Alveolar macrophages from humans and rodents selectively inhibit T-cell proliferation but permit T-cell activation and cytokine secretion.

Alveolar macrophages (AM) are thought to play a key role in the regulation of immune responses within the lung. While it is well established that AM inhibit T-cell proliferation in vitro, it is unclear whether other aspects of the T-cell activation process are also inhibited. The present study demonstrates that AM from rat, mouse and human differ markedly in the potency with which they inhibit mitogen-induced T-cell proliferation, although in humans the degree of inhibition approaches that observed in the animal systems, if antigen (as opposed to mitogen) is employed as the T-cell activating agent. Rodent and human AM also differ in the mechanisms employed to achieve this inhibition; rodent AM appear to utilize reactive nitrogen intermediates, while this does not appear to be the case for human AM. Despite these differences, T cells stimulated in the presence of AM display a similar phenotype in all species examined, i.e. CD3 down-modulation, up-regulation of interleukin-2 receptor (IL-2R) expression and IL-2 production, but inability to respond to IL-2. Thus, AM appear to allow T-cell activation and expression of T-cell effector function, while selectively inhibiting T-cell proliferation.     

Alveolar macrophages. II. Inhibition of lymphocyte proliferation by purified macrophages from rat lung.

Macrophages were prepared from the lung, peritoneal cavity and blood of normal, unstimulated rats from a number of strains. The macrophages were purified by adherence, and characterized via surface markers, enzyme activity and phagocytic capacity, and subsequently tested for activity in cultures of mitogen-stimulated syngeneic lymphocytes. Peritoneal macrophages and blood monocytes were mildly stimulatory, or ineffective in modulating mitogen-induced DNA synthesis; peritoneal macrophages reconstituted the blastogenic responses of macrophage-depleted lymph node cell cultures to normal limits. In contrast, alveolar macrophages were markedly inhibitory to lymphocyte proliferation; in some instances inhibitory activity was demonstrable when added alveolar macrophages comprised only 0.04% of the total cells in culture. Lymphocyte proliferation induced by T-cell mitogens was more susceptible to this inhibition than was proliferation induced by the B-cell mitogen LPS. Alveolar macrophages recovered from SPF rats, while less in number, exhibited comparable inhibitory activity. These results form part of an emerging picture picture of the normal alveolar macrophage as a potential 'suppressor' of T-cell activity in the lung.     

Resident salivary gland macrophages function as accessory cells in antigen-dependent T-cell proliferation.

The function of salivary gland macrophages in the induction of local immunity in secretory organs was investigated in Fischer 344 rats. Macrophages obtained from dispersed submandibular gland (SMG) cells were characterized and examined for their ability to present antigen to T cells. Populations of SMG-adherent cells contained approximately 80% macrophages, of which 46-62% were I-A+ cells. These numbers were from five to 10-fold greater than the I-A+ cells in macrophage populations from peritoneal exudates (5-11%). SMG macrophages functioned effectively as antigen-presenting cells. Antigen presentation was antigen specific, macrophage dose dependent and inhibitable by monoclonal anti-I-A antibodies. These studies suggest that a functional salivary-gland immune-response pathway exists that can function independently of a gut-associated lymphocyte-homing mechanism.     

Alveolar macrophages. VI. Regulation of alveolar macrophage-mediated suppression of lymphocyte proliferation by a putative T cell.

Alveolar macrophages (AM) from normal rats suppressed antigen- or mitogen-stimulated blastogenic responses in cultures of splenic or lymph node lymphocytes, high levels of suppression often being observed when added AM comprised as few 0.6% of the total cells in culture. The efficiency of AM-mediated suppression of spleen cell blastogenesis declined with the age of the spleen cell donors, was severely curtailed by pretreatment of donors with low levels of cyclophosphamide, and was depleted by adult thymectomy coupled with thoracic duct drainage. The suppressive activity of AM was most obvious at high cell density, was unaffected by the presence of indomethacin in the cultures, or by prior X-irradiation of the spleen cells. Fractionation of spleen cells by velocity sedimentation yielded cell populations of greatly varying sensitivities to AM-mediated suppression, from small splenocytes (sedimentation velocity 1.1-2.8 mm/h) which were almost totally refractory to AM-suppression when assayed in isolation from the remainder of the spleen cell population, to larger cells (sedimentation velocity greater than 3.,5 mm/h) exhibiting high levels of sensitivity. Fractionation of spleen cells by glass wool adherence indicated decreased sensitivity to AM-suppression in the effluent population. Examination of the suppressive activity of individual subpopulations of AM separated by velocity sedimentation indicated that the larger macrophages were the most active in vitro. Suppressive activity of this nature was not seen with unstimulated peritoneal macrophages, but was observed when 'activated' peritoneal exudate cells were tested. These data are discussed in terms of a two-cell model for suppression of blastogenesis, the ultimate effector cell being a macrophage, the activity of which is controlled by a long-lived, recirculating lymphocyte, which we have provisionally designated as a T lymphocyte.     

The Chediak-Higashi gene in humans. III. Studies on the mechanisms of NK impairment

Lymphocytes from six Chediak-Higashi (CH) patients were markedly depressed in their ability to lyse tumour cell targets in both 51Cr release and single cell cytotoxicity assays. The frequency of lymphocytes bearing the OKM1 marker and the frequency of T3+, T4+, T8+, Ia+, Mo1+, Mo2+ and B1+ cells was normal among sheep erythrocyte rosetting (E+) and non-rosetting (E-) peripheral blood leucocytes analysed by flow cytofluorography. Cells expressing the NK shared markers, OKM1, mac-1, FcR, and the characteristic large granular lymphocyte (LGL) morphology of NK cells were also present in normal numbers in the highly enriched NK fraction separated on Percoll density gradients. This fraction did not contain detectable numbers of cells expressing the Mo2 marker of human monocytes. Therefore most of the cells stained by monoclonal OKM1 and mac-1 in this fraction are likely NK cells, rather than monocytes, and we conclude that the size of the NK pool in CH patients is probably normal. The capacity of CH lymphocytes to recognize and bind to tumour cells was also normal as was the subsequent burst of oxygen intermediates produced by the NK cells in a chemiluminenscence assay. We have shown elsewhere that O2- generation is directly involved in activating subsequent steps in the NK cytolytic pathway. These results suggest that NK cells in CH patients are present in normal frequency but are blocked at some post-recognition, post-activation step in the cytolytic pathway subsequent to the burst of oxygen intermediates but preceding the lethal hit.     

Alveolar macrophages. V. Comparative studies on the antigen presentation activity of guinea-pig and rat alveolar macrophages.

The addition of syngeneic alveolar macrophages (AM) to mitogen-stimulated lymphocyte cultures from the rat and the guinea-pig resulted in markedly dissimilar effects upon in vitro blastogenesis, guinea-pig AM stimulating the response and rat AM exhibiting strong suppressive activity. The capacity of guinea-pig and rat AM to initiate antigen-specific blastogenesis was also examined. Ovalbumin-immune lymph node cells from guinea-pigs were passed through nylon wool columns to deplete macrophages. This process abolished their capacity to respond to the antigen via blastogenesis. The addition of ovalbumin-pulsed AM to these cultures restored their blastogenic responsiveness, and did so with considerably greater efficiency than was observed employing peritoneal macrophages from the same animals. Identical manoeuvres in the rat again yielded opposite results; the addition of rat AM to syngeneic antigen-stimulated lymphoid cell cultures consistently suppressed blastogenesis, an effect not seen employing peritoneal macrophages from the same species.     

Macrophage subpopulations regulate intrathymic T-cell development. I: Ia-positive macrophages augment thymocyte proliferation

The contribution of H2-Ia-positive thymic macrophages (Ia 1 thymic M phi) to intrathymic lymphopoiesis was investigated. An isolation method yielding cell suspensions highly enriched for Ia+ thymic M phi was performed. Cocultivation of these Ia+ thymic M phi with thymocytes showed that, while not affecting spontaneously proliferating thymocytes, the Ia+ thymic M phi strongly augmented the mitogen-induced proliferation of thymocytes by about 200%. This effect was dependent on the number of Ia+ M phi added as well as on the degree of thymocyte maturity: stronger augmentation occurred at higher concentrations of M phi and immature thymocytes showed highest susceptibility to the Ia+ thymic M phi-mediated effect. Cytochalasin B was employed to prove that cellular interaction is an important prerequisite for the proliferation amplifying effect of Ia+ thymic M phi. Additionally, humoral factors produced by Ia+ thymic M phi after induction with LPS are also involved in the described phenomenon. Furthermore, the use of interleukin preparations in the thymocyte-Ia+ M phi cocultures demonstrated that humoral factors support or probably regulate the interaction of these cells. The implications of these findings in view of the proliferation and differentiation events of thymocytes within the thymus are discussed.     

The additive effect of certain drugs on the cyclosporine A inhibition of human T-cell proliferation. Studies using theophyllamine, warfarin, verapamil and dipyridamol

The inhibitory effect of Cyclosporine A (CsA) when combined with theophyllamine, warfarin, verapamil or dipyridamol on in vitro proliferation of human T lymphocyte was investigated. All drugs caused an additional inhibition of PHA activation compared with CsA alone, also when the CD8-negative and -positive subsets were tested separately. This effect was dose-dependent for all drugs. Theophyllamine, verapamil and warfarin had to be added early during PHA-stimulated T-cell activation to cause maximal inhibition. Also when testing the proliferative response in mixed lymphocyte cultures, the drugs showed a dose-dependent additional inhibition compared with CsA alone. CsA alone caused no inhibition of lymphokine-dependent growth of T-cell lines, whereas all four drugs caused a weak inhibition in this test system.     

Tumor-associated macrophages promote tumor cell proliferation in nasopharyngeal NK/T-cell lymphoma

Objective: To explore the relationship between the number of tumor-associated macrophages (TAMs) and proliferative activity of tumor cells and the relationship between two macrophage biomarkers CD68 and CD163 in nasopharyngeal NK/T-cell lymphoma. Methods: Immunohistochemistry was used to reconfirm the diagnosis of nasal NK/T-cell lymphoma and detect the numbers of TAMs and the ki-67 label index of the tumor cells in all 31 cases. In addition, 12 cases of inflammatory cases were collected as controls, for which the immunostaining of CD68 and CD163 were done as well. Then staining results were analyzed with Pearson correlation and t test. Results: The number of TAMs was positively correlated with tumor proliferative activity (P = 0.024) in nasopharyngeal NK/T-cell lymphoma. The expression of CD68 and CD163 was closely related (P = 0.009), and the positive rate of CD68 was generally higher than CD163, however there is no statistical significance. Conclusion: The increase in numbers of TAMs in nasopharyngeal NK/T-cell lymphoma is related to higher proliferative index, indicating the TAMs play an important role in tumor proliferation. Meanwhile both CD68 and CD163 might be the markers for TAMs but CD163 would be the better one.     

Cell intrinsic mechanisms of T-cell inhibition and application to cancer therapy

Summary: Establishing a balance between the rapid generation of effective immunity and the production of overly exuberant or excessively prolonged responses is critical to the maintenance of the equilibrium between health and disease. The preservation of homeostasis and prevention of inappropriate activation of immunity is safeguarded by systems integrating the influences of T-cell receptor signaling, pro-inflammatory danger signals, and positive costimulatory signals on the one hand with those of several layers of both cell-intrinsic and cell-extrinsic inhibitory checkpoints on the other. Evolution has thus provided an immunological system capable of clearance of pathogens and infected cells but which generally avoids the severe collateral damage that is associated with failure to control immunity. Central tolerance to self-antigens constitutes the first line of defense against self-destruction. Because central tolerance mechanisms fail to eliminate all self-reactive immune effector cells, other immune-regulating (peripheral tolerance) mechanisms are required to prevent excessive immune responses. Dysfunction of these inhibitory pathways in terms of reduced activity can result in the unmasking of self-directed responses and a variety of autoimmune morbidities. Conversely, enhanced inhibitory activity can restrict the generation of clinically useful immunity to cancers and to chronic infectious pathogens. This may manifest not only as inhibition of immunity directed towards what are largely aberrantly or overexpressed ‘self’ targets on malignant cells but also additional exaggeration of inhibitory pathway activity mediated via upregulation on tumor cells or stromal tissues of the ligands for inhibitory receptors expressed by lymphocytes. The selective pressures exerted by immuno-editing will favor the outgrowth of such immuno-evasive malignant clones. These pathways therefore represent significant hurdles to the generation of therapeutic anti-cancer responses. The most active of the T-cell intrinsic inhibitory pathways belong to the immunoglobulin superfamily, which occupies a central importance in the coordination of immune responses. The CD28/cytotoxic T-lymphocyte antigen-4 (CTLA-4):B7-1/B7-2 receptor/ligand grouping represents the archetypal example of these immune regulators. Therapies aimed at overcoming these mechanisms of peripheral tolerance, in particular by blocking the inhibitory checkpoints, offer the potential to generate anti-tumor activity, either as monotherapies or in synergism with other therapies that directly or indirectly enhance presentation of tumor epitopes to the immune system.     

The mechanisms of inhibitory effects of liver extract on lymphocyte proliferation. I. The extracellular mechanism of the inhibition

Liver aqueous extract (LEx) can powerfully inhibit phytohaemagglutinin (PHA)-induced lymphocyte proliferation. The extracellular mechanism of inhibition by LEx was studied. There are three possibilities. (1) The possibility of extracellular inactivation of PHA by LEx was excluded by incubating cells with PHA first, followed by washing, and then incubating cells with LEx. The result was that LEx was still able to inhibit cell proliferation completely. (2) The possibility of competition for cell surface PHA receptors by LEx was excluded by the above experiments plus the use of N-acetyl-D-galactosamine to remove surface-bound PHA. Following this treatment, LEx was still able to inhibit cell proliferation completely. (3) The possibility of arginase-induced arginine depletion resulting in lymphocyte suppression was supported by the following experiments. Cells were incubated in media in which arginine was depleted either by reacting with LEx, and the media were afterwards bound with anti-arginase antibody, or by amino acid constituted media without arginine. The degrees of proliferation inhibition were similar in both treatments. These results indicate the important role of arginine-depletion by LEx, and may account for the LEx-induced lymphocyte inhibition.     

Attempts to characterize the mechanisms involved in mycobacterial growth inhibition by gamma-interferon-activated bone marrow macrophages.

Bone marrow-derived murine macrophages are able to inhibit the growth of Mycobacterium bovis and of some strains of M. tuberculosis after stimulation with either recombinant gamma interferon (rIFN-gamma) or lymphokines from antigen-specific T-cell clones. To elucidate the mechanism(s) involved in antimycobacterial activity, macrophages were infected with M. bovis in the presence of agents thought to influence the antimicrobial effects of phagocytes. Scavengers of toxic oxygen metabolites failed to influence the capacity of IFN-gamma-activated bone marrow macrophages to inhibit the growth of M. bovis. Suramin slightly affected mycobacterial growth in IFN-gamma-activated macrophages, and chloroquine markedly induced growth inhibition of M. bovis in unstimulated macrophages. We conclude that growth inhibition of M. bovis by IFN-gamma-activated macrophages is an oxygen-independent process.     

Inhibition of macrophage-induced, antigen-specific T-cell proliferation by poly I:C role of suppressor macrophages.

Poly I:C treatment can inhibit the ability of macrophages (M phi) to induce antigen-specific T-cell proliferation. This study investigated whether this inhibition is the result of suppressor or cytotoxic activity. Pretreatment of M phi with indomethacin in vivo, in vitro or both failed to reverse the inhibition of T-cell proliferation induced by poly I:C-treated, keyhole limpet haemocyanin (KLH)-pulsed M phi, suggesting that prostaglandin production does not mediate the inhibition of T-cell proliferation. The transfer of supernatants from cultures containing poly I:C-treated, KLH-pulsed M phi to cultures containing saline-treated, KLH-pulsed M phi and T cells did not inhibit T-cell proliferation, suggesting that the inhibition of T-cell proliferation by poly I:C is not mediated by the production of soluble suppressor factors. As addition of poly I:C-treated, KLH-pulsed M phi to cultures containing saline-treated, KLH-pulsed M phi did not significantly inhibit KLH-specific T-cell proliferation, the inhibition of T-cell proliferation is also not mediated by direct cell contact or short-range soluble suppressor factors. In addition, poly I:C-treated, KLH-pulsed M phi did not induce cytolysis of syngeneic T cells. These results indicate that cytotoxic or suppressor effector functions of M phi are not involved in the mechanism by which poly I:C inhibits M phi-induced, antigen-specific T-cell proliferation.     

Studies on the mechanisms of macrophage activation. III. Investigations on the interaction of macrophage-activating factor with the macrophage membrane

A macrophage-activating factor (MAF), produced by concanavalin (Con A)-stimulated spleen lymphocytes, was tested by its capacity to induce mouse peritoneal exudate macrophages to destroy the intracellular parasite Leishmania enriettii. MAF could be removed from active media by adsorption on macrophages at 37°C and 4°C; the adsorption was not specific, as cells from various origins could similarly be used to deplete supernatants of their activity. Treatment of macrophages with trypsin resulted in loss of response to MAF, presumably due to inactivation of a membrane receptor for the lymphokine. In addition, trypsin treatment of macrophages, following a 6-h pulse, but not a 9-h-pulse, of MAF-rich medium, inhibited activation suggesting that triggering of the biochemical processes of activation required interaction between macrophages and the lymphokine of sufficient duration, and that once these processes were initiated, parasite destruction could proceed to completion in the absence of further exogenous lymphokine. MAF activity was enhanced by treatment of macrophages with al-antitrypsin suggesting a role for membrane-associated serine esterases in modulating the cell response to the lymphokine. High concentrations of Con A also impaired MAF-induced activation, presumably through binding of the lymphokine by the lectin.     

Regulation of T-cell activation in the lung: alveolar macrophages induce reversible T-cell anergy in vitro associated with inhibition of interleukin-2 receptor signal transduction.

Alveolar macrophages (AM) are recognized as archetypal 'activated' macrophages with respect to their capacity to suppress T-cell responses to antigen or mitogen, and this function has been ascribed an important role in the maintenance of local immunological homeostasis at the delicate blood:air interface. The present study demonstrates that this suppression involves a unique form of T-cell anergy, in which 'AM-suppressed' T cells proceed normally through virtually all phases of the activation sequence including Ca2+ flux, T-cell receptor (TCR) modulation, cytokine [including interleukin-2 (IL-2)] secretion and IL-2 receptor (IL-2R) expression. However, the 'suppressed' T cells fail to up-regulate CD2, and do not re-express normal levels of TCR-associated molecules after initial down-modulation; moreover, they are unable to transduce IL-2 signals leading to phosphorylation of IL-2R-associated proteins, and remained locked in G0/G1. The induction of this form of anergy is blocked by an NO-synthase inhibitor, and is reversible upon removal of AM from the T cells, which then proliferate in the absence of further stimulation. We hypothesize that this mechanism provides the means to limit the magnitude of local immune responses in this fragile tissue microenvironment, while preserving the capacity for generation of immunological memory against locally encountered antigens via clonal expansion of activated T cells after their subsequent migration to regional lymphoid organs. In an accompanying paper, we demonstrate that a significant proportion of T cells freshly isolated from lung exhibit a comparable surface phenotype.     

Distinct signaling mechanisms for apoE inhibition of cell migration and proliferation

Over the years, the vascular protective role of apolipoprotein (apo) E has been attributed to the ability of apoE to induce cholesterol efflux from macrophage foam cells and its transport of extrahepatic cholesterol to the liver for excretion out of the body. Recently, apoE has been shown to protect against vascular disease by additional mechanisms that are independent of its cholesterol transport functions. This review summarizes data demonstrating apoE binding to specific cell surface receptors and proteoglycans in smooth muscle cells triggers distinct signalling pathways that result in inhibition of cell migration, proliferation, and excessive extracellular matrix deposition. apoE binding to the low density lipoprotein receptor-related protein is responsible for inhibition of cell migration, due to the induction of cyclic AMP accumulation and protein kinase A activation. apoE inhibition of cell proliferation is mediated by its binding to proteoglycans and the resulting activation of inducible nitric oxide synthase. apoE also inhibits excessive extracellular matrix protein synthesis. The receptor responsible for this latter apoE function remains to be identified.     

实验性肺癌泡巨噬细胞扫描电镜观察与增殖抑制...

Alveolar macrophages (AM) from rats with lung cancer induced by 3,4-benzo(a)pyrene were collected and examined under SEM. According to changes of the surface ultrastructure, AMs were classified into activated and unactivated groups. Different groups of cells were incubated with Hela cells as the target, and cytostatic activity of AM was studied in vitro, including observation of the morphological changes as well as determination of the cytostatic index (CI). Results showed that AMs from non-tumor-bearing and control groups had spontaneous cytotoxicity, which might be related to the existence of a certain proportion of activated macrophages in the host, while the activity of AM from the tumor-bearing group was obviously inhibited.     

Stimulation of bystander T-cell proliferation by tumor necrosis factor produced by HIV-1-infected macrophages

OBJECTIVE: Cocultivation of the CD4+CD95+ T-cell line (MT4) with monocyte-derived macrophages (MDMs) infected with the HIV-1 resulted in costimulation of proliferation and apoptosis after 20 hours of contact. This study sought to determine whether tumor necrosis factor (TNF) produced by HIV-1-infected MDMs was involved in the costimulation of cell proliferation, the apoptotic pathway, or both. STUDY DESIGN/METHODS: MT4 cells were cocultivated with infected or noninfected MDMs in the presence or absence of soluble TNF receptors (sTNFRs) or antagonistic anti-Fas antibody (ZB4). Cell proliferation was assessed by measuring thymidine incorporation. Apoptosis was monitored by using flow cytometry and enzyme-linked immunosorbent assay (ELISA). RESULTS: Thymidine incorporation was higher in cells cocultivated with HIV-infected or noninfected MDMs than it was in controls grown in culture medium. It also was higher in cells cocultivated with HIV-infected MDMs than it was in cells cocultivated with noninfected MDMs. sTNFRs blocked the increase of thymidine incorporation specifically induced by HIV-infected MDMs. They did not inhibit apoptosis at 20 hours. Cells recovered from cocultures involving HIV-infected or noninfected MDMs exhibited decreased sensitivity to apoptosis induced through the Fas receptor. CONCLUSION: TNF produced by HIV-infected MDMs acts as an accessory T-cell growth factor that synergizes with an as yet unidentified growth-inducing signal or signals produced by HIV-infected and noninfected MDMs. Stimulation of cell proliferation by MDMs induces transient resistance to Fas-induced apoptosis.     

TNFR1 signalling is a critical checkpoint for developing macrophages that control of T-cell proliferation

Macrophages (Mϕ) are professional antigen-presenting cells, but when they accumulate at sites of inflammation, they can inhibit T-cell proliferation. In experimental autoimmune uveoretinitis, this limits the expansion of T cells within the target organ. To define requirements for the elaboration of this outcome, we have generated populations of Mϕin vitro that could also regulate T-cell responses; stimulating CD4⁺ T-cell activation and cytokine production, but simultaneously suppressing T-cell proliferation. When T cells are removed from the influence of such cells, normal T-cell responses are restored. We show that tumour necrosis factor 1 (TNFR1) signalling is a critical checkpoint in the development of such Mϕ, as TNFR1^−/− Mϕ are unable to suppress T-cell proliferation. This deficit in antigen-presenting cells results in a lack of production of prostaglandin E₂ (PGE₂) and nitric oxide, which are critical effector mechanisms that inhibit T-cell division. However, TNFR1 signalling is not required for the inhibitory function of Mϕ because we could circumvent the requirement for this receptor, by maturing Mϕ in the presence of exogenous interferon-γ and PGE₂. This produced TNFR1^−/− Mϕ that inhibited T-cell proliferation and indicates that TNFR1 delivers a signal that is necessary for the development but not the execution of this function.