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Murine contact sensitivity (CS) reaction could be antigen-specifically regulated by T CD8(+) suppressor (Ts) lymphocytes releasing microRNA-150 in antibody light-chain-coated exosomes that were formerly suggested to suppress CS through action on macrophages (Mφ). The present studies investigated the role of Mφ in Ts cell-exosome-mediated antigen-specific suppression as well as modulation of Mφ antigen-presenting function in humoral and cellular immunity by suppressive exosomes. Mice depleted of Mφ by clodronate liposomes could not be tolerized and did not produce suppressive exosomes. Moreover, isolated T effector lymphocytes transferring CS were suppressed by exosomes only in the presence of Mφ, demonstrating the substantial role of Mφ in the generation and action of Ts cell regulatory exosomes. Further, significant decrease of number of splenic B cells producing trinitrophenyl (TNP) -specific antibodies with the alteration of the ratio of serum titres of IgM to IgG was observed in recipients of exosome-treated, antigen-pulsed Mφ and the significant suppression of CS was demonstrated in recipients of exosome-treated, TNP-conjugated Mφ. Additionally, exosome-pulsed, TNP-conjugated Mφ mediated suppression of CS in mice pre-treated with a low-dose of cyclophosphamide, suggesting de novo induction of T regulatory (Treg) lymphocytes. Treg cell involvement in the effector phase of the studied suppression mechanism was proved by unsuccessful tolerization of DEREG mice depleted of Treg lymphocytes. Furthermore, the inhibition of proliferation of CS effector cells cultured with exosome-treated Mφ in a transmembrane manner was observed. Our results demonstrated the essential role of Mφ in antigen-specific immune suppression mediated by Ts cell-derived exosomes and realized by induction of Treg lymphocytes and inhibition of T effector cell proliferation., (© 2015 John Wiley & Sons Ltd.)

Macrophages (Mφ) as efficient phagocytes able to present the antigen and playing an effector role induce and orchestrate the immune response also through the release of soluble factors. Recently described T CD8+ cell-derived suppressive exosomes carrying miRNA-150, that act antigen-specifically, seem to inhibit murine contact sensitivity reaction indirectly by affecting antigen presenting cells, especially Mφ. Present studies investigated the influence of suppressive exosomes on secretory activity of Mφ assessed as their ability to generate reactive oxygen intermediates (ROIs), nitric oxide, cytokines as well as their viability and expression of antigen phagocytosis and presentation markers. Interestingly, in vivo and in vitro treatment of Mφ with assayed hapten-specific exosomes affected only ROIs generation, significantly enhancing their production. Current results suggest that ROIs may participate in antigen-specific tolerance mechanism mediated by suppressive T lymphocyte-derived exosome-influenced Mφ, by inhibition of effector T cell proliferation and induction of T regulatory lymphocytes.

Testicular macrophages (TMf) are located in the interstitial tissue of the male gonad. Highly purified TMf populations can be prepared either by the mechanical shaking of dispersed testicular tissues or by enzymatic digestion with collagenase followed by cell adherence, rosetting and gradient centrifugation. TMf obtained by the enzymatic procedure produced significantly more cytokines (IL-6, IL-10 and TNF-alpha) than TMf isolated by the mechanical method and this effect is long-lasting. Our results indicate that isolation of tissue macrophages by enzymatic digestion may influence their functional activity, and suggest that critical evaluation of the method used to obtain these cells should be the regular practice.

Testicular macrophages (TMs) are located in the interstitial tissue of male gonad. These phagocytic cells take part in forming the organ-specific functional blood-testis barrier and participate in the regulation of the local hormonal balance. In the present study, we isolated TMs from testicular tissues using previously described methods--mechanical (M-TMs) or enzymatic, by treatment with collagenase (E-TMs) and then we studied production by these cells of several cytokines and reactive oxygen intermediates (ROI's). Similarly treated oil-induced peritoneal macrophages (PMs) were used as control cells. PMs had a higher baseline level of production of TNF-alpha, IL-6, IL-10 and IL-12 than M-TMs and collagenase treatment increased the production of these cytokines (except IL-12) by both cell populations. This effect was significantly more expressed in TMs. In contrast to PMs, TMs produced little ROI's when stimulated by zymosan. We conclude that in the case of local inflammation in the testis, ROI-negative TMs do not contribute to the tissue damage and instead may direct the local immune response into humoral pathway.

Problem: Testicular macrophages (TMf) participate together with Sertoli cells in formation of blood-testis barrier. The present experiments were aimed to test their immunoregulatory functions in vivo and in vitro., Method of Study: TMf were purified by glass adherence, rosetting with opsonized erythrocytes and fractionation on discontinuous Percoll gradient (over 95% purity). Their antigen-presenting capacity in humoral and cell-mediated responses was tested in vitro (Mishell-Dutton cultures, proliferation assay) and in vivo (induction of contact sensitivity reaction)., Results: TMf represent a heterogeneous cell population. Heavier Percoll fractions produce little transforming growth factor (TGF)-beta and are efficient antigen-presenting cells in humoral and cell-mediated immune responses. Lighter fractions produce high amounts of TGF-beta and are rather tolerogenic than immunogenic. Their immunosuppressive activity can be prevented by treatment of TMf donors with cyclophosphamide or in vitro by anti-TGF-beta monoclonal antibody. In non-separated TMf population the immunosuppressive activity prevails., Conclusions: Subpopulation of TMf able to trigger specific immune responses is present in the testis but remains under control of other TMf subpopulation which minimizes the risk of development of autoimmune reactions.

Inflammatory foci are rich in proteases released by neutrophils (serine proteases) and macrophages (metalloproteases). These enzymes can degrade extracellular matrix proteins and cell membrane bound proteins thus contributing to the development and progression of inflammatory reaction. In this study we have investigated the influence of collagenase (metalloprotease) and trypsin (serine protease) on murine resident and oil-induced peritoneal macrophages (Mf). Short in vitro treatment of Mf, not affecting cell viability, significantly reduced the release of reactive oxygen intermediates (ROIs) and at the same time triggered the increase of IL-6 production and to lesser extent of TNF-alpha production. Both these effects were dependent on enzyme concentration used and were particularly well pronounced in resident macrophages. In addition both enzymes cleaved a number of cell-membrane molecules, including CD23, CD14, CD95L, and Mac-3. We hypothesize that the enzymatic digestion of certain Mf surface receptor proteins in inflammatory foci may be responsible for modification of cell behaviour either by preventing the generation of specific signal or alternatively by delivering a mock substitute signal to the cell interior. In effect inhibition of ROIs production limits their destructive effects and the increase in the secretion of IL-6 stimulates the synthesis of acute phase proteins and triggers other anti-inflammatory mechanisms thus directing Mf present in inflammatory foci into regulatory pathway rather than allowing them to perform solely the effector function.

Cells isolated enzymatically from the interstitial tissue of mouse male gonads are composed of macrophages, Leydig cells, and myofibroblasts. They can be separated on density gradients, either by sedimentation (Ficoll) or flotation (Percoll) into several fractions according to the different buoyant densities of the different cells in the mixture. Macrophages (Fc gamma R+, esterase+) present in cell mixtures can by highly enriched (to 95% purity) in a single step by rosetting with opsonized erythrocytes followed by sedimentation on Lymphoprep. Separate fractions of highly purified (over 95%) macrophages obtained by the successive use of density gradients and rosetting differ significantly in the production of cytokines, e.g. cells from fractions at lower density produce little IL-6, cells from fractions at higher density are poor producers of TNF-alpha, while testicular macrophages (TMf) in intermediate fractions produce significant amounts of both cytokines. These differences may suggest that particular subpopulations of testicular macrophages play different biological roles in the testis.

The purpose of the study was to examine the response of macrophages and the concentration of selected released cytokines following contact with a new carbon-polylactide composite. The macrophages were grown on samples of the materials and on each of its components separately. Viability of the cells as well as concentrations of interleukins IL-6, IL-10, IL-12 and TNF-alpha were then determined. Some differences in the viability of the cells were demonstrated. They varied according to the kind of material used. After incubation with the serum, the composite and its components induced the release of IL-6, IL-12 and TNF-alpha which did not differ significantly from one another.

The increased incidence of bacterial and mycotic infections in poorly controlled diabetic patients or animals is frequently attributed to impaired activities of professional phagocytes (granulocytes, macrophages) in hypoinsulinaemic milieu. We measured production of monokines (IL-6 and tumour necrosis factor-alpha (TNF-alpha)), active NO and reactive oxygen intermediates (ROIs), as well as expression of several cell surface adhesion molecules (Mac-1, -2 and -3, intercellular adhesion molecule-1 (ICAM-1) and Fc gammaRII), by thioglycollate medium-induced peritoneal macrophages of normoglycaemic and alloxan diabetic CBA/J mice (blood glucose level in the range 300 or 500 mg/dl). Macrophages of animals with moderate diabetes (300 mg/dl) produced significantly more IL-6 and TNF-alpha and ROIs than cells of control mice and showed an increased expression of all cell surface molecules, except Mac-3. NO/NO2 production was not affected. Administration of insulin restored enhanced values to normal levels, except for the production of ROIs which remained unusually high. We conclude that two separate mechanisms influence macrophage physiology in diabetes--lack of saturation of insulin receptors on macrophages and an indirect effect due to formation of advanced glycosylation endproducts (AGE) on their surfaces. The latter is possibly responsible for increased generation of ROIs, since it cannot be down-regulated by prolonged insulin treatment. How the increased activity of macrophages of moderately diabetic mice (enhanced production of proinflammatory monokines and oxygen radicals as well as expression of molecules) is related to their ability to kill bacteria is now under investigation.

Alkylating agents, cyclophosphamide (CY) and the related compound mechlorethamine (NM), significantly increase in vivo the blood level of IL-6 but not of IL-1. Since in vitro CY is inactive we have used in our experiments NM, a compound structurally and functionally related to phosphoramide mustard, the natural biologically active metabolite of CY. Thioglycollate or oil-induced peritonal macrophages (Mf) of four different mouse strains treated with NM produce significantly more IL-6 than the non-treated cells. In contrast, under these conditions, the production of IL-1, TNF alpha and NO/NO2 radicals is not affected. The NM-induced elevated production of IL-6 by Mf could not be further increased by the treatment of cells with LPS, which may suggest that both agonists stimulate the same signalling pathway. The antineoplastic activity of the alkylating agents like CY or NM is usually attributed to the interstrand cross-linking of the DNA of the dividing cells. Our experiments can be tentatively interpreted as the induced overplus of IL-6 can also contribute to the stimulation of the cytotoxic NK-cells.

We examined the isoenzyme patterns of alpha and beta naphtyl acetate esterase and the IL6 production of two macrophage cell lines, which were cloned from a single fusion of macrophage tumor cells and spleen adherent cells. These clones were phenotypically indistinguishable but differ functionally as line 59 presents antigen to Th 1 lymphocytes while line 63 induces suppressor T lymphocytes. Cell extracts of these lines exhibit different isoenzyme patterns of alpha and beta naphtyl acetate esterase at both pH 7.5 and 5.8 but do not differ significantly in the level of produced IL6. Treatment with nitrogranulogen (NG), a derivative of cyclophosphamide, changes the isoenzyme pattern in line 59 and decreases severalfold IL6 production, while in similarly treated line 63 cells isoenzyme pattern remains unaffected but the production of IL6 is significantly increases. We assume that the observed differences between these two cell lines are due to distinct intracellular translation of the membrane signal delivered by NG. The different behaviour of these two parameters can thus be used as a useful tool to further delineate different macrophage subpopulations. We regard it likely that nonspecific esterases may play a role in intracellular processing or trafficking of antigen.

We have tested the ability of trinitrophenyl (TNP)-labelled antigen-presenting cells (Langerhans cells (LC) and peritoneal macrophages (Mφ)) administered intravenously to induce cells that mediate and regulate contact sensitivity, TNP-Mφ fail to induce contrasuppressor cells (Tcs) but activate efferent T suppressor (Ts) cells. However, the activity of immune cells can be recovered after removal of Ly 2 Ts cells. In sharp contrast, TNP-substituted purified LC produced a significant contact sensitivity reaction, which is roughly equivalent to that achieved by skin sensitization with picryl chloride. Immune cells from these animals were relatively resistant to suppression by antigen-specific Ts cells, and we have found that their resistance is due to the presence of Ly 1, Vicia villosa lectin adherent, I-J+ cells. The Tcs cell induced by TNP-LC is indistinguishable by surface markers and function from Tcs cell found in mice injected with antigen-antibody complexes. Both these Tcs cells are capable of protecting immune cells from the effects of suppression by antigen-specific Ts cells if added in the proper sequence. Although they have identical surface phenotypes, it is not known at present whether or not Tcs cells induced by two different antigen presentations are identical. The possible reasons why LC are such potent inducers of contrasuppression are discussed. [ABSTRACT FROM AUTHOR]