The induction of lymphocytes with the capacity to render macrophages cytotoxic in an allogeneic murine system.

Sensitized spleen and peripheral lymph node lymphocytes were tested after different types of immunization with allogeneic tumour cells for their capacity to induce macrophage cytotoxicity in vitro. The macrophages were rendered cytotoxic either by direct contact with lymphocytes and tumour cells (activation of macrophages) or by a factor (macrophage arming factor, MAF), released by the sensitized lymphocytes incubated with tumour cells (arming of macrophages). Both types of reactions are T-cell dependent. Macrophage activation is a more sensitive way to detect lymphocytes with the capacity to render macrophages cytotoxic than arming of macrophages. The route of immunization subcutaneously (s.c.) or intraperitoneally (i.p.) with allogeneic cells did not influence the induction of lymphocytes with the capacity to render macrophages cytotoxic. However, the tumour cells had to be intact as disrupted cells (suspended in Freund's complete adjuvant, FCA) did not induce macrophages activating lymphocytes. The adjuvant dimethyl dioctadecyl ammonium bromide (DDA) did not increase the lymphocyte response. Intact allogeneic tumour cells were needed in vitro when used for secondary antigenic stimulation. This secondary stimulation was independent of antigen presentation by macrophages. This suggests that also in vivo the primary response is independent of macrophage antigen presentation. Delayed-type hypersensitivity and antibody responses against the allogeneic tumour cells were comparable after s.c. and i.p. immunization and after immunization with FCA and DDA.     

Differences in the induction of macrophage cytotoxicity by the specific T lymphocyte factor, specific macrophage arming factor (SMAF), and the lymphokine, macrophage activating factor (MAF)

Specific T cell factors, such as specific macrophage arming factor (SMAF), are involved in the initiation of the immune response. Induction of SMAF-producing T lymphocytes in vivo and of SMAF production by T lymphocytes in vitro is dependent on the presence of intact tumor cells, and is independent of antigen presentation by macrophages. SMAF renders peritoneal macrophages cytotoxic for tumor cells. The armed peritoneal macrophages expressed a specific cytotoxicity. However, antigen-presenting cells can trigger lymphokine-producing T lymphocytes. These T lymphocytes produce lymphokines (e.g. macrophage activating factor (MAF] that activate macrophages. The MAF-activated macrophages express a non-specific tumoricidal activity. In the present study, we investigated the difference in the induction of macrophage cytotoxicity by SMAF and MAF. The following differences were found: 1) SMAF renders peritoneal resident macrophages cytotoxic, whereas MAF could only render peritoneal exudate macrophages cytotoxic. 2) SMAF requires only a 4-h incubation with macrophages, whereas MAF activates macrophages optimally after 12 h. 3) SMAF-armed macrophages recognize only the specific target cell(s), and thus, the cytotoxicity is specific in its expression. MAF activated macrophages were non-specifically cytotoxic. 4) Lipopolysaccharide (LPS) in the culture medium did not enhance the cytotoxicity of SMAF-armed macrophages. In contrast, MAF induced tumoricidal activity was enhanced by adding LPS to the culture medium. 5) After adsorption chromatography with anti-murine interferon-gamma (IFN-gamma), the arming capacity of SMAF supernatant was not reduced, whereas the activating capacity of the MAF supernatant was significantly reduced or abrogated. After immunization of mice with allogeneic tumor cells, SMAF-producing lymphocytes were detected in the draining lymph nodes already 4 days after immunization and up to 12 days. Lymphocytes with the capacity to produce MAF were present in the draining lymph nodes 14-24 days after immunization. Our data indicate that the T cell factors SMAF and MAF can both render macrophages cytotoxic, but act in a different way and during different stages of the cellular immune response against allogeneic tumor cells.     

Interferon-induced enhancement of macrophage-mediated tumor cytolysis and its difference from activation by lymphokines

Mouse peritoneal macrophages expressed increased cytolytic activity against tumor cells upon in vitro exposure to partially purified L cell interferon (IFN-beta). In contrast, treatment with human IFN or with mock IFN preparations did not enhance macrophage tumoricidal capacity. Macrophage activation by IFN was optimal with long exposure times and high IFN concentrations. Treatment with polymyxin B sulfate did not affect IFN-induced macrophage cytotoxicity, thus excluding the possibility that bacterial lipopolysaccharide contaminants were responsible for macrophage activation. Conversely, treatment with a highly specific anti-IFN antiserum completely abolished IFN effect on macrophages, but had no effect on lymphokine-induced cytolysis. IFN and the lymphokine macrophage-activating factor (MAF) were compared for their ability to provide the sequence of activation signals to macrophages from the normal responder C3H/HeN mice and from C3H/HeJ mice, which are defective for several macrophage responses. Like MAF, IFN was incapable of inducing tumoricidal activity in C3H/HeJ macrophages. However, whereas MAF provided the first "priming" signal to macrophages of both strains, IFN acted as first signal only for C3H/HeN macrophages, being inactive for cells of the defective C3H/HeJ strain. Furthermore, IFN was not capable of providing the second "expression" signal to "primed" macrophages. These data suggest two different macrophage activation pathways for IFN and MAF.     

Mechanisms by which activated normal macrophages destroy syngeneic rat tumour cells in vitro: Cytokinetics, non-involvement of T lymphocytes, and effect of metabolic inhibitors

Analysis of the kinetics of the in vitro interaction between nonimmune activated macrophages and syngeneic tumour cells (induced in inbred DA rats by polyoma virus, dimethylbenzanthracene or methylcholanthrene) has shown that a distinctive series of events can be clearly separated. The earliest consequence of interaction detectable by objective means is a marked decrease in tumour cell proliferation as reflected in the reduction of the capacity of tumour cells to incorporate DNA precursors such as [³H]thymidine. By 3—4 hours, the cytostatic effect is strongly marked, and is essentially established after 12 hours of interaction. Shrinkage, agglutination and decrease in the number of tumour cells as examples of the morphological consequences of cytostatic target cell damage accomplished by activated macrophages are rarely perceptible before 10–12 hours although the tumour cells have completely disappeared after 24–48 hours.

Under the experimental conditions employed, occasional tumour cells escaped interaction with activated macrophages. The fact that such recovery of targets was fully reversed by adding further activated macrophages indicates that tumour cell revival reflects a decrease in macrophage effector functions during prolonged incubation. The possibility that some tumour cells might be resistant to macrophage effects thus seems excluded. Activated macrophages from normal and from congenitally athymic nude mice are equally effective in reducing tumour cell proliferation. Thus T lymphocytes and/or their soluble mediators are not essential for the macrophage function under investigation.

Pretreatment of activated macrophages with an extensive array of metabolic inhibitors and agents known to affect distinct cellular functions yields much data but does not yet permit a simple comprehensive interpretation. However, the findings are compatible with the thesis that macrophage activation is accompanied by the de novo or enhanced synthesis of the cytostatic principle. Once possessed of this mechanism, other basic functional capacities of macrophages, such as membrane activity, movement or endocytosis are no longer essential for the mediation of the cytostatic effects.

     

Growth inhibition of Marek's disease T-lymphoblastoid cell lines by chicken bone-marrow-derived macrophages activated in vitro

Studies have been performed on the induction of cytostatic activity of cultured chicken bone-marrow-derived macrophages. Cultured macrophages were exposed to supernatants of ConA-stimulated spleen cell cultures (lymphokines), lipopolysaccharide (LPS), ConA or infectious bursal disease virus (IBDV). Cytostatic activity of macrophages was examined by testing their growth-inhibiting effect on T-lymphoblastoid Marek's disease cell lines RP-1, HP-2 and MSB-1 as target cells. Lymphokines, LPS and IBDV caused considerable enlargement of adherent macrophages. Results of cytostasis assays suggested that morphological signs of macrophage activation were not correlated with the cytostatic potency of activated macrophages. Lymphokine-activated macrophages caused at least an 80% growth inhibition of target cells, whereas LPS, ConA or IBDV-activated macrophages exhibited only a marginal cytostatic effect in the range of 20%. Attempts to detect soluble cytostatic factors in supernatants of activated macrophage cultures failed or had equivocal results. Untreated macrophages were rarely cytostatic at an effector:target cell ratio of I:1 to 4:1, and they stimulated RP-1 cell growth if these cells were seeded at suboptimal concentrations. The results suggest that the suppressor activity of macrophages from Marek's disease virus-infected chickens, as demonstrated by other authors in vitro, is probably a result of nonspecific immunomodulation in vivo where lymphokines may also play a major role.     

Comparison of Various Assays to Quantitate Macrophage Activation by Biological Response Modifiers

Macrophages treated with various compounds that enhance host antitumor resistance exhibit measurable changes in metabolism, function, and surface antigens. In this study, murine peptone-induced peritoneal macrophages were stimulated in vitro by bacterial lipopolysaccharide (LPS), muramyl dipFtide (MDP), and polyI-polyC. They were subsequently compared in their ability to release superoxide and act as tumoristatic and tumoricidal effectcr cells. Superoxide generation was assayed by the reduction of ferricytochrome C. All three compounds failed to induce significant O_&2bar; release, unless the cells were also treated with phorbol myristate acetate (PMA). MDP was most active in potentiating the PMA response. In the tumor growth inhibition assay, cytostatic activity was comparable for all three compounds and did not exceed 32 percent. The combination of subthreshold levels of these compounds and hybridoma-derived MAF acted synergistically to induce potent cytostatic activity. In the chromium release assay, LPS and polyI-polyC rendered macrophages cytolytic for P815 target cells at concentrations ≥ 1 μglml. In contrast, significant cytolysis was observed with MDP only at 100 μglml. Defining precisely the effect of various biological response modifiers on several parameters of macrophage function may facilitate use of these agents in cancer therapy.     

Stimulation by a bacterial extract (Broncho-Vaxom) of the metabolic and functional activities of murine macrophages

Peritoneal and bone marrow-derived macrophages of the C57BL/6 and DBA/2 mouse strains were exposed in vitro to increasing concentrations of the bacterial lysate Broncho-Vaxom (BV), in the presence or absence of macrophage-activating factor (MAF)-rich media. Two metabolic pathways and two functional activities of the macrophages were studied. First, oxidative metabolism was found to increase sharply in macrophages incubated with BV, as measured by the catabolism of glucose via the hexose monophosphate shunt pathway, and by the production of the superoxide anion (O2-). Both effects were further increased by co-stimulation of macrophages with MAF. Second, exposure to BV together with MAF (or with recombinant murine interferon-gamma) led to acquisition by macrophages of the capacity to destroy the intracellular parasite Leishmania enriettii; such activated macrophages were also lytic towards P815 mastocytoma indicator target cells. These cytotoxic properties failed to develop in the absence of MAF. The BV-dependent increase in metabolic and functional activities was of the same magnitude as that induced by incubation of macrophages with 10 ng/ml of bacterial lipopolysaccharide (LPS). Residual contamination of BV by endotoxin was however much lower. In addition, polymyxin B, a LPS inhibitor, blocked the effect of LPS without significantly affecting macrophage stimulation by BV. These experiments indicate that BV can markedly stimulate macrophage metabolic and functional parameters that are important for host defense against pathogens and tumors.     

Cellular requirements for lymphokine secretion by rainbow trout Salmo gairdneri leucocytes

The ability of different populations of rainbow trout blood leucocytes to produce MAF following stimulation with Con A/PMA was assessed by the amount of NBT reduction in target macrophages. The effect of varying lymphocyte or macrophage number on MAF production in the presence of a constant number of macrophages or lymphocytes respectively, showed that in both cases MAF activity initially increased with increasing cell number and then plateaued. Macrophages alone did not produce MAF whereas some MAF activity was produced by macrophage-depleted lymphocytes, although significantly lower than in the presence of macrophages. Separation of leucocytes into sIg- and sIg+ cells by panning showed that only sIg- lymphocytes could produce MAF and that macrophages were necessary as accessory cells. These results support the contention that fish lymphocytes can be divided into sIg- T cells and sIg+ B cells.     

Intracellular activation of human and rodent macrophages by human lymphokines encapsulated in liposomes

Cell-free culture supernatant fluids rich in macrophage-activating factor (MAF) activity were obtained from mitogen-stimulated human peripheral blood mononuclear leukocytes (MNL). The MAF preparations were incubated with human peripheral blood monocytes, rat alveolar macrophages (AM), and mouse peritoneal exudate macrophages (PEM). Only human monocytes were rendered tumorilytic against the human A375 melanoma cells, whereas rat AM or mouse PEM were not activated to lyse their respective syngeneic tumor targets. In contrast, once entrapped in multilamellar liposomes, the human MAF activated the human and rodent macrophages to become tumoricidal. The MAF activity was not due to contamination with endotoxins nor to the presence of gamma interferon. These data suggest that in contrast to macrophage surface receptors, which are species specific, the intracellular target sites for human MAF may cross species barriers.     

Lysis of herpesvirus-infected cells by macrophages activated with free or liposome-encapsulated lymphokine produced by a murine T cell hybridoma.

Thioglycolate-induced mouse peritoneal macrophages were activated in vitro by the lymphokine designated macrophage-activating factor (MAF) produced by a murine T cell hybridoma to lyse herpes simplex virus type 2 (HSV-2)-infected murine target cells. Comparison of uninfected BALB/c 10E2 cells with HSV-2-infected 10E2 cells showed that macrophages activated with MAF selectively destroyed HSV-2-infected cells and left uninfected cells unharmed, as measured by an 18-h 51Cr-release assay. In contrast, macrophages treated with medium were as efficient as MAF-activated macrophages in suppressing the production of HSV-2 from virus-infected cells. These findings suggest that macrophages must attain an activated state to lyse HSV-2-infected cells. Finally, incubation of macrophages with liposomes containing MAF was shown to be a highly efficient method for activation of macrophages against HSV-2 infected cells. The ability to selectively destroy herpesvirus-infected cells in vitro by macrophages activated with liposome-encapsulated MAF suggests that the therapeutic efficacy of this treatment in vivo should be evaluated.     

Lymphocyte-induced macrophage cytotoxicity. III. Induction of specific macrophage cytotoxicity is independent of lipopolysaccharide

The induction of specific macrophage cytotoxicity by allo-sensitized T cells in vitro is shown to be independent of the presence of lipopolysaccharide (LPS). This contrasts with the induction of macrophage cytotoxicity by Macrophage Activating Factor (MAF). The specific macrophage cytotoxicity could be induced in LPS-free medium (less than l ng/ml). Addition of LPS to the macrophages did not increase the cytotoxicity. Addition of LPS-binding polymyxin B to the macrophages before and during the induction of cytotoxicity did not reduce the specific macrophage cytotoxicity. Macrophages obtained from the LPS-unresponsive mouse strain C3H/HeJ were rendered cytotoxic by the allo-sensitized lymphocytes to the same extent as the macrophages from LPS-responsive C3HeB/Fe and C57BL mice. This indicates that the induction of macrophage cytotoxicity by MAF is different from the induction of specific macrophage cytotoxicity by Specific Macrophage Arming Factor (SMAF).     

Lead inhibits intracellular killing of Leishmania parasites and extracellular cytolysis of target cells by macrophages exposed to macrophage activating factor

Activation of Leishmania enriettii-infected mouse macrophages in vitro by treatment with macrophage activating factor (MAF)-rich media supplemented with lipopolysaccharide (LPS) leads to rapid killing of the microorganism. When exposed to MAF + LPS in the presence of 30-100 microM lead acetate, however, macrophages failed to destroy the parasites. This effect was not due to lead toxicity for macrophages. Decreased microbicidal activity correlated with depressed respiratory burst as determined by measurements of glucose oxidation through the hexose monophosphate shunt (HMPS). Lead had little effect on intracellular parasite killing induced by exposure of macrophages to the electron carrier methylene blue; HMPS in such cells was similarly little affected, indicating that chemical triggering of this pathway bypassed the lead-imposed blockade. Lead also abolished macrophage activation measured by the lysis of tumor target cells in vitro. The metal failed, however, to interfere with target-cell lysis by macrophages activated in lead-free medium, suggesting that lead inhibited the acquisition of the activated state rather than the functional expression of such state. Lead did not prevent the binding of radiolabelled interferon-gamma to macrophages; it did, however, slow down receptor turnover and degradation of bound interferon. Lead also inhibited the LPS-triggered cytotoxicity in macrophages previously exposed to interferon-gamma in lead-free medium, suggesting that depressed intracellular killing might result from an effect on both the priming (interferon or MAF-dependent) and the triggering (LPS-dependent) steps of activation.     

Tumor necrosis factor and macrophage activation are important in clearance of Nocardia brasiliensis from the livers and spleens of mice.

The roles of tumor necrosis factor (TNF) and macrophage activation in clearance of Nocardia brasiliensis from BALB/c mouse livers and spleens were evaluated. TNF activity was detectable in sera from animals at all stages of infection. Treatment of infected mice with an antiserum against TNF significantly enhanced the experimental infection as judged by enumeration of CFU in the spleens and livers of infected mice. In another set of experiments, a population of activated macrophages from the peritoneal cavities of N. brasiliensis-infected mice was studied by using a cytostatic assay. The observed cytotoxic activity of these activated macrophages against L929 cells was mediated by TNF, since this activity was inhibited by anti-TNF antiserum treatment. The level of TNF activity generated in vitro in the presence of lipopolysaccharide (LPS) by peritoneal macrophages from infected mice was higher than that of adherent peritoneal cells obtained from normal mice after challenge with LPS. When the nocardiacidal activity of peritoneal cells from N. brasiliensis-infected mice was estimated in vitro, a significant decrease in the number of CFU recovered was observed. Moreover, nocardiacidal activity of peritoneal cells obtained from N. brasiliensis-infected mice previously treated with anti-TNF antiserum was significantly reduced compared with the activity of cells obtained from infected mice previously treated with normal rabbit serum and that of cells from uninfected mice. These data suggest a role for TNF in resistance to N. brasiliensis infection.     

Transforming growth factor-beta 1 inhibits activation of macrophage cell line RAW 264.7 for cell killing.

Transforming growth factor beta-1 (TGF-beta) is a multi-potent immunoregulatory peptide that has effects on numerous cell types. Here we report that human TGF-beta inhibits the activation of the macrophage cell line RAW 264.7 for killing of the L1210 tumour cell line. RAW 264.7 cells, like normal macrophages, require sequential interaction with priming and triggering stimuli for full activation of cytolytic activity. TGF-beta inhibits this cytotoxicity in a dose-dependent manner at both the priming and the triggering stage. Addition of as little as 1 ng/ml TGF-beta when added with either the priming signal, recombinant interferon-gamma (IFN-gamma), or the triggering signal, bacterial lipopolysaccharide (LPS), completely abrogated tumouricidal activity. Incubation with TGF-beta also inhibited the morphological changes normally observed in activated RAW 264.7 cells. However, TGF-beta was unable to inhibit the cytotoxic activity of RAW 264.7 cells against the target cell line WEHI 164, which is sensitive to tumour necrosis factor. In contrast to the effects on cytotoxic activity, the cytostatic activity of activated RAW 264.7 cells was not inhibited by TGF-beta at doses of up to 5 ng/ml. In addition, pretreatment of the L1210 target cells with TGF-beta made them refractory to both the cytostatic and cytotoxic effects of RAW 264.7 cells. These data suggest that TGF-beta may be an important mediator in the regulation of macrophage tumouricidal activity.     

Leukotriene D4 and indomethacin enhance additively the macrophage cytostatic activity in vitro towards MOPC-315 tumour cells

Leukotriene C4, the precursor of leukotriene D4 (LTD4), was earlier shown to activate macrophages, as indicated by lysosomal enzyme discharge. This event is limited by LT-induced secretion of PGE2, which inhibits the functions of these cells. The circumstance that activated macrophages may exert cytostatic activity towards tumour cells prompted us to study the effect of LTD4 and indomethacin on the cytostatic activity of macrophages as expressed by inhibition of [3H]thymidine incorporation in MOPC-315 plasmacytoma tumour cells. Decrease in incorporation, caused by separate administration of the two substances, was additively enhanced upon exposure of the co-culture of macrophages and tumour cells to the two substances simultaneously. Tumour cells alone are not affected by the substances, but the low rate of thymidine incorporation by macrophages alone was increased by LTD4 in the presence of indomethacin. Decrease of thymidine incorporation into tumour cells co-cultured with macrophages is attributed to a macrophage-mediated, rather than to a direct, influence of LTD4 and indomethacin on tumour cells. An increase in macrophage-mediated cytostasis by LTD4 requires the inhibition of endogenous PGE2 production in macrophages.     

Endogenous lipoxygenase metabolites mediate A23187 induced macrophage cytostasis towards P815 tumor cellsin vitro

The calcium ionophore A23187 stimulated cytostatic activity of peritoneal macrophages towards P815 tumor cells in coculture served as a model for macrophage activation. A macrophage enriched preparation, separated on the basis of cell size in a discontinuous fetal calf serum gradient column, expressed cytostatic activity when stimulated by A23187. This was inhibited dose-dependently, by AA-861 but not by nordihydroguaiaretic acid (NDGA). AA-861 inhibited the 5-lipoxygenase specifically, NDGA inhibited both lipoxygenase- and cyclooxygenase activity: The ratio cyclooxygenase/lipoxygenase products increased with AA-861 but not with NDGA. These results show that lipoxygenase products are necessary for expression of cytostatic activity of these arachidonic acid metabolite producing macrophages and that the ratio of cyclooxygenase/lipoxygenase metabolites plays an important role in macrophage activation.     

Deglycosylation of Serum Vitamin D3-Binding Protein by α-N-Acetylgalactosaminidase Detected in the Plasma of Patients with Systemic Lupus Erythematosus

A serum glycoprotein, Gc protein (vitamin D₃-binding protein), can be converted by β-galactosidase of B cells and sialidase of T cells to a potent macrophage-activating factor (MAF), a protein withN-acetylgalactosamine as the remaining sugar moiety. Thus, Gc protein is the precursor for MAF. Treatment of Gc protein with immobilized β-galactosidase and sialidase generates a remarkably high titered macrophage-activating factor (GcMAF). When peripheral blood monocytes/macrophages (designated macrophages) of 33 systemic lupus erythematosus patients were incubated with GcMAF (100 pg/ml), the macrophages of all patients were activated as determined by superoxide generation. However, the precursor activity of patient plasma Gc protein was lost or reduced in these patients. Loss of the precursor activity was the result of deglycosylation of plasma Gc protein by α-N-acetylgalactosaminidase activity found in the patient plasma. Levels of plasma α-N-acetylgalactosaminidase activity in individual patients had an inverse correlation with the MAF precursor activity of their plasma Gc protein. Deglycosylated Gc protein cannot be converted to macrophage-activating factor. The resulting defect in macrophage activation may lead to an inability to clear pathogenic immune complexes. Thus, elevated plasma α-N-acetylgalactosaminidase activity resulting in the loss of MAF precursor activity and reduced macrophage activity may play a role in the pathogenesis of systemic lupus erythematosus.     

Macrophage Involvement in the Antitumor Activity of A Synthetic Acyltripeptide (Fk-565) Against Experimental Lung Carcinoma Metastases

Resident peritoneal macrophages can be activated to develop cytotoxicity against P815 mastocytoma target cells following incubation in vitro with either D-lactoyl-L-alanyl-γ-D-glutamyl-(L)-meso-diaminopimelyl-(L)-glycine (FK-156), heptanoy1-γ-D-glutamyl-(L)-meso-diaminopimelyl-(D)-alanine (FK-565), or bacterial lipopolysaccharide (LPS) at a minimum concentration of 10 µg/ml. Subthreshold levels of hybridoma-derived macrophage activating factor (MAF) markedly potentiated this activity. In an experimental metastasis model, subcutaneous or intraperitoneal treatment with FK-565 (1 to 10 mg/kg) markedly inhibited lung metastasis formation when administered 2-4 days prior to i.v. tumor inoculation. Moreover, this protective activity could be abrogated by the selective macrophage inhibitor, 2-chloroadenosine, suggesting that activated macrophage were responsible for the antimetastatic activity of FK-565.     

Alveolar macrophages. III. Studies on the mechanisms of inhibition of T-cell proliferation.

Unlike macrophages from the peritoneal cavity or the peripheral blood, rat alveolar macrophages actively inhibit mitogen-induced T-cell proliferation. Studies on the characteristics of this inhibitory activity revealed the following: the macrophages must be live, but mitomycin C does not block their activity; they must be added to lymphocyte cultures soon after the initiation of the cultures, and prolonged pre-incubation of macrophages in vitro diminishes their cytostatic activity; suppressive activity is most obvious in cultures of rapidly proliferating lymphocytes, and the lymphocytes themselves may be syngeneic or allogeneic; the suppressive activity may be duplicated by a low molecular-weight dialysable component of macrophage culture supernatants, and suppression of RNA synthesis is as readily demonstrable as suppression of DNA synthesis in target cells; the cytostatic effects of alveolar macrophages in lymphocyte cultures do not appear to result from target cell destruction. Studies involving repeated endobronchial lavage of rats revealed the presence of two alveolar macrophage subpopulations, one (weakly adherent in vivo) supportive to T-lymphocyte proliferation, and another (strongly adherent) strongly inhibitory; the latter population comprises the majority of alveolar macrophages in normal rats.