Influence of Secretome of Different Functional Phenotypes of Macrophages on Proliferation, Differentiation, and Collagen-Producing Activity of Dermal Fibroblasts In Vitro.

We studied the effect of conditioned media of GM-CSF-differentiated human macrophages polarized in M1(LPS), M2a(IL-4), M2c(dexamethasone), and M2(low serum) phenotypes on proliferation, differentiation, and collagen-producing activity of dermal fibroblasts. It was found that M1(LPS) and M2a(IL-4) were characterized by moderate influence on functional activity of fibroblasts. At the same time, soluble factors of M2c(dexamethasone) significantly enhanced the proliferative response of fibroblasts, but not their differentiation and type I collagen production. On the contrary, M2(low serum) generated under conditions of growth factors deficiency had a pronounced stimulating effect on the differentiation of fibroblasts and production of type I collagen by these cells, but moderately stimulated the fibroblast proliferation. Thus, the secretory activity of various functional phenotypes of macrophages is an important mechanism of fibrogenesis regulation.

Effect of M2 Macrophage-Derived Soluble Factors on Proliferation and Apoptosis of SH-SY5Y Cells.

Macrophages play the key role in the regulation of neuroregeneration. For evaluation of the neuroregenerative potential of M2 macrophages, we studied the effect of macrophages polarized with IL-4 (M2a (IL-4)) and by efferocytosis under conditions of serum deprivation (LS, Low Serum; M2(LS)) on proliferative activity and apoptosis of SH-SY5Y cells under conditions of deficiency of growth/serum factors. Conditioned media of both M2(LS) and M2a(IL-4) stimulated proliferation of SH-SY5Y cells. Moreover, soluble factors of M2(LS) and M2a(IL-4) reduced the degree of early apoptosis of SH-SY5Y cells and the protective effect of M2(LS) was observed at earlier terms of culturing. Our findings suggest that M2 macrophages have high neuroregenerative potential that is mediated through soluble factors and manifests itself both in stimulation of proliferation and inhibition of apoptosis of SH-SY5Y cells.

Efferocytosis Modulates Arginase-1 and Tyrosine Kinase Mer Expression in GM-CSF-Differentiated Human Macrophages.

We studied the expression of arginase-1 (Arg1) and tyrosine kinase Mer (MerTK) in GMCSF-differentiated human macrophage populations М0, М1(IFNγ), М2а(IL-4), and М2(low serum) generated under conditions of growth/serum factor deficiency. The maximum relative content of Arg1+ and MerTK+ cells was found in М2 macrophage populations: М2а(IL-4) and М2(low serum). As the uptake of apoptotic cells is the key mechanism of M2 polarization during M2(low serum) generation, we performed a special series of experiments and showed that incubation with allogeneic apoptotic neutrophils significantly increased the percentages of CD206+ macrophages co-expressing Arg1 and MerTK.

Effect of Apoptotic Neutrophils on the Production of Erythropoietin, MMP-9, and TIMP-1 in Cultures of Human Macrophages.

We studied the effect of apoptotic neutrophils on the production of erythropoietin, MMP-9, and TIMP-1 by GM-CSF-induced human macrophages. GM-CSF-induced macrophages spontaneously produce erythropoietin and secrete MMP-9 and TIMP-1. Polarization of these macrophages towards the M2-like phenotype after exposure to apoptotic neutrophils considerably increased the production of erythropoietin; the MMP-9/TIMP-1 ratio tended to increase under these conditions due to a decrease in TIMP-1.

Allostimulatory activity as a criterion of the functional phenotype of human macrophages.

The functional phenotype of macrophages (Mφ) is determined by both differentiation factors and polarization stimuli. In mouse Mφ could be easily divided into the distinct Mφ subtypes. However, the identification of human M1 and M2 cells is much more difficult due to the lack of M1- or M2-specific markers. We assumed that the Mφ capacity to induce T cell proliferation in mixed leukocyte culture, or allostimulatory activity, may be a marker of Mφ functional phenotype. We compared the allostimulatory activity of Mφ differentiated with GM-CSF or M-CSF and polarized into M1, M2a, M2c subtypes using appropriate stimuli. GM-CSF-differentiated M1 Mφ showed pronounced allostimulatory activity whereas the polarization into M2a and M2c of GM-CSF-differentiated Mφ was associated with decreased allostimulatory activity. M-CSF-differentiated M1 Mφ demonstrated the moderate increasing of allostimulatory activity but its level has never reached that of GM-CSF-activated M1. The level of allostimulatory activity of M2a and M2c M-CSF-induced Mφ was comparable to that of GM-CSF-induced M2a and M2c Mφ. Thus, low allostimulatory activity is a common property of human M2a and M2c macrophages regardless of the differentiating factor and a polarizing stimulus and can be used to distinguish between M1 and M2 phenotypes.

Effects of Anti-CD206 Antibodies on Macrophage Functions in Male CBF1 Mice with Lipidemia.

The effects of anti-CD208 antibodies (mannose receptor) on functional characteristics of peritoneal macrophages were studied in intact mice and mice with lipidemia induced by poloxamer-407. Lipidemia was associated with suppression of phagocytosis and increase in spontaneous proliferative potential and NO production by macrophages. Anti-CD206 antibodies suppressed NO production by macrophages in mice with lipidemia.

The Phenotypic and Functional Features of Human M2 Macrophages Generated Under Low Serum Conditions.

The phenotypic and functional features of human M2 macrophages, in particular, their immunosuppressive activity, can considerably vary depending on M2 polarizing stimulus. This study was aimed at the investigation of cytokine production and pro-apoptogenic/inhibitory molecule expression in macrophages generated with GM-CSF using either standard conditions (M1) or deficiency of serum/growth factors (M2-LS cells). In contrast to M1, M2-LS cells were characterized by an enhanced content of CD206(+), B7-H1(+), FasL(+) and TRAIL(+) cells along with a decreased production of IFN-γ, IL-5, IL-6, IL-13, TNF-α, IL-17 and MCP-1. In addition, M2-LS exhibited a lower T cell stimulatory activity in MLC that was associated with the higher numbers of apoptotic and the lower numbers of proliferating T cells. B7-H1 plays a key role in M2-LS-mediated cytotoxic effects as the neutralization of B7-H1 reduces the apoptosis-inducing activity of M2-LS, while the blocking of CD206 and TRAIL reduces the cytostatic activity of M2 macrophages.

Contribution of dehydroepiandrosterone sulfate and progesterone to in vitro regulation of tolerogenic activity of IFN-α-induced dendritic cells.

Dehydroepiandrosterone sulfate and progesterone exhibited an immunomodulatory effect on the tolerogenic characteristics of IFN-α-induced dendritic cells. The hormone effects depended on the initial level of allostimulatory activity of dendritic cells in mixed lymphocyte culture. However, dehydroepiandrosterone sulfate significantly more often stimulated allostimulatory activity by attenuating the tolerogenic properties of dendritic cells, while progesterone potentiated their tolerogenic potential. The capacity of the hormones (dehydroepiandrosterone sulfate and progesterone) to attenuate tolerogenic activity of dendritic cells was associated with reduction of FasL expression on these cells, while the increase in tolerogenic activity was associated with the increase in the percentage of CD123(+) dendritic cells, and under conditions of modification with dehydroepiandrosterone sulfate it was associated with increased B7-H1 expression. Possible contribution of indolamine-2,3-dioxygenase and prostaglandin E2 to stimulation of tolerogenic characteristics of dendritic cells modified with dehydroepiandrosterone sulfate and progesterone, respectively, was demonstrated.

[Characteristics of A-interferon-generated dendritic cells in patients with pulmonary tuberculosis].

The phenotype of the dendritic cells (DC) generated from the adhesion fraction of mononuclear cells in the presence of GM-CSF and alpha-interferon was studied in patients with pulmonary tuberculosis. Despite the absence of significant differences in the count of mature CD83+DCs in the groups of patients (n = 38) and healthy donors (n = 30), elevated CD14(+)-monocyte levels and few activated CD25(+)-DCs were indicative of the impaired process of DC maturation/generation in patients with pulmonary tuberculosis, particularly in a subgroup of patients with a low T-cell proliferative response against PPD (PPD-anergy, n = 10). The patients with tuberculosis showed the lower relative levels of CD11c(-)-CD123(+)-DC and the normal levels of myeloid CD11c(+)D123(-)DCs. However, in patients with PPD-anergy, the content of myeloid CD11c(+)CD123(-)-DCs was significantly higher than that in PPD-reactive patients. Moreover, the patients with PPD-anergy were characterized by the elevated peripheral blood levels of CD14+CD16(+)-monocytes, which was associated with the high suppressive activity of monocytes (r(s) = 0.53; p < 0.05). The impaired process of DC generation/maturation in patients with pulmonary tuberculosis is believed to be associated with the changes in the phenotypic and functional properties of monocytes and to be a cause of an inadequate antigen-specific response in tuberculous infection.

[Interleukin-2 in the correction of T-cell anergy in patients with pulmonary tuberculosis].

The clinical and immunomodulating effects of lymphotropic administration of interleukin-2 (IL-2) were studied in the combine treatment of patients with pulmonary tuberculosis. The patients with tuberculosis were shown to have the low levels of monocytes with the intracellular expression of tumor necrosis factor-alpha (TNF-alpha) and the high count of CD14+ CD16+ monocytes with the intracellular expression of IL-10. The changes in the monocytic link were most pronounced in patients with PPD-induced anergy appeared as the low proliferation and production of alpha-interferon (alpha-INF). During clinical trials, 19 patients received tuberculostatic therapy in combination with IL-2 (Roncoleukin) (a study group) whereas 16 patients had tuberculostatic therapy alone (a control group). The administration of Roncoleukin statistically significant increased a proliferative response to PPD and normalized the count of CD14+ CD16+ monocytes with anti-inflammatory and immunosuppressive activities. The restoration of a PPD response was recorded more frequently in the study group than in the control one (75% vs 30%; p = 0.045). The magnitude of positive X-ray changes was also higher in the study group than in the control one (63% vs 25%; p = 0.026). The findings suggest the clinical and immunomodulating effects of IL-2 (Roncoleukin) in the combined therapy for tuberculosis.

[T-cellular energy deficiency in the pathogenesis of immunodeficiency in pulmonary tuberculosis]. / T-kletochnaia anergiia v patogeneze immunnoi nedostatochnosti pri tuberkuleze legkikh.

The phenotypic and functional properties of T cells were evaluated in patients with tuberculin anergy and a possible role of anergic T cells in the development of immune deficiency in pulmonary tuberculosis (PT) was studied. The profound decrease (more than 50%) depressed T-cell proliferation in PPD-stimulated cultures was shown to be recorded in 44% (59/134) patients with PT. PPD hyporesponsiveness was associated with the low proliferation of anti-CD3 and SEB-stimulated proliferation of a healthy donor's mononuclear cells evidencing the enlargement of anergic T cells with a suppressive activity in PT. A PPD-stimulated response in healthy donors was under the negative control of CG25-positive cells. In the PPD-anergic patients, there was a significant increase in CD4+CD25+ T cells that were inversely correlated with the PPD-induced proliferative response. The development of tuberculin anergy was more pronounced in patients with drug resistance. The intensity of a PPD-stimulated response in patients with tuberculin anergy may be restored in the presence of exogenous interleukin-2.

[T cell subsets undergoing apoptosis and anergy in patients with pulmonary tuberculosis]. / Subpopuliatsionnaia prinadlezhnost' T-kletok, podverzhennykh anergii i apoptozu u bol'nykh tuberkulezom legkikh.

T-cell apoptosis and anergy as possible causes of impaired antigen specific responses and their subpopulation targets in patients with pulmonary tuberculosis were investigated. A decrease in PPD-stimulated proliferative responses were revealed in 43% of the examinees. The impaired PPD response was shown to be associated with both increased lymphocytic apoptosis and the arrest of cell cycle progression. CD4 and CHD8 T cells underwent apoptosis in PPD-stimulated cultures. Whereas a moderate apoptosis of CD4 cell could occur in PPD-reactive patients, accelerated apoptosis of CD8 cells developed only in PPD-unresponsive group. Both T-cell subpopulations displayed a decreased count of cells in S,G2/M phases of a cell cycle. Similar to apoptosis, the anergy of CD8 T cells was typical of PPD-unresponsive patients. Elevated apoptosis and anergy of CD4 and CD8 T cells in vitro were accompanied by a decline in the proportion of T cells and their subpopulations in patients with impaired PPD responses.

[Involvement of nitric oxide in the development of tuberculin anergy in patients with pulmonary tuberculosis]. / Uchastie oksida azota v razvitii tuberkulinovoi anergii u bol'nykh tuberkulezom legkikh.

The production of nitric oxide (NO) and the magnitude of an antigen-specific proliferative response of the human lymphocytes stimulated by M. tuberculosis antigen [a purified protein derivative (PPD)] were investigated. PPD-reactive T lymphocytes were found in the peripheral blood of healthy donors. Normal values (mean values, the range of the minimum and maximum values) of PPD-induced proliferation and NO production were determined. Patients with pulmonary tuberculosis were found to have different levels of PPD-stimulated proliferation and NO production. The lymphocytes are shown to preserve their PPD reactivity in patients with normal NO production whereas the PPD-induced proliferative response was significantly decreased in those with high NO production. Patients with reduced tuberculin reactivities and high NO production were less responsive to treatment. The findings suggest that nitric oxide is involved in the development of tuberculin anergy with pulmonary tuberculosis.

[Specific features of immunity in patients with different forms of pulmonary tuberculosis]. / Osobennosti immuniteta u bol'nykh s razlichnymi formami tuberkuleza legkikh.

The cellular immunity was studied in 59 patients with pulmonary tuberculosis. The development of tuberculous infection was ascertained to be accompanied by decreases in the relative counts of CD9, CD8, and CD72 lymphocytes, as well as monocytes, expressing class II histocompatibility antigens (DR). The patients with tuberculosis were found to have suppressed proliferative T-cell activity and IL-2 production, moderately decreased IL-1 production and increased TNF alpha secretion.

Role of nitric oxide in activation of human T lymphocytes induced by bacterial superantigen.

The involvement of nitric oxide (NO) in the regulation of human T cell response to bacterial superantigen (staphylococcal enterotoxin B) was studied. It was shown that stimulated T lymphocytes are the main source of NO. This superantigen markedly increased NO production and triggered the proliferative response of mononuclear cells from healthy individuals; the degree of apoptosis was low. In patients with purulent surgical diseases with high spontaneous and induced NO production, superantigen enhanced apoptosis of lymphocytes and induced anergy of T cells to enterotoxins. Increasing the concentration of NO in cultured cells from healthy individuals in the presence of NO donors also stimulated apoptosis and inhibited proliferative activity. These data suggest that NO regulates T lymphocyte response to superantigens. The increased production of NO probably contributes to the development of immunosuppression during bacterial infection.

[Secondary immunodeficiency of medical staff contacting with tuberculous and nonspecific infection]. / Vtorichnyi immunodefitsit meditsinskogo personala, kontaktiruiushchego s tuberkuleznoi i nespetsificheskoi infektsiei.

The present paper analyzes immunological parameters and the incidence of secondary immunodeficiency (SID) in physicians and medium-levelled medical staff contacting with tuberculous and non-specific infection. Suppressed cell immunity was recorded in 44% of the medical staff of a pulmonary surgical tuberculosis hospital, with increased length of service there was a rise in the number of patients diagnosed as having immunodepression. The clinical manifestations of SID were recorded in 56% in this group and they were most pronounced in a group of long-working personnel. The proportion of persons with immunodepression proved to be twice higher among nurses than among physicians. Nurses are at the highest risk for immunopathological states. This common occurrence of SID among medical staff is an indicator to make an obligatory regular immunological examinations of the staff for the prevention and immunotherapy of SID.

[Preparation of hybrid proteins consisting of human interleukin-2 and the cytotoxic A-subunit of Shigella toxin]. / Poluchenie gibridnykh belkov, sostoiashchikh iz interleuikina-2 cheloveka i tsitotoksicheskoi A-sub''edinitsy toksina shigelly.

Recombinant plasmids providing the synthesis of chimeric proteins consisting of amino acid sequences of human interleukin-2 (IL-2) and Shiga toxin cytotoxic A-subunit (ILA and AIL chimeric toxins) were constructed. The ILA and AIL chimeric toxins were shown to inhibit protein synthesis in the rabbit reticulocytes cell-free system. These chimeric toxins displayed two opposite activities of the constituent parts of their molecules on T-lymphocytes from the peripheral blood of healthy volunteers. Hybrid protein AIL (approximately 10(-6) g/ml) has caused the most significant depression of T-lymphoblast proliferation.

[Effective expression of genes for interleukin-2 and its mutant analogs in E. coli cells]. / Effektivnaia ékspressiia v kletkakh E. coli genov interleikina-2 cheloveka i ego mutantnykh analogov.

Recombinant plasmids were constructed for the efficient expression in E. coli cells of the human interleukin-2 (HIL-2) gene and two its mutant analogues obtained by of chemical-enzymic synthesis and polymerase chain reaction (deletion of 14 C-terminal amino acids and a change of the codon for Trp121 to Phe). The recombinant HIL-2 but not the mutant analogues were shown to be biologically active. Both analogues obtained were weak antagonists to HIL-2.

[The role of intercellular interactions in the activation of T-lymphocyte colony precursors]. / Rol' mezhkletochnykh vzaimodeistvii v protsesse aktivatsii predshestvennikov T-limfotsitarnykh kolonii.

The influence of the previous cultivation of human peripheral blood mononuclear cells in suspension with mitogen on the consequent PHA-stimulated response in suspension and agar cultures was studied. It has been established that proliferative response of mononuclear cells in suspension with mitogen is not changed, but the intensity of T-cell colony formation in two-layer agar systems in increased after preincubation mainly at the expense of increasing the number of type II colonies and clusters. It is concluded that the free cell-cell contacts are needed to activate T-lymphocyte colony precursors.