Modulation of anti-Candida activity of human alveolar macrophages by interferon-gamma or interleukin-1-alpha

The fungicidal and bactericidal activities of human alveolar macrophages (AM) and peripheral blood monocytes (PBM) from 18 healthy volunteers were evaluated. The results showed that AM were able to phagocytize and kill Candida albicans, Pseudomonas aeruginosa, and Staphylococcus aureus. However, killing of the bacteria was already complete in 2 h, whereas killing of Candida required 4 to 6 h despite an early phagocytosis of yeast cells. The fungicidal activity of freshly collected AM and PBM was also tested after effector cell exposure to interferon-gamma (IFN-gamma), interleukin-1-alpha (IL-1 alpha), endotoxin lipopolysaccharide (LPS), or interleukin 2 (IL-2). It was found that treatment with IFN-gamma, IL-1 alpha, or LPS significantly augmented macrophage and PBM candidacidal activity, whereas the addition of IL-2 was ineffective. We also evaluated killing of C. albicans by AM cultured in vitro for different times. While phagocytosis was apparently unaffected, the candidacidal activity progressively decreased over the in vitro culture period, an effect that was largely reversed by cell exposure to IFN-gamma, IL-1 alpha, or LPS. In an experimental model in which mice infected with an agerminative C. albicans strain (PCA-2) resisted lethal microbial challenge, freshly harvested AM showed increased cytotoxic activity to Aspergillus fumigatus in vitro as well as enhanced IL-1 production. In conclusion, present data confirm the crucial role of AM in the surveillance of bacterial and fungal infections and indicate that treatment of these cells with IFN-gamma or IL-1 alpha is able to enhance their antimicrobial capability.     

Effect of histamine on chemotaxis and phagocytosis of human alveolar macrophages and blood monocytes

We studied in vitro the effect of histamine on the chemotactic and phagocytic abilities of human blood monocytes and alveolar macrophages. The chemotactic response to activated autologous serum, leukotriene B4 or N-formyl-methionine-L-phenylalanine was similar for macrophages and monocytes. Incubation of monocytes with histamine in picomolar concentrations caused a significant chemotactic inhibition (about 25%). This effect was antagonized by cimetidine but not by promethazine. Histamine did not have an effect on alveolar macrophages chemotaxis or phagocytosis. Thus, histamine, in minute concentrations, exerts, in vitro, a partial inhibitory effect on monocyte chemotaxis through activation of H2-type receptors.     

Antigenic specificity of human alveolar macrophages and blood monocytes studied by an immunofluorescence technique

Lung alveolar macrophages (AM) and blood monocytes (BM) are part of the mononuclear phagocyte system (MPS) and have been shown to be composed of several functionally and biochemically distinct subsets. AM and BM were each fractionated into four subfractions by centrifugation over Percoll, 95 per cent of the monocytes having a higher buoyant density than the high-density AM fraction. Antigen distribution within the cell subfractions was studied by an immunofluorescence technique using a panel of ten monoclonal antibodies (MoAbs) reacting with separate antigenic determinants previously found on mononuclear phagocytes. Several antigens were present on both types of cells (Ki-M1, Ki-M6, Leu-M3, Leu-M5, 1D5, Dako-Ma, and HLA-DR), while other antigens were expressed exclusively on BM (MAS 072 and MAS 081) or on AM (Ki-M8). Ki-M1, Leu-M3, Leu-M5, and HLA-DR were expressed to a greater degree on the surface of AM (95-96 per cent of cells) than on BM (56-68 per cent), while an inverse relationship was noted for 1D5 antigen (present on 41 per cent of BM and 11 per cent of AM). Ki-M6 and Dako-Ma MoAbs recognized an intracellularly restricted antigen present in both AM and BM. Ki-M1, Ki-M8, MAS 072, MAS 081, Leu-M3, Leu-M5, 1D5, and HLA-DR were expressed both in the cytoplasm and at the cell surface. Some antigens (1D5, Leu-M3, MAS 072, MAS 081, and Dako-Ma) showed an uneven distribution among subsets of both AM and BM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracellular survival ofHaemophilus somnusin bovine blood monocytes and alveolar macrophages

The mechanisms used byHaemophilus somnusto survive and multiply within bovine mononuclear phagocytes are not fully understood. In order to study the interaction between bovine mononuclear phagocytes andH. somnus, a colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was developed to assess the survival ofH. somnuswithin cultured bovine blood monocytes (BBM). Using this system, it was found thatH. somnuswas able to survive within BMMin vitro, and the kinetics of its survival were similar to that seen in BBM isolated from experimentally infected cattle. Using ultrastructural studies, it was possible to demonstrate the survival ofH. somnusin freshly isolated bovine mononuclear phagocytes in membrane-bound vacuoles. To determine if activation of macrophage function would result in elimination of intracellularH. somnus, BBM were treated withE. colilipopolysaccharide (LPS) or recombinant bovine (rBo) cytokines, interferon-γ(IFN-γ), granulocyte macrophage colony stimulating factor (GM-CSF), tumour necrosis factor-α (TNF-α) or interleukin-1β (IL-1β). Treatment of BBM with rBoIFN-γ, rBoGM-CSF orE. coliLPS resulted in decreased intracellular survival ofH. somnusat 18 and 48 h, whereas BBM treated with rBoTNF-α or rBoIL-1β had reduced intracellular survival ofH. somnusonly at 18 h. However, none of these treatments resulted in complete elimination of the intracellular bacteria. The ability ofH. somnusto survive and multiply in both freshly isolated and cytokine-treated cultured BBM demonstrated the capability ofH. somnusto escape from macrophage killing mechanisms. This capability may play a role in the dissemination ofH. somnusinfection in the body.     

Bleomycin-stimulated hamster alveolar macrophages release interleukin-1.

The capacity of alveolar macrophages (AM) of bleomycin-instilled hamsters to proliferate mouse thymocytes (interleukin-1 activity) and hamster fibroblasts (fibroblast proliferation (FP) activity was studied. Using bleomycin-instilled hamsters, the FP activity of AM culture supernatants was increased significantly on days 1, 5, and 10 after instillation of bleomycin. The interleukin-1 (IL-1) activity, however, was increased significantly on day 1 only as compared with saline-treated hamsters. Next, normal AM were stimulated in vitro by bleomycin. After being fractionated by chromatography, their culture supernatant showed IL-1 activity, which also indicated FP activity. These results suggest that bleomycin directly stimulates AM to release IL-1 in the fibrogenic responses.     

Alpha 1-acid glycoprotein potentiates lipopolysaccharide-induced secretion of interleukin-1 beta, interleukin-6 and tumor necrosis factor-alpha by human monocytes and alveolar and peritoneal macrophages.

Although the physiological role of alpha 1-acid glycoprotein (AGP), an acute-phase protein, is poorly understood, several lines of evidence support a modulatory action on the immune response. In this study, we investigated the effect of AGP on the production of interleukin (IL)-1 beta, IL-6 and tumor necrosis factor (TNF)-alpha by human monocytes, macrophages and the monocytic THP-1 cell line. AGP significantly enhanced (2- to 7-fold) the production of these cytokines in monocytes induced by suboptimal concentrations of lipopolysaccharide [E. coli lipopolysaccharide (LPS): 100 ng/ml] in serum-free conditions, whereas it had little or no effect in the absence of LPS. The potentiating effect of AGP was inhibited by specific antibodies. It was concentration dependent and the greatest enhancement was observed with 250-500 micrograms/ml. Moreover, AGP only potentiated the effect of suboptimal concentrations of LPS. AGP did not alter the time course of LPS-induced IL-1 beta, IL-6 or TNF-alpha secretion. AGP acts as a co-inducer and could also potentiate cytokine secretion triggered by Neisseria meningitidis LPS and muramyl dipeptide. The glycan moiety of AGP did not seem to be involved in its potentiating effect, since both its major glycoforms and asialo-AGP potentiated the effect of LPS to the same extent as native AGP. Possible differences in the effect of AGP according to cell maturation were investigated using isolated human macrophages: AGP potentiated LPS-induced cytokine production by both peritoneal and alveolar macrophages. These data suggest that AGP can modulate monocyte/macrophage functions, thereby contributing to the amplification and regulation of immune and inflammatory responses.     

DR framework antigens and 63D3 antigens on human peripheral blood monocytes and alveolar macrophages

The expression of surface antigens on human peripheral blood monocytes and alveolar macrophages was compared using the monoclonal antibodies 63D3, which react with monocytes, and OKIa, which reacts with DR framework antigens. Fluorescence-activated cell-sorter analysis revealed that 71.5 +/- 4.6% of peripheral blood monocytes reacted with 63D3 and showed a uniform distribution of binding. Alveolar macrophages also reacted with 63D3 and displayed uniform binding. Furthermore, the relative fluorescence intensity was very similar to that of monocytes. These data suggest that 63D3 antigen expression is similar on peripheral blood monocytes and alveolar macrophages and may be a stable marker in the differentiation of monocytes to macrophages. Analysis showed that 45.8 +/- 4.9% of peripheral blood monocytes reacted with OKIa and showed a nonuniform distribution of binding, while 74 +/- 8.4% of alveolar macrophages reacted with OKIa and exhibited uniform binding. The relative fluorescence intensity of alveolar macrophages which were reacted with OKIa was significantly greater than that of blood monocytes (P less than 0.001). Size analysis suggested that alveolar macrophages express approximately five times more DR antigens per unit surface area than do peripheral blood monocytes. The expression of DR antigens on the alveolar macrophage surface suggests an important role for macrophages in antigen presentation in the lung.     

Pulmonary interstitial macrophages: isolation and flow cytometric comparisons with alveolar macrophages and blood monocytes

Pulmonary interstitial macrophages (IM) were isolated from rat lungs by an Fc gamma receptor-based affinity technique coupled with multiparameter flow cytometry. Single cell suspensions obtained by collagenase digestion of extensively perfused and lavaged lungs were applied to carpets of opsonized sheep red blood cells (SRBC-IgG) bound to plastic tissue culture flasks. At 0-4 degrees C, optimal binding of lung cells occurred within 60 min at plating densities of 1-2 X 10(6) lung cells/cm2 when the SRBC substrate was opsonized with 10 micrograms/ml anti-SRBC IgG. Nonadherent cells were removed by gently rinsing the plates and adherent cells were recovered by lysing the SRBC-IgG substrata. By light microscopy, the mixture of adherent cells was comprised of mononuclear cells (approximately 54%), many of which appeared to be macrophages, lymphocytes (approximately 20%), polymorphonuclear leukocytes (approximately 15%), plasma cells (approximately 8%), eosinophils (approximately 2%), and mast cells (approximately 0.5%). The cells which adhered to the SRBC-IgG monolayers were further resolved into subpopulations by multiparameter flow cytometry and sorted according to their electro-optical characteristics. One subpopulation appeared morphologically to be macrophages, and greater than 90% of these cells readily phagocytized SRBC-IgG in vitro. Peroxidase staining of this population was minimal, indicating that these cells were not blood monocytes (BM). Using a method by which alveolar macrophages (AM) were prelabeled with SRBC-IgG in situ, we demonstrated that alveolar macrophages constituted only approximately 5% of the total adherent cell population. We concluded from these observations that the macrophage population harvested in this manner were neither BM nor AM, but, rather, were harvested from the lung's interstitial compartment. Flow cytometric analyses indicated that the IM exhibited electro-optical characteristics intermediate between those of BM and AM, which is consistent with the concept of the lung's interstitium as a maturation compartment for the BM prior to migration into the alveolar compartment. However, the IM more closely resembled the BM than the AM, indicating that if the IM is in fact a precursor to the AM population, substantial maturation or differentiation must occur subsequent to its migration into the alveolar compartment. This isolation technique will be useful for harvesting highly purified IM for in vitro investigations.     

111Indium-labeled human alveolar macrophages and monocytes: Function and ultrastructure

Human alveolar macrophages and peripheral blood mononuclear cells were labeled with indium-111 (¹¹¹In)-oxine in ethanol. The efficiency of labeling averaged 84% for macrophages and 74% for mononuclear cell preparations. Phagocytosis by macrophages, the release of colony stimulating factor activity (CSF) by macrophages, and chemotaxis by monocytes in response to human C5-derived chemotactic factor (C5_fr) and formyl-methionyl-leucyl-phenylalanine (f-MLP), were indistinguishable between labeled and unlebeled cells. Microscopically, lebeled cells looked similar to control cells. Radioautography demonstrated ¹¹¹In throughout the labeled cells, but there was a disproportionately high quantity over nuclei. These findings add to the growing literature indicating that ¹¹¹In labels cells efficiently and that cells can retain function after being labeled with ¹¹¹In.     

Differential prostaglandin production by unfractionated and density-fractionated human monocytes and alveolar macrophages

Mononuclear phagocyte elaboration of E series prostaglandins (PGE) may be important in the regulation of inflammatory and fibrotic reactions. Mononuclear phagocytes are morphologically and functionally heterogeneous cells. To further understand the processes controlling inflammation and fibrosis, in particular that in the human lung, we characterized the ability of unfractionated and density-fractionated human alveolar macrophages and blood monocytes to elaborate PGE. Alveolar macrophages and blood monocytes constitutively elaborated small amounts of PGE, and their elaboration of PGE was increased with lipopolysaccharide (LPS) stimulation. Monocytes elaborated more PGE than autologous alveolar macrophages. In addition, denser monocytes (specific gravity greater than 1.055) and denser alveolar macrophages (specific gravity greater than 1.044) elaborated more PGE than less dense monocytes and alveolar macrophages, respectively. When monocytes were incubated in vitro, their constitutive PGE elaboration decreased with time. However, in vitro incubation did not cause monocytes to lose their capacity to elaborate PGE in response to LPS. Thus, mononuclear phagocyte populations differ in their ability to elaborate PGE. These differences can be only partially attributed to differences in cell maturation.     

Interleukin 1 secretion by human monocytes and macrophages

Interleukin 1 (IL-1) is generally regarded as a major regulator of T lymphocyte proliferation. Macrophages from animals and cloned tumor cell lines have been shown to produce this monokine in response to a variety of stimuli. The ability of human monocytes and macrophages to generate IL-1 is much less well characterized. We previously demonstrated that human monocytes cultured for 1-6 days transformed to macrophages but retained their capacity to support concanavalin A-driven T cell proliferation. However, cultured macrophage capacity to support antigen-driven T cell proliferation began to decline after 3 days of culture and was markedly deficient by 6 days of culture. To determine if this loss of accessory cell function was due to the inability to secrete IL-1, we measured the monokine produced by normal fresh human monocytes and macrophages cultured in vitro from monocytes. IL-1 was assayed by the mouse thymocyte proliferation method. Fresh monocytes secreted IL-1 readily in response to lipopolysaccaride and latex particles. Macrophages cultured from fresh monocytes, however, lost this ability after greater than or equal to 2 days in culture. Mixing experiments failed to demonstrate an inhibitor present in the macrophage supernatants that would suppress thymocyte proliferation. Stimulated T cells incubated with monocytes and 3-day cultured macrophages failed to prolong or promote IL-1 secretion.     

Quantitative analysis of maedi-visna virus DNA load in peripheral blood monocytes and alveolar macrophages

Viral load may be an important indicator of disease progression in sheep infected with maedi-visna virus (MVV). To assess this variable accurately in MVV-infected sheep, a quantitative competitive-polymerase chain reaction (QC-PCR) was developed. A conserved region of the MVV pol gene was selected. The RT-PCR MVV pol product was cloned and mutagenised in vitro by PCR to produce a competitor template reduced in length from 217 to 192 bp, but which retained the original flanking MVV pol PCR primers. The competitor template was quantified accurately and in an optimised QC-PCR protocol serial dilutions of this template were co-amplified with known amounts of sample DNA. MVV DNA levels in peripheral blood monocytes and alveolar macrophages from MVV-infected sheep (n=12) were assessed by QC-PCR. Viral DNA load in alveolar macrophages was significantly higher than that in peripheral blood monocytes when the animals were compared overall. A comparison was also made between alveolar macrophages from the lungs of seropositive animals with or without histopathological evidence of pulmonary lesions. The load of MVV DNA in alveolar macrophages was low in sheep without histopathological evidence of lesions in the lung. In contrast, in alveolar macrophages from sheep with histopathological lesions in the lung, there was a significantly higher level of MVV DNA. The correlation of MVV load with pulmonary lesions suggests that infected alveolar macrophages play a key role in the pathogenesis of this lymphoid interstitial pneumonia.     

Expression of the VEP13 antigen (CD16) on native human alveolar macrophages and cultured blood monocytes

Human alveolar macrophages (AM phi) from thirteen patients, who were suffering from various lung diseases were harvested by bronchoalveolar lavage. Peripheral blood monocytes from eight healthy donors were isolated by Ficoll-Hypaque gradient centrifugation and adherence to plastic surface. To detect the VEP13 antigen (CD16) on these cells, a rosette assay employing ox erythrocytes coated by the CrCl3 method with purified VEP13 monoclonal antibody (Eo-VEP13) was used. A mean of 31.3% of freshly isolated AM phi and 3.9% of blood monocytes formed Eo-VEP13 rosettes. Monocytes cultured for 3 or 6 days in the presence of a supernatant from mouse L929 cells, which had been shown previously to improve long-term viability of human monocytes in culture, showed 12.5% and 25.3% Eo-VEP13 rosettes, respectively. No significant increase in VEP13 antigen expression was noted by culturing monocytes without L929 cell supernatant. The factor in L929 supernatant that induces VEP13 antigen expression has not been identified. Tunicamycin at 10 micrograms/ml inhibited significantly VEP13 antigen expression on monocytes. In contrast, IgG rosette formation was not reduced by tunicamycin. Our data show that subpopulations of native human AM phi and peripheral blood monocytes cultured in presence of a supernatant of L929 fibroblasts containing mainly murine CSF may express the CD16 antigen, which is normally found on large granular lymphocytes (LGL). Suppression by tunicamycin indicates that Fc receptor glycosylation takes place during a later differentiation step of mononuclear phagocytes.     

Patients with chronic pancreatitis have an impaired oxidative burst ability of blood monocytes

The destruction by phagocytic cells of ingested microorganisms is best ensured by an intact respiratory burst with production of superoxide anion and other metabolites. The aim of the present study was to investigate the status of the reticulo-endothelial system, as assessed by superoxide anion generation of blood monocytes, in 18 patients with chronic pancreatitis (CP) as compared to 15 with chronic renal failure (CRF), 14 with diabetes mellitus (DM) and to 20 healthy volunteers. Macrophage suspensions (1 × 10⁶) were prepared from blood samples withrawn after an overnight fast were tested for conventional phagocytosis function (carbon partical ingestion: percent and intensity) and for superoxide anion generation (chemiluminescent activation: cpm of light integrated intensity). Conventional phagocytosis function tests did not show any differemce among the groups examined. However, the peak value of generated susperoxide anion (× 10⁴ cpm) was significantly lower in CP patients as compared to either healthy controls (14.9 ± 4.3 vs. 28.3 ± 4.7, P < 0.001) and CRF (24.8 ± 3.9, P < 0.01) and DN patients (22.8 ±_4.9, P < 0.05). Further, the time elapsed to generate such a peak was significantly longer in CP patients (374 ± 106 s) than in controls (208 ± 35 s, P < 0.01). This study suggests that patients with CP present a defective monocytes burst ability which is unrelated to flare-up of the disease and to the underlying malnutrition. Further, this phenomenon appears to occure to a far greater extent as compared to other long-standing chronic illnesses.     

Complement biosynthesis in human breast-milk macrophages and blood monocytes.

The availability of techniques for establishment of primary, long term monolayers of breast milk macrophages and blood monocytes permitted a direct comparison of biosynthetic functions of human tissue macrophage and its progenitor. In addition to previously described morphological differences, four specific characteristics of the breast milk macrophage were identified: (i) a reduced rate of total protein secretion relative to the monocyte; (ii) abrogation of the 3 day lag in complement secretion regularly observed in blood monocyte cultures; (iii) an increase in the secretion of complement proteins C2 and factor B; and (iv) an increase in the ratio of C2 : factor B secretion. These differences did not result from cell-cell interactions between monocytes and macrophages, stable factors elucidated by monocyte or macrophage monolayers, or heat-stable factors in breast milk. In addition, both monocytes and macrophages synthesized and secreted C3 in an apparently native but haemolytically inactive form. The differences observed suggest that macrophages may modulate local availability of complement proteins in tissues or in the early phase of an inflammatory response.     

Rat resident testicular macrophages have an alternatively activated phenotype and constitutively produce interleukin-10 in vitro

The ability of the rodent testis to tolerate graft alloantigens and spermatogenic cell autoantigens is well known. The mechanisms underlying this "immune privilege" are poorly understood, but the numerous resident TMs have been implicated. Although it has been assumed that TMs display a phenotype consistent with immune privilege, this has not been formally established. Consequently, TMs were isolated from adult rats and cultured under basal conditions and following stimulation with LPS and IFN-γ (classical activation) or IL-4 (alternative activation). BMMs matured in vitro were used as control. Expression of the classical (proinflammatory) activation markers TNF-α, IL-1β, iNOS, IL-6, RANTES, IL-12p40, and SOCS3 and alternative (immunoregulatory) activation markers IL-10, TGF-β1, CXCL2, and SOCS1 was measured by QPCR or ELISA. In culture, TMs were characterized by poor expression of classical activation genes and TGF-β1 but constitutively high IL-10 production and reduced costimulatory activity in a polyclonal T cell activation assay. This pattern of gene expression was associated with TMs expressing the scavenger receptor CD163, which is characteristic of tissue resident macrophages and alternative activation. By contrast, CD163-negative TMs displayed reduced inflammatory gene expression but did not constitutively produce IL-10. These data indicate that under the influence of the testicular environment, macrophages adopt an alternatively activated phenotype, involving reduced capacity for proinflammatory gene expression, constitutive IL-10 production, and impaired ability to support T cell activation, consistent with a role in maintaining testicular immune privilege.     

Normal and sarcoid alveolar macrophages differ in their ability to present antigen and to cluster with autologous lymphocytes.

Human bronchoalveolar macrophages from normal individuals function poorly as accessory cells for the presentation of common recall antigens. In sarcoidosis, alveolar macrophages (AM) are reported to be effective accessory cells for the presentation of such antigens. In this study normal and sarcoid AM were compared with blood monocytes for their ability to act as accessory cells in presenting tuberculin purified protein derivative (PPD) and streptokinase-streptodornase (SKSD) to autologous T lymphocytes, or to form spontaneous, antigen- or mitogen-induced clusters with the T cells. When compared to autologous monocytes, normal AM failed to present the two recall antigens effectively. Likewise normal AM formed very few clusters with T lymphocytes when compared to monocytes, even in the presence of antigens or the mitogen phytohaemagglutinin (PHA). In contrast, sarcoid AM presented both antigens as effectively, and were equally effective as monocytes in forming clusters with T lymphocytes, spontaneously and in further response to antigen or mitogen. The results suggest that in sarcoidosis enhanced accessory cell function and enhanced cluster formation may be related features of bronchoalveolar macrophage populations.     

Suppressive effect of anti-rheumatic drugs on interleukin-1 beta release from human peripheral blood monocytes

We developed an ELISA system for human IL-1 alpha and -beta release from silica-stimulated monocytes from healthy volunteers and tested the effect of several anti-rheumatic drugs including nonsteroidal anti-inflammatory drug (Ibuprofen). Anti-rheumatic drugs including Auranofin and Sulphasalazine suppressed IL-1 beta release significantly at therapeutic concentrations, whereas Bucillamine, Lobenzarit, D-Penicillamine and Ibuprofen did not. These results suggest a possible immunotherapeutic effectiveness of some anti-rheumatic drugs on rheumatoid arthritis through their inhibition of IL-1 beta release.