Studies on the mechanism of phagocytosis. II. The interaction of macrophages with anti-immunoglobulin IgG-coated bone marrow-derived lymphocytes

We have examined the effect of the distribution of anti-immunoglobulin IgG molecules on the surface of bone marrow-derived lymphocytes upon the interaction of these cells with macrophages. Lymphocytes which were diffusely coated with antibodies to surface immunoglogulin were ingested by macrophages. Lymphocytes which had the same number of anti-immunoglobulin IgG molecules redistributed to one pole of the surface bound to the macrophages' Fc receptors but were not ingested. These results confirm our previous hypothesis that ingestion of an immunologically coated particle requires the sequential, circumferential binding of specific receptors on the plasma membrane of a phagocytic cell to immunologic ligands distributed over the entire particle surface. Macrophages which had bound capped lymphocytes by the macrophages' Fc receptors removed the immune complex caps from the lymphocyte surface without destroying the lymphocytes. These lymphocytes remained attached to the macrophage surface. The finding that macrophages can phagocytize immune complexes from the surface of a cell without destroying the cell to which these complexes are attached may be important in understanding the effects of antigens and antibodies on cells participating in a humoral immune response, in identifying the mechanisms by which chronic viral infections are established, and in defining the roles of blocking antibodies in tumor immunity.     

The interaction of human macrophages and lymphocytes in the phytohemagglutinin-stimulated production of interferon

In studies of 13 normal adults to determine the blood cell types responsible for interferon production induced by phytohemagglutinin, the following observations were made. (a) In cultures containing 96-100% pure macrophages derived from blood monocytes, no interferon was detected in either the presence or the absence of phytohemagglutinin for up to 92 hr. (b) In cultures of 99.5-100% pure lymphocytes, low levels of interferon were detected in the presence, but not in the absence, of phytohemagglutinin. (c) An average fivefold increase in interferon titers occurred when pure lymphocytes were combined with the macrophages in culture with phytohemagglutinin. The peak response of interferon occurred at 68 hr after the initiation of the combined cultures. For maximum response, phytohemagglutinin was required for the duration of the culture, and both cell types in association were necessary. Medium from phytohemagglutinin-stimulated macrophages or lymphocytes could not substitute for the corresponding intact cell. However, frozen-thawed macrophages in combination with lymphocytes and phytohemagglutinin produced an intermediate interferon response. An increase in either cell type produced an increased response in the range studied: lymphocytes, 0.45-1.8 × 10⁶ per ml; and macrophages, 0.5-2.1 × 10⁵ per ml. Syngeneic fibroblasts, HeLa cells, or mouse macrophages could not substitute for the human macrophages in the combined cultures with phytohemagglutinin. (d) Although all cultures producing interferon showed some degree of transformation (thymidine-³H incorporation into deoxyribonucleic acid), no direct correlation between the degree of phytohemagglutinin-induced lymphocyte transformation and the interferon titers was observed.     

In vitro interaction of scrapie agent and mouse peritoneal macrophages

Scrapie brain homogenate was mixed with mouse peritoneal macrophages in vitro. After 2 h of incubation at 37 degrees, a portion of the scrapie infectivity was associated with macrophages. In contrast, very little infectivity was associated with kidney cells that had been exposed to scrapie brain homogenate. After incubation of the scrapie brain homogenate-macrophage mixture at 4 degrees rather than 37 degrees, a reduced quantity of infectivity was associated with the macrophages. These result show that in vitro incubation, the scrapie agent was associated with macrophages, and the data suggest that phagocytic activity was involved.     

Differential kappa-opioid receptor expression on mouse lymphocytes at varying stages of maturation and on mouse macrophages after selective elicitation.

The combination of indirect immunofluorescent labeling and flow cytometry has proven to be a sensitive method for labeling of the kappa-opioid receptor on mouse thymocytes. In the present study, this labeling procedure was applied, along with phenotypic analysis, to mature immune cell populations to determine whether kappa-opioid receptor expression is present after immune cell maturation. Unfixed primary splenocytes from 6- to 8-week-old C57BL/6ByJ male mice were incubated with the fluorescein-containing, kappa-selective ligand fluorescein-conjugated 2-(3, 4-dichlorophenyl)-N-methyl-N-[1-(3-aminophenyl)-2-(1-pyrrolidinyl)eth yl]acetamide (FITC-AA). Amplification of FITC-AA binding to the kappa-opioid receptor was attained by adding a biotin-conjugated antifluorescein antibody, followed by extravidin-R-phycoerythrin. It has been shown previously that greater than 60% of immature thymocytes (CD4(+)/CD8(+)) demonstrated specific kappa-opioid receptor labeling. However, the present report shows that less than 25% of either T-helper or T-cytotoxic splenic lymphocytes expressed the kappa-opioid receptor. Likewise, only 16% of all splenic B lymphocytes were labeled for the kappa-opioid receptor. These findings demonstrate a decrease in kappa-opioid receptor expression on maturation of mouse lymphocytes. Interestingly, resident peritoneal macrophages showed a greater magnitude of specific receptor labeling, compared with either thymocytes or splenocytes, and approximately 50% of the resting Mphi expressed the kappa-opioid receptor. However, elicitation of Mphi with thioglycollate resulted in the complete loss of the expression of this receptor. Taken together, these findings demonstrate the diversity in the expression of the kappa-opioid receptor on immune cells at varying stages of differentiation, with preferential expression demonstrated by resident, peritoneal macrophages.     

A possible role of activated macrophages in the adoptive immunotherapy using CD4+ T lymphocytes.

Potent anti-tumor T lymphocytes with CD4+8- phenotype were obtained in peritoneal exudate cells by immunizing mice with irradiated tumor cells and OK-432. These effector cells were used in adoptive immunotherapy for tumor-bearing mice. Admixed administration of effector T cells with irradiated relevant tumor cells resulted in a marked enhancement of anti-tumor activity against local tumor and lymph node metastasis compared with the immunotherapy by effectors alone. The activating state of macrophages inoculated with viable tumor cells had much relevance with the implementation of immunotherapy. Innocent bystander lysis was not observed in this immunotherapy. Interleukin-2 given instead of stimulant tumor cells caused no enhancement, while interleukin-1 emerged stronger enhancement than stimulant tumor. In this case, activating state of macrophages had no relevance with the effectiveness of the therapy. These results suggest that macrophages in tumor play a role to secrete interleukin-1 to enhance anti-tumor activity of specific T cells.     

The specific binding of Listeria monocytogenes-immune T lymphocytes to macrophages. I. Quantitation and role of H-2 gene products

A system was developed to study the binding of Listeria monocytogenes-specific T cells to L. monocytogenes-pulsed macrophages as an analogue of the initial phase of T-cell activation: antigen recognition. Specific binding, demonstrable after a brief (1 h) contact, was quantitated by the depletion of L. monocytogenes-specific T-cell activity in the cells nonadherent to L. monocytogenes-pulsed macrophage monolayers. L. monocytogenes-specific T-cell function was measured by its ability to activate L. monocytogenes-pulsed macrophages, both to secrete a protein mitogenic for thymocytes and to effect nonspecific tumoricidal activity. These manifestations of T-cell function are known to be regulated by products of I region of the H-2 gene complex. Studies designed to determine the role of H-2 gene products in specific T-cell-macrophage binding have revealed the following. T cells bind specifically to syngeneic macrophages and poorly to allogeneic macrophages. The binding ability appears to map to the K end of the H-2 gene complex (K through I-E). At least two distinct populations of B6AF1 T cells with binding avidity for L. monocytogenes presented on parental macrophages can be identified. Finally, the binding of a given parental-reactive B6AF1 T-cell clone can be specifically inhibited by pretreatment of the antigen-pulsed B6AF1 binding macrophage with anti-H-2 (anti-Ia) antibodies reactive with the appropriate parental haplotype. These results strongly suggest that H-2 gene products play a direct role in mediating the specific binding of T cells to macrophages and imply that the antigen-dependent physical interaction between T cells and macrophages is the initial, and determining, event in some forms of H-2 gene control of immune reactivity.     

Macrophage-lymphocyte interaction. II. Antigen-mediated physical interactions between immune guinea pig lymph node lymphocytes and syngeneic macrophages

The effect of specific antigen on the development of physical interactions between lymph node lymphocytes (LNL) obtained from animals which had been immunized to that antigen and macrophages was examined. We found that the presence of antigen, either limited to the macrophage () or free in the medium, profoundly increased the degree of ) or free in the medium, profoundly increased the degree of Mphi-LNL interaction observed. This enhanced interaction was dependent on the coincidence in the cultures of Mphi bearing antigen and LNL from animals specifically immunized to that antigen. Although antigen-independent interactions developed equally well between syngeneic and allogeneic combinations of lymphocytes and macrophages, antigen mediated interactions required that macrophages and lymphocytes be syngeneic. Prolongation of antigen-mediated Mphi-LNL interactions resulted in the induction of LNL DNA synthesis, initially involving those lymphocytes physically associated with antigen-bearing Mphi. These studies are interpreted to indicate that physical interaction between immune lymphocytes and antigen-bearing Mphi represents a morphological correlate of the functional activation of immune lymphocytes. Further, it is suggested that the physical events involved in lymphocyte proliferation may proceed sequentially from antigen-independent reversible binding of lymphocytes by macrophages to prolonged antigen-stabilized interaction eventuating in the triggering of specifically immune lymphocytes.     

Rejection of syngeneic tumor cells by the interaction of Lyt-1+ T lymphocytes and macrophages

A high degree of cytostatic activity was detected in peritoneal exudate cells (PEC) from BALB/c mice immunized with mitomycin C-treated Meth A cells (MMC-MA) given intraperitoneally (i.p.) While this cytostatic activity was not detected in a nonadherent fraction of immune PEC, the addition of starch-induced PEC to the nonadherent immune cells led to a cytostatic activity. Treatment of these nonadherent cells with anti-Thy-1 X 2 or anti-Lyt-1 X 2 antibody plus complement abolished the restored cytostatic activity. Normal PEC were also rendered cytostatic after addition of the culture supernate of a nonadherent fraction of immune PEC mixed with MMC-MA. These findings suggest that macrophages are rendered cytostatic by immunization with tumor antigen and that these cytostatic macrophages evolve after activation of sensitized Lyt-1 positive T lymphocytes.     

The mechanism of the interaction between amiodarone and warfarin in humans.

Amiodarone decreased the total body clearance of both (R)- and (S)-warfarin in normal subjects but did not change volumes of distribution. Warfarin excretion products were quantified and clearance and formation clearance values calculated. Amiodarone and metabolites inhibited the reduction of (R)-warfarin to (R,S)-warfarin alcohol-1 and the oxidation of both (R)- and (S)-warfarin to phenolic metabolites. Inhibition of warfarin hydroxylation by amiodarone in human liver microsomes was compared with the in vivo results. In agreement, the in vitro data indicates that amiodarone is a general inhibitor of the cytochrome P450 catalyzed oxidation of both enantiomers of warfarin, but the metabolism of (S)-warfarin is more strongly inhibited than that of (R)-warfarin. These data suggest that the enhanced anticoagulant effect observed when amiodarone and warfarin are coadministered is attributable to inhibition of P4502C9, the isozyme of P-450 primarily responsible for the conversion of (S)-warfarin to its major metabolite, (S)-7-hydroxywarfarin.     

Restricted replication of lentiviruses. Visna viruses induce a unique interferon during interaction between lymphocytes and infected macrophages

Lentivirus infections are characterized by a persistent, restricted type of virus replication in tissues. Using sheep and goat lentiviruses, whose target cells in vivo are macrophages, we explored virus-host cell interactions to determine whether an interferon (IFN) is produced during virus replication in vivo which causes restricted replication. We show that the lentiviruses were incapable of inducing IFN directly in any infected cell, including macrophages and lymphocytes. However, after infection with these viruses, sheep and goat macrophages acquired a factor that triggered IFN production by T lymphocytes. Only sheep/goat lentiviruses were capable of inducing the factor and, although these viruses replicated productively in various cell cultures of the natural host animal, only infected macrophages developed the IFN-inducing factor. The factor was produced continuously and was strictly cell associated, requiring direct contact with lymphocytes. The lymphocytes responded with a single, sudden release of IFN beginning 7 h after cocultivation and reaching peak values at 48 h, after which they ceased production and became refractory. IFN production was not immunologically specific and did not require histocompatibility between donors of the two cell types. The IFN is a nonglycosylated protein of molecular weight 54,000-64,000, and is stable to heat and acid treatments. These findings identify a unique IFN and a new method for virus induction of IFN. The novel two-stage process of induction provides a mechanism for local amplification and continuity of production of IFN in vivo. This is compatible with infection in the animal whose lentivirus-induced pathologic lesions consist of accumulations of lymphocytes and infected macrophages in target tissues.     

The role of macrophages in the generation of T-helper cells. II. The genetic control of the macrophage-T-cell interaction for helper cell induction with soluble antigens

Helper cell induction to nonparticle antigens in vitro requires the cooperation of T cells and macrophages, but does not occur if the macrophages are allogenic. The reasons for this were investigated. Malfunction of allogenic macrophages was excluded by cultures with their syngenic T cells; suppressor cell induction was excluded by admixture experiments. Thus, T cells and macrophages only cooperated if they were genetically similar. The genetic locus (loci) involved was mapped. Using congenic lines differing only at the H-2 complex, the genetic control of T-macrophage interaction was localized in the H- 2 region. Mice with intra H-2 recombinants were used to map the T- macrophage interaction locus in the I-A region of the H-2 complex (formerly known as poly-D, L-ala-poly-L-lys. Recombinants were also used to exclude the presence of another T-macrophage locus either the K, I-B, or I-C, SS-Slp, or D regions of the H-2 complex. Genetic restrictions for T-macrophage interaction in helper cell induction was shown in mice of the H-2-k, d, b, q, s genotypes as well as in H-2 recombinants. The possible mechanisms and significance of this genetic restriction are discussed.     

The ingestion and digestion of human lactoferrin by mouse peritoneal macrophages and the transfer of its iron into ferritin

Human lactoferrin (Lf) labeled with 125I and/or 59Fe was found to be ingested in vitro by mouse peritoneal macrophages (MPM). The uptake measured after 15 h incubation reached a saturation point at a concentration of 200 microgram/ml in the culture medium, whatever was the iron content of Lf. In such conditions, the uptake of transferrin (Tf) used as a control was 10 times lower. At a concentration of 80 microgram/ml in the medium, one cell picked up about 0.7 X 10(6) molecules of Lf per hour, and 0.13 X 10(6) molecules of Tf per hour. Iron-saturated Lf disappeared from MPM with a half life of 14.5 h, whereas the halflife of iron-free Lf was 4.2 h. Concomitant with the intracellular digestion of Lf, the iron was transmitted to ferritin. These data provide additional support for the hypothesis that Lf plays a key role in iron turnover, especially at the level of the reticuloendothelial system where iron is recovered from the catabolism of erythrocytes.     

Contact sensitivity to azobenzenearsonate and its inhibition after interaction of sensitized cells with antigen-conjugated cells

Painting mice on the skin with the diazonium salt of p-arsanilic acid elicited two types of T cell activity. One was restricted by the I region of the major histocompatibility complex and was responsible for the transfer of azobenzenearsonate (ABA) sensitivity to naive mice. The other was H-2K restricted and could be demonstrated by its ability to interact specifically with ABA-coupled cells in vitro and to inhibit nonspecifically the transfer of sensitivity by cells sensitized either to ABA or to another antigen. Free antigen, or antibody directed against the cross-reactive idiotype on the anti-ABA antibodies of A/J mice, could inhibit the H-2K-restricted suppressive activity induced in the ABA immune A/J cells.     

The crucial role of macrophages in lymphangiogenesis

Lymphangiogenesis is associated with pathological processes such as the metastatic spread of carcinoma cells and organization of immunologically active lymphocytic infiltrates following organ transplantation. It has not yet been established whether expansion of the lymphatic vascular meshwork is driven by incorporation of progenitor cells or by local endothelial cell division. In this issue of the JCI, Maruyama et al. provide evidence that after mouse corneal transplant, CD11b+ macrophages infiltrate the corneal stroma and transdifferentiate into lymphatic endothelial cell clusters that join existing lymphatic vessels. In complementary in vitro experiments, murine peritoneal macrophages expressed lymphatic endothelial markers and formed vessel-like protrusions. These findings add yet another facet to the plasticity of macrophages, which are already known to transform from naive monocytes into VEGF-C-producing cells. Thus, macrophages support lymphangiogenesis in 2 different ways, either by transdifferentiating and directly incorporating into the endothelial layer or by stimulating division of preexistent local lymphatic endothelial cells.     

The role of alveolar macrophages in Pneumocystis carinii degradation and clearance from the lung.

Although studies indicate that alveolar macrophages participate in host defense against Pneumocystis carinii, their role in organism degradation and clearance from the lung has not yet been established. We, therefore, quantified the uptake and degradation of 35S-labeled P. carinii by cultured macrophages, demonstrating significant degradation of P. carinii over 6 h. We further evaluated the role of macrophages in elimination of P. carinii from the living host. Rats received either intratracheal PBS, liposomal PBS (L-PBS), or liposomal dichloromethylene diphosphonate (L-Cl2MDP), a preparation which leads to selective depletion of macrophages. Over 72 h, L-Cl2MDP-treated animals had loss of > 85% of their alveolar macrophages. In contrast, L-PBS-treated rats had cellular differentials identical to rats receiving PBS. Macrophage-depleted rats and controls were next inoculated with P. carinii and organism clearance was determined after 24 h. P. carinii elimination was evaluated with both cyst counts and an ELISA directed against glycoprotein A (gpA), the major antigen of P. carinii. Both assays indicated that macrophage-depleted rats had substantial inpairment of P. carinii clearance compared to L-PBS- or PBS-treated rats. These data provide the first direct evidence that macrophages mediate elimination of P. carinii from the living host.     

Immunomodulatory role of PPAR-gamma in alveolar macrophages.

The lung is constantly exposed to inhaled pathogens and toxins yet totally dependent on the integrity of a delicate alveolar-capillary interface for its function. Much of the balance between protection and collateral damage rests on the alveolar macrophage, which not only phagocytoses inhaled particles but also modulates the activity of both innate and acquired immune systems to limit unnecessary or exuberant inflammation. In its resting state, the alveolar macrophage secretes anti-inflammatory mediators while limiting antigen presentation to the adaptive immune system. The alveolar macrophage's state of activation is regulated by a variety of factors, including the activity of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR-gamma). Peroxisome proliferator-activated receptor gamma agonists reduce the ability of inflammatory stimuli to activate the alveolar macrophage while simultaneously stimulating phagocytosis of both opsonized and unopsonized particles, via the Fcgamma and CD36 receptors, respectively. All known endogenous PPAR-gamma ligands are fatty acid derivatives, and macrophage-specific knockout of the enzyme that converts esterified fatty acids to free fatty acids results in severe lung inflammation. Peroxisome proliferator-activated receptor gamma expression is reduced in alveolar macrophages from patients with pulmonary sarcoidosis and alveolar proteinosis, suggesting that the deficiency may play a role in pathogenesis of these diseases. In summary, these observations point to PPAR-gamma in the context of the alveolar macrophage as a crucial factor in limiting excessive and possibly injurious inflammation in the lung.