions from the fetal mouse yolk sacs, with a mouse bone marrow stromal cell clone (ST2) particularly with those at 8 days of gestation. In the soft agar or liquid culture of yolk sac cells with LP3-conditioned medium, monocyte-macrophage colonies were effectively induced, but not fetal macrophage colonies. The results provide evidence for the existence, in yolk sac hematopoiesis, of two distinct macrophage populations: a fetal macrophage population and a monocyte-derived macrophage population. The data indicate an obvious difference in development and differentiation between the two populations and the temporal precedence of fetal macrophages appearing before monocyte-macrophages.

Monokine secretion in aging and protein malnutrition.

Abstract: Aged and protein-malnourished hosts have diminished febrile responses and increased morbidity and mortality from infection that could be due to deficiencies in the production of certain monokines. In this study, the ability of peritoneal macrophages from aged and protein-malnourished rats to produce IL-1 and TNF was explored. Aged rats fed a standard diet produced less IL-1 and TNF, as measured by the thymocyte proliferation and L929 cytotoxicity assays, than young and middle-aged rats. Monokine production was not diminished by protein malnutrition in any age group. No synergistic decline in IL-1 or TNF production was seen with increasing age in malnourished rats. Diminished IL-1 and TNF production may partially explain the severity of infection seen in the elderly patient, but not the malnourished host. The role of other cytokines such as IL-6 and cytokine inhibitors in aging and malnutrition should be explored.

Dexamethasone modulation of in vivo effects of endotoxin, tumor necrosis factor, and interleukin-1 on liver cytochrome P-450, plasma fibrinogen, and serum iron

Abstract: Treatment of mice with endotoxin (lipopolysaccharide, LPS) and the two LPS-induced monokines, tumor necrosis factor (TNF) and interleukin-1 (IL-1), caused a depression of liver cytochrome P-450 and related drug-metabolizing enzymes, as well as other acute-phase changes including increase in plasma fibrinogen levels and hypoferremia. However, only IL-1, not TNF or LPS, depressed cytochrome P-450 in cultured hepatocytes, suggesting that the effect of TNF in vivo might be mediated by a second mediator. TNF- or LPS-stimulated monocytes released a factor capable of depressing cytochrome P-450 in cultured hepatocytes. This factor was inhibited by anti-IL-1 antiserum, and its synthesis, like that of IL-1, was inhibited by dexamethasone (DEX). Pretreatment of mice with DEX protected against the depression of liver cytochrome P-450 by LPS or TNF but not by IL-1, suggesting that IL-1 directly depresses cytochrome P-450 and that DEX acts by inhibiting IL-1 synthesis in vivo induced by LPS or TNF. However, DEX did not inhibit two other effects of LPS and TNF in vivo: increase of plasma fibrinogen levels and decrease of plasma iron, suggesting that these might not be mediated by IL-1. Therefore, the effect of DEX in vivo, although supporting the hypothesis that depression of liver cytochrome P-450 by LPS and TNF is mediated by IL-1, indicates the existence of IL-1-independent pathways in the acute-phase response.

Species variation in neutrophil biochemistry and function.

Abstract: ies of neutrophil physiology and function require close scrutiny of the methods to disclose which species is being examined. The relevance of studies of neutrophils from one species to disease in another species obviously depends upon the degree to which the basic physiologic processes are comparable. However, this comparability can be very difficult to determine. Most studies of neutrophil physiology and function use cells from only a single species, and differences in collection, isolation, and processing complicate any attempt at comparison of different studies. Whenever an animal model is used to study the pathogenesis of human infection or to justify clinical trials of antimicrobial therapy, tacit assumptions are made about the nature of the host response. Among these assumptions is that neutrophil response in the animal model is analogous to that in the human or that responses of other

Differential release of mediators from human basophils: differences in arachidonic acid metabolism following activation by unrelated stimuli.

Abstract: We have examined the release of histamine and LTC4 from purified human basophils challenged with several different stimuli, both physiological and nonphysiological. Basophils (n = 16) challenged with 0.1 .tg/mI anti-IgE released 38±4% of their available histamine and 39±12 ng LTCI/106 basophils within 15-30 mm. F-Met peptide (n = 8) caused the release of 54±8% histamine and 42±25 ng LTC�/106 basophils within a period of 2-5 mm. C5a caused the release of 22±3% histamine from selected donors but failed to initiate any LTC4 release unless combined with D20 or 5 mM extracellular calcium. The two nonphyslologlcal stimuli A23187 and TPA caused extensive histamine release, 67±8 and 82±11%, respectIvely, and while A23187 initiated a large and rapid release of leukotrlene, TPA failed to release any LTC4 even when combined with D20 or 2-5 mM extracellular calcium. Increased concentrations of extracellular calcium enhanced antiIgE and f-Met peptide induced release of LTC4 but inhibited the A23187 induced release of leukotriene. A single peak of immunoreactive leukotriene C4 that comigrated with the authentic standard was Identified using HPLC followed by radloimmunoassay. No LTD4 or LTE4 could be detected. Purified human basophils incubated with 0.2 �M [3H]AA Incorporated 290 pmol/10 cells, or 32±5% of the available label within 60 mm. The [3H]AA was taken principally into the phospholipids (73±5%), wIth 20±3% as neutral lipid, and only 5±2% remaining as the free acid. Three phospholipid subclasses, phosphatldylcholine, PC (24±2%), phosphatidylinositol, P1 (22±1%), and phosphatidylethanolamine,

Induction of interferon-gamma and tumor necrosis factor by Legionella pneumophila: augmentation of human neutrophil bactericidal activity.

Abstract: We have previously reported that Legionella pneumophila antigens can induce interferon-gamma (IFN-gamma) and tumor necrosis factor (TNF) in vitro and in vivo in mice. Furthermore, treatment of murine polymorphonuclear leukocyte (PMN) cultures with these cytokines resulted in augmented killing of the bacteria in vitro. The purpose of the present study was to determine if these findings could be extended to human responses. Here we report that Legionella antigens induced IFN-gamma and TNF in nonimmune human leukocytes cultures, and that these cytokines were able to stimulate the bactericidal activity of isolated PMN against L. pneumophila in vitro. Furthermore, optimal production of IFN-gamma was found in cultures which were enriched for large granular lymphocytes (LGL). The phenotype of IFN-producing cells was determined to be CD11+, CD16+, CD2+, and negative for CD4, CD8, CD14, and Leu 7. Additionally, Legionella-infected monocytes were found to produce TNF in a dose-dependent response to the number of infecting bacteria, and the addition of recombinant IFN-gamma to infected monocytes resulted in augmented production of TNF in a synergistic manner. Finally, treatment of PMN with recombinant IFN-gamma and recombinant TNF augmented their bactericidal activity against Legionella in a dose-dependent response. Thus, cytokines which can be induced by L. pneumophila antigens are able to stimulate PMN function in vitro, suggesting that resistance to infection results from a complex interaction of cytokines and cell responses.

Separation of potent and poorly functional human lung accessory cells based on autofluorescence.

Abstract: Human alveolar macrophages obtained by bronchoalveolar lavage are usually poor accessory cells in in vitro lymphoproliferation assays. However, we recently described a subpopulation of pulmonary mononuclear cells, obtained from minced and enzyme-digested lung, which were potent stimulators of allogeneic T-lymphocyte proliferation. These cells were enriched in loosely adherent mononuclear cell (LAM) fractions, but further study of these accessory cells was hampered by the heterogeneous nature of LAM. It was observed that in the majority of lung tissue sections, most alveolar macrophages were autofluorescent, whereas most interstitial HLA-DR positive cells were not. Therefore autofluorescence was utilized to fractionate LAM in an attempt to remove alveolar macrophages and selectively purify interstitial accessory cells. LAM were separated by flow cytometry using forward and side scatter to exclude lymphocytes, and red autofluorescence to obtain brightly autofluorescent (A pos) and relatively nonautofluorescent (A neg) mononuclear cells. Although both populations contained over 80% HLA-DR positive cells, A pos cells were poor accessory cells, whereas A neg cells were extremely potent stimulators of a mixed leukocyte reaction at all stimulator ratios tested. When A pos cells were added to A neg cells, T-cell proliferation was markedly suppressed in the majority of experiments. Morphologically, A pos cells appeared similar to classical alveolar macrophages with 95% of the cells being large and intensely nonspecific esterase positive. In contrast, the majority of A neg were smaller, B-cell antigen-negative, nonspecific esterase negative, and had a distinctive morphology on Wright-stained smears. We conclude that fractionation of LAM based on autofluorescence is a powerful tool to isolate and characterize lung mononuclear cells that act either as stimulators or as suppressors of immune responses in the lung.

Selenium and the immune response: 2. Enhancement of murine cytotoxic T-lymphocyte and natural killer cell cytotoxicity in vivo

Abstract: An inverse correlation between cancer incidence and dietary intake of the trace mineral element selenium has been well established in epidemiological and experimental studIes. The mechanisms for this chemoprotective effect are unresolved. Much attention has been focused on the antiprollferative effects of selenium on various normal and neoplastlc cell types. However, dietary selenium supplementation can also enhance the expression of various humoral and cellular immune responses. In examining the effects of dietary selenium on cell-mediated immunity in mice, we observed that selenium supplementation caused the enhanced expression of spontaneous natural killer (NK) cytotoxicity in spleen cells and of specific cytotoxic T-lymphocyte (CTL) cytotoxicity In peritoneal exudate cells (PEC). NK activity of spleen-cell suspensIons from seleniumsupplemented mice increased an average of 70% over that of the control group (basal diet). Cytotoxic activity of PEC from mice injected with tumors Intraperitoneally peaked earlier In selenium-supplemented animals, and the appearance of cells staining positively for Thy I .2 surface antigen in selenium-supplemented animals also preceded the values observed in control animals. We propose here that enhancement of in vivo cytotoxic mechanisms, is likely to act synergistically with tumor growth inhibition In the reduction of tumor Incidence associated with selenium Intake.

Characterization of the luminol-amplified light-generating reaction induced in human monocytes.

Abstract: Increased production of oxidative metabolites following interaction between mononuclear phagocytes and soluble stimuli can be measured as luminol-amplified chemiluminescence (CL). The effects of superoxide dismutase (SOD), catalase, and azide on the monocyte CL response were investigated. Azide, a myeloperoxidase (MPO) inhibitor, reduced the CL reaction by more than 80%, which indicates that the CL reaction is dependent on the granule enzyme MPO. Because SOD and catalase only partly inhibited the monocyte CL response, the authors propose that part of the monocyte CL response is of intracellular origin. This conclusion is further supported by the effects on the CL response obtained by adding extra peroxidase and the lack of correlation with techniques measuring only extracellular generated metabolites. However, it should be pointed out that the relation between extracellular and intracellular activity is stimulus dependent. Furthermore, even if quantitative differences exist between monocyte and granulocyte CL, the mechanism for the light-generating reaction seems to be the same in both cell types.

Both recombinant interleukin-1 (beta) and purified human monocyte interleukin-1 prime human neutrophils for increased oxidative activity and promote neutrophil spreading.

Abstract: Both purified human monocyte interleukin-1 and recombinant interleukin-1 (beta) primed neutrophils for increased superoxide production and chemiluminescence in response to f-met-leu-phe. In addition, purified human monocyte interleukin-1 and recombinant interleukin-1 (beta) altered neutrophil shape. Recombinant interleukin-1 (alpha) used at the same concentration of interleukin-1 (beta) did not prime neutrophils for increased superoxide production after stimulation with f-met-leu-phe. Interleukin-1 expressed by monocytes in response to endotoxin stimulation could act as a modulator of neutrophil function.

Mechanisms of tumor necrosis factor alpha-induced lymphopenia, neutropenia, and biphasic neutrophilia: a study of lymphocyte recirculation and hematologic interactions of TNF alpha with endogenous mediators of leukocyte trafficking.

Abstract: Tumor necrosis factor alpha (TNF) induces lymphopenia, neutropenia, and biphasic neutrophilia after intravenous injection of 3,000 U TNF in Lewis rats. The mechanism of TNF-induced lymphopenia was investigated by means of thoracic duct cannulation. Hourly measurements of lymphocyte recirculation via the thoracic duct failed to reveal any significant decrease in lymphocyte recirculation in TNF-treated vs. control rats, suggesting that a decrease in lymphocyte recirculation through the thoracic duct is not the mechanism for TNF-induced lymphopenia. The mechanism of TNF-induced neutropenia was investigated by administering TNF to rats in whom a neutrophilia had been induced with interleukin-1 (IL-1). In rats with neutrophilia, TNF resulted in a sharp decrease in the circulating neutrophil pool, demonstrating that TNF induces neutropenia by causing neutrophils to leave the circulating pool rather than decreasing neutrophil release from the marrow. The mechanism of neutropenia was furthermore shown to be due to the transient intravascular margination of neutrophils by administering epinephrine concomitantly with TNF. Epinephrine, which causes neutrophilia solely by demargination, abrogated the TNF-induced neutropenia and actually resulted in a neutrophilia that was greater than the neutrophilia occurring in epinephrine alone-treated rats, demonstrating both that TNF had already caused release of marrow neutrophils at the time of peripheral neutropenia, and that the paradoxical neutropenia was due to the transient intravascular margination of neutrophils. The known property of epinephrine to cause neutrophilia exclusively by demargination was proved by examination of the bone marrow of epinephrine-treated rats in whom no decrease in marrow neutrophils was observed (in contrast to TNF- and IL-1-treated rats in whom neutrophilia is accompanied by a depletion of marrow neutrophils). The mechanism of TNF-induced neutrophilia was investigated by modulating the magnitude of both the first and second peaks of neutrophilia by priming of rats with daily injections of IFN gamma for 2 days prior to administration of TNF. The first peak of neutrophilia in IFN gamma-primed TNF-treated rats was decreased in comparison to TNF alone-treated rats because of the well-known neutropenic and myelosuppressive effect of IFN gamma, which resulted in a decrease in the number of neutrophils that could be recruited to cause neutrophilia.(ABSTRACT TRUNCATED AT 400 WORDS)

Phagocytosis and killing of Cryptococcus neoformans by rat alveolar macrophages in the absence of serum.

Abstract: The in vitro interaction between yeast cells of Crypt ococcus neoformans and Lewis rat alveolar macrophages (AM4) was studied in the absence of serum. AM+ were harvested by lung lavage, and monolayers of adherent cells were established In wells of microtiter plates. Radlolabeled yeast cells were added to fresh AM4 monolayers, the plates were incubated at 37#{176}C under 5% C02, nonadherent yeasts were removed, and phagocytosis (I.e., attachment or ingestion) was determined by measuring adherent radioactivity. AM� were able to bind or ingest, and kill, encapsulated strains of C. neoformans in the absence of serum. Serum-free phagocytosis was suboptimal by comparison with phagocytosls in the presence of serum. The mechanism of serum-free phagocytosls involves a

Rat bone marrow and monocyte cultures: influence of culture time and lymphokines on the expression of macrophage differentiation antigens.

Abstract: A set of seven monoclonal antibodies (moabs) has been shown to discriminate in situ between distinct subpopulations of macrophages in the rat. It is still controversial if this heterogeneity is caused by the existence of different lineages or by differentiation of a common precursor. In both cases, the differentiation process might be regulated by microenvironmental factors. The present study examines the expression of the macrophage markers recognized by the seven ED-moabs in bone marrow and monocyte cultures. Furthermore, the impact of culture time and stimulating factors on the antigen expression in these cultures was tested. The expression of the ED3 antigen is highly inducible in bone marrow cultures. Factors that might be responsible for the increased ED3 expression are investigated. This strong ED3 expression by bone marrow-derived macrophages is nearly absent by monocyte-derived macrophages. This implies that the ability to express ED3 is blocked before the macrophage precursor cells enter the circulation to become monocytes. The ED2 expression cannot be induced under the tested circumstances bone marrow macrophages in vivo do not express these antigens. In culture, these macrophages stain positive for these markers already after the first day of culturing. The other three antigens are expressed on all macrophages under all tested circumstances.

Proliferation of macrophage subpopulations in the adult rat: comparison of various lymphoid organs.

Abstract: Adult male Lewis rats received a single intravenous injection of 5-bromo-2'-deoxyuridine (BRDU) to label all proliferating cells in the S-phase of the cell cycle. Various lymphoid organs were removed 1 and 24 hr after injection to assess local proliferation and migration of newly formed cells, respectively. In cell suspensions, surface staining was performed for macrophage subsets (ED1, ED2, ED3), and the DNA label BRDU was detected by a monoclonal antibody. Local proliferation of ED1+ macrophages occurred in all organs investigated with the exception of the blood. Bone marrow outweighed the other organs by far; in addition to the proliferating ED1+ promonocytes, the bone marrow also contained BRDU-labeled ED2+ macrophages. Newly formed ED1+ monocytes migrated into lymphoid organs such as the mesenteric lymph nodes and spleen where they comprised about 90% of newly formed macrophages. In the spleen, ED3+ macrophages seemed to be renewed by local proliferation, whereas in the mesenteric lymph nodes these cells were replaced by immigration. The heterogeneity of macrophages was further demonstrated by the different renewal of splenic macrophages. ED1+ and ED3+ cells were replaced in a matter of days, whereas it would probably take several months to renew ED2+ cells.

Beta-carotene stimulates human leukocytes to secrete a novel cytokine

Abstract: The effect of beta-carotene on cytokine production by human peripheral blood leukocytes was tested. Beta-carotene stimulated the secretion of a novel cytotoxic cytokine when peripheral blood cells were exposed to carotenoid concentrations between 10(-6) and 10(-10) M. Beta-carotene-treated supernatants caused the cytolysis of four out of the six human tumor cell lines tested. Low level toxicity was also observed when normal diploid fibroblast lines were exposed to beta-carotene-treated leukocyte supernatants. The cytotoxic activity elicited by beta-carotene was found to be distinct from characterized cytokines based on both antisera neutralization and target cell specificity studies. This study demonstrates that beta-carotene can induce human leukocytes to secrete one or more cytokines that can manifest cytotoxic activity against human tumor cells in vitro.

Mononuclear cells from human lung parenchyma support antigen-induced T lymphocyte proliferation.

Abstract: We have previously demonstrated that there is a subpopulation of loosely adherent pulmonary mononuclear cells that can be isolated from minced and enzyme-digested lung tissue with a potent capacity to stimulate allogeneic T lymphocyte proliferation. We now demonstrate that these cells are also capable of stimulating an autologous mixed leukocyte reaction (AMLR) and presenting antigen to autologous T lymphocytes. These loosely adherent mononuclear cells (LAM) were more effective than either alveolar macrophages or monocytes as antigen-presenting cells. Depletion of phagocytic or Fc receptor-positive cells from the LAM population enhanced the stimulation of an reaction AMLR while preserving antigen-induced T lymphocyte proliferation. These results indicate that there are nonphagocytic, Fc receptor-negative accessory cells in human lung parenchyma capable of activating resting T cells in an AMLR and supporting antigen-specific T lymphocyte proliferation. The identity of these cells is uncertain, but the data strongly suggest that the cell is not a classical monocyte-derived macrophage. These antigen-presenting cells may be critical in the initiation of immune responses within the lung.

Activation of human T lymphocytes by crosslinking of anti-CD3 monoclonal antibodies.

Abstract: The proliferation of human T lymphocytes induced by anti-CD3 monoclonal antibodies (mAb) is used as a model for antigen-induced activation via the T cell receptor-CD3 complex. Since both systems are accessory cell (AC)-dependent, an understanding of the role of AC in anti-CD3-induced proliferation may provide an understanding of physiological activation via the T cell receptor. Previous work has implicated receptor crosslinking as an important AC function. To determine its necessity in anti-CD3-induced lymphocyte proliferation, we prepared highly purified T lymphocytes and found that these cells did not respond to the anti-CD3 mAb UCHT1, either alone or with interleukin 1 (IL1), interleukin 2 (IL2), or tetradecanoyl phorbol acetate (TPA). However, the response, as measured by appearance of IL2 receptors and proliferation, was restored by crosslinking with immobilized goat anti-mouse antibodies (GAM) and did not require the addition of IL1, IL2, or TPA. Thus, crosslinking of CD3 receptors was a sufficient signal for proliferation of these cells. Cyclosporine A (CsA) inhibited the activation induced by immobilized UCHT1. Since macrophages are the principle targets of CsA-mediated suppression of mitogen-induced proliferation, but macrophages do not participate in the response to immobilized anti-CD3, this may indicate that CsA was inhibiting crosslinking or a signal generated by it.