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In vitro culture of murine resident peritoneal macrophages with lymphokine (LK)-rich leukocyte culture fluids induces enhanced microbicidal activity against amastigotes of the protozoan parasite Leishmania tropica. Macrophages infected with Leishmania and treated with LKs after infection acquire the capacity to kill the intracellular parasite within 72 h. When compared with control macrophage cultures treated with medium lacking LKs, 80 to 90% fewer macrophages treated with LKs contained amastigotes. In experiments designed to test liposome delivery of LKs to infected macrophages, addition of multilamellar liposomes composed of phosphatidylcholine and phosphatidylserine (molar ratio, 7:3) completely abrogated LK-induced microbicidal activity. Liposomes containing only phosphatidylcholine were not inhibitory. Inhibition of LK activity by the liposomes occurred regardless of whether the liposomes contained LKs. Liposomal inhibition of activated macrophage effector activity was limited to intracellular killing; LK-induced macrophage extracellular cytolysis (i.e., tumor cytotoxicity) was not affected by liposome treatment. These data indicate that elucidation of the effects of liposome composition on acquired host defense mechanisms may be useful for the design of drug delivery systems that allow expression or augmentation of immunologically induced mechanisms for the intracellular destruction of infectious agents.

The mononuclear cell chemotaxis assay was adapted for use with normal mouse peritoneal cells. Mouse peritoneal macrophages responded well to endotoxinactivated mouse serum and to chemotactic factors produced by mouse spleen cell cultures stimulated with mitogens or specific antigen. The assay was quantitative, reproducible, and applicable to several mouse strains.

Tumor-specific antigens of cells of the diethylnitrosamine-induced hepatomas in strain-2 guinea pigs were extracted with 3 M KCl. Immunization of normal animals with the extracted tumor antigens in adjuvant protected them against a subsequent challenge with viable tumor cells. Extracted tumor-specific antigens were less effective immunogens than viable tumor cells for both of two antigenically distinct lines.

Macrophage activation factor (MAF) was isolated from PPD-stimulated, BCG-immune mouse spleen cell culture fluids. In nine gel filtration runs on Sephadex G-100 or G-200, MAF was eluted in a single peak corresponding to a MW of 55,000 ± 1600. Recovery of activity was about 65%. Since the relative concentration curve of eluted MAF was wider than that of a single protein, MAF activity may be due to more than one protein with similar molecular weights. This possibility is strengthened by a broad elution range on DEAE cellulose chromatography, from a specific conductance of 3.5 to 8.5 mmho/cm, at pH 7.9. MAF was labile at both pH 4 and 10, and was destroyed by proteolytic enzymes. Eighty percent was destroyed by heating at 56 °C for 30 min. In affinity chromatography experiments, MAF did not bind to Con-A Sepharose; but it was bound to insolubilized Cibacron-blue and was eluted by an increase in ionic strength.

Non-tumorigenic and ‘spontaneously’ transformed tumorigenic paired cell lines derived from mouse, rat, or hamster embryonic tissues were examined for their susceptibility to killing by mouse peritoneal macrophages activated in vivo by infection with Mycobacterium bovis, strain Bacillus Calmette-Guerin (BCG). Susceptibility to killing was measured by the extent of clearing in a confluent layer of target cells overlying an area of adherent macrophages or by the release of 3H counts from target cells prelabelled with [3H]-thymidine. Only tumorigenic lines showed complete clearing by activated macrophages; the reaction occurred regardless of species differences between macrophages and target cells. The degree of clearing increased with increasing ratio of macrophages to target cells. Normal macrophages from uninfected mice did not produce a clearing reaction. The macrophage reaction discriminated between non-tumorigenic and tumorigenic cells with short tumor latent periods (

Peritoneal macrophages recovered from mice infected with intracellular parasites such as Mycobacterium bovis, strain BCG,Toxoplasma gondii or Listeria monocitogenes exert non specific cytotoxic activity against tumor cells in vitro (1 ,2).Macrophage cytotoxicity is selectively expressed against neoplastic cells.Tumor cells,derived from viral or chemical carcinogen-induced or spontaneous syngeneic and non syngeneic tumors,were found to be quantitatively more susceptible to macrophage cytophatic effects than were non neoplastic cells.Tumor cell killing requires cell to cell contact;soluble cytotoxic factors released by macrophages or by macrophage-tumor cell interactions have not been described.A cinemicrographic analysis revealed that macrophage-tumor cell interactions are characterized by repeated and relatively short (2 hr.) contacts inducing permanent tumor cytostasis and degenerative morphological changes (3).The development of a cytotoxicity assay based on radioisotope release facilitated quantitative analysis of macrophage tumoricidal activity (4).Tumor cell death was evaluated as release of tritiated thymidine from prelabeled target cells into supernatant culture fluids BCG-activated macrophages induced significant release of 3HTdR from tumor cells by 24 hours;however,optimal measurement of tumor cytotoxicity (50% total counts) was observed after 48–72 hours.Macrophages from uninfected mice or from oil-, starch- or thioglycollate induced peritoneal exudates had little effect on labeled tumor mono-layers.

Macrophages from C3H/HeN mice treated in vivo with Mycobacterium bovis, strain BCG or in vitro with supernatants from antigen-stimulated leukocyte cultures (lymphokines) were cytotoxic to tumor cells in vitro. Macrophage tumoricidal activity, however, did not develop after identical in vivo or in vitro treatment of cells from endotoxin (LPS)-unresponsive C3H/HeJ mice. Increasing time of macrophage incubation in lymphokines, concentration of lymphokines or number of lymphokine-treated macrophages added to tumor cells did not evoke tumoricidal activity. Similarly, varying time of macrophage collection after BCG infection, numbers of BCG organisms in the infectious inoculum or numbers of macrophages from BCG-infected mice added to tumor cells also did not evoke cytotoxic activity. The tumorical defect of macrophages from C3H/HeJ mice appeared highly selective: inflammatory responses to BCG infection in C3H/HeN and C3H/HeJ mice were indistinguishable; moreover, no difference was detected between these strains in production of macrophage activation factors. Thus, despite normal inflammatory reactions, normal production of lymphokines and extensive experimental manipulation of single activation stimuli, macrophages from C3H/HeJ mice did not express tumoricidal activity in vitro. Nevertheless, macrophages from C3H/HeJ mice could develop tumoricidal activity under appropriate conditions. Macrophages from in vivo immune reactions (BCG infection, Con A injection), but not from irritant-induced peritoneal exudates, developed full cytotoxic activity after exposure to certain in vitro stimuli. These stimuli include microgram/ml concentrations of LPS and certain factors in lymphokine supernatants or supernatants from a T-cell lymphoma line. The effect of LPS but not that of lymphokines or lymphoma culture supernatants was abrogated by polymyxin B. These data suggest that the ultimate expression of macrophage cytotoxicity may depend upon certain signals from the milieu of immune reactions. These expression signals, derived from bacterial organisms or from soluble mediators of immune responses, provide the necessary and final stimulus for macrophage activation. The tumoricidal defect of macrophages from C3H/HeJ mice reflects a genetic inability of these cells to respond to environmental expression signals.

A cloned variant of the EL-4 murine T-cell line treated with phorbol myristate acetate (PMA) releases a factor that activates macrophages for nonspecific tumor cytotoxicity. This macrophage activation factor (MAF) is both physicochemically (Mr 25,000; pH 2 stable) and biologically different from interferon-gamma (IFN-gamma). However, EL-4 MAF may represent a breakdown product or otherwise altered fragment of IFN-gamma. We examined this possibility with a unique pair of hamster monoclonal antibodies against different epitopes of murine IFN-gamma. Both antibodies inhibited IFN-gamma-induced fibroblast antiviral activity; H21 but not H1 antibody also inhibited lymphokine (LK)-induced macrophage-mediated tumor cytotoxicity. Neither antibody, however, had any effect on the EL-4 MAF throughout a broad dose response. Moreover, passage through a H21 immunoaffinity chromatography column or addition of staphylococcal protein A and antibody completely inhibited LK-induced macrophage tumoricidal activity but did not affect the activity in EL-4 MAF. Identical effects in both fluid and solid phase were observed with polyclonal rabbit antisera to murine IFN-gamma. Results with all of these antibodies strongly suggest that the EL-4 MAF and murine IFN-gamma are antigenically distinct.

Analysis of Listeria infection in mice (Mackaness, 1962) clearly defined host-cell interactions that occur during resolution of this infectious disease: T lymphocytes develop specific immune responses to the invading microorganism, but macrophages are the proximate effector cells that eliminate the pathogen. Parenthetically, these early studies also showed that macrophages recovered from infected animals can destroy microorganisms antigenically unrelated to Listeria. In fact, these “activated” macrophages develop the capacity to kill a variety of intracellular and extracellular targets. The observation that mediators released by specifically sensitized T lymphocytes confer enhanced but entirely nonspecific killing properties on macrophages focused attention on a new group of important immunoregulatory mechanisms. Initial attempts to demonstrate activation of macrophages for enhanced microbicidal activities in vitro,however, were frustrated by a variety of technical difficulties inherent in these facultative intracellular bacteria: adherence to macrophage surfaces, extracellular replication, and short generation times obscured important details of intracellular events. Only recently have we and several other groups developed techniques to analyze macrophage antimicrobial activities in vitro (Anderson and Remington, 1974; Hinrichs and Jerrels, 1976; Nogueira and Cohn, 1978; Mauel et al.,1978; Nacy and Meltzer, 1979; Nacy et al., 1981b). The key to success of these later studies is the target cell: obligate intracellular microorganisms. The use of obligate intracellular parasites circumvents the major problem associated with facultative microorganisms—extracellular replication—and permits quantitative analysis of events that lead to macrophage activation for microbicidal activities.

Histologic changes and therapeutic effects seen after a single injection of emulsified trehalose- 66' -dimycolate, a mycobacterial glycolipid, into growing dermal implants of a mouse fibrosarcoma were similar to those seen after injection of endotoxin and of emulsified turpentine. Intralesional injection of any of these agents caused the tumors to regress in a significant number of animals. The number of tumor free mice capable of resisting the growth of tumor cells in a challenge inoculum, however, was greater for animals treated with trehalose- 6,6' -dimycolate than those treated with turpentine or endotoxin. Granuloma formation was not a requirement for tumor regression in this mouse model.

Four lines of transgenic mice containing the HIV LTR linked to the bacterial gene encoding chloramphenicol acetyltransferase (CAT) were constructed. In each line, a characteristic tissue pattern of CAT expression was observed with detectable levels present in the eye, heart, spleen, thymus, and tail. Low levels of CAT were present in circulating lymphocytes, but CAT activity in these cells could be augmented following treatment with the mitogen phytohemagglutinin (PHA). Likewise, CAT expression was present at only low levels in circulating monocytes, but higher levels of CAT were observed in macrophages grown in the presence of various cytokines (CSF-1, GM-CSF, IL-1 alpha, IL-4, and IL-2). Furthermore, Langerhans cells recovered from skin showed higher levels of CAT activity than those observed in other cells of monocyte-macrophage lineage. These results indicate that LTR-CAT expression in cells of monocyte-macrophage lineage may increase in proportion to the degree of differentiation of these cells. These animals may be useful in the study of cell-specific determinants of LTR-directed gene activity and may serve to identify exogenous cofactors that promote the progression of HIV-related disease in vivo.

Phytohemagglutinin (PHA)-stimulated peritoneal exudate (PE) cells from strain-2 guinea pigs were more cytotoxic in culture to syngeneic tumorigenic cells than to syngeneic nontumorigenic cells. Cytotoxicity was measured by the release of 3H-thymidine from prelabeled target cells. Tumor-producing guinea pig fetal cells transformed in culture by chemical carcinogen released up to 70 percent of their label in the presence of PHA-stimulated PE cells. Non-tumor-producing cells, regardless of their morphologic characteristics, were less affected by PHA-stimulated PE cells. Nontumorigenic cultures included untreated early passage and long-term cultures previously treated with a carcinogenic or noncarcinogenic chemical. Differential cytotoxicity to tumorigenic cultures were best distinguished from nontumorigenic cultures when 0.5-1 times 10-6 PE cells and 50-100 mug PHA were incubated with 1 times 10-4 3H-thymidine-labeled target cells for 48 hours.

BALB/c mice develop both nonhealing cutaneous lesions and fatal systemic disease following subcutaneous (sc) inoculation of the protozoan parasite, Leishmania major. Several interesting features characterize this fulminant infection, and set BALB/c mice apart from other susceptible strains: (1) fatal disease in the BALB/c is virtually independent of infective parasite dose (Howard, Hale & Chan-Liew 1980), (2) a macrophage defect(s) appears to underlie the innate susceptibility of BALB/c mice to this strain of Leishmania (Behin, Mauel & Sordat 1979; Handman, Ceredig & Mitchell 1979; Nacy, Fortier, Pappas & Henry 1983; Mitchell, Anders, Brown, Handman, Roberts-Thomson, Chapman, Forsyth, Kahl & Cruise 1982) and (3) immunosuppression attributed to either lymphocytes (Howard, Hale & Liew 1980) or macrophages (Scott & Farrell 1981) develops in the later stages of infection, possibly the net result of vigorous parasite proliferation in defective macrophages.

Peritoneal macrophages (M phi) from mice became cytotoxic after incubation with lymphokine (LK); tumoricidal activity was evident with M phi treated with LK for 4 hr, became maximal after 8-12 hr of incubation, and decreased to control levels by 24-36 hr. LK induced marked changes in M phi lipid composition: cellular content of cholesterol (CHOL) and polyunsaturated fatty acid (UFA) content of cellular lipids (especially 18:3) increased two- to threefold after 8 hr when the cells showed maximal tumoricidal activity. Cellular lipid and fatty-acid content returned to control levels by 24 hr when the M phi had lost tumoricidal activity. These changes were not observed with equal numbers of M phi cultured in control supernatants. To analyze the role of CHOL and UFA in M phi tumor cytotoxicity, casein-induced peritoneal M phi were enriched in CHOL or linolenic acid (18:3) and then tested for their ability to kill 1023 tumor cells. The 18:3-enriched cells were markedly tumoricidal, whereas controls cultured in delipidized medium alone or enriched with saturated fatty acid (18:0) were not cytotoxic. CHOL-enriched M phi were not tumoricidal; indeed, these cells were inhibited in their killing after treatment with LK compared to M phi cultured in delipidized medium with LK alone. The effects of 18:3 and CHOL enrichment of the M phi on their metabolic status, inflammatory function, and tumor cell-binding capacity were tested. The 18:3-enriched M phi were depressed in their ability to synthesize protein and in phagocytic activity compared to controls; these cells showed a transient increase in superoxide release. M phi cultured with 18:3 for 48 hr were also cytotoxic for P815 tumor cells, but did not show an enhanced capacity for P815 binding compared to controls. CHOL-enriched M phi were similar to control cells in their protein synthesizing and phagocytic activities; these cells also showed an early transient increase in superoxide release. CHOL-enriched M phi were not cytotoxic for P815 cells, but bound the tumor cells more readily than did the 18:3-enriched M phi. The data suggest that endogenous levels of 18:3 and CHOL can regulate M phi tumor cytotoxicity, but not through regulation of M phi protein synthesis, oxidative metabolism, or augmented capacity for tumor target binding.

Chemotactic factor was produced in vitro by antigen or mitogen stimulation of mouse spleen cells. Activity was eluted from Sephadex columns in a major peak at 38,000 daltons. Small amounts of activity sometimes occurred in the 13,000 molecular weight region. Total recovery in the eluted fractions ranged from 30 to 60% of the applied material. The molecular weight of mouse lymphocyte-derived chemotactic factor (LDCF) is different from that of human, guinea pig, and chicken, all of which have reported molecular weights of 12,500 to 15,000. Mouse LDCF was eluted from DEAE columns at a specific conductance of about 15 mmho/cm, which indicates an apparent net charge different from chemotactic factor produced by stimulated human peripheral blood leukocytes. Thus, among lymphocyte-derived chemotactic factors hitherto described, the 38,000 MW molecule produced by mouse spleen cells is unique.

Peritoneal cells from Mycobacterium bovis BCG-infected C3H/HeN mice produced nitrite (NO2-, an oxidative end product of nitric oxide [NO] synthesis) and inhibited the growth of Francisella tularensis, a facultative intracellular bacterium. Both NO2- production and inhibition of bacterial growth were suppressed by NG-monomethyl-L-arginine, a substrate inhibitor of nitrogen oxidation of L-arginine, and monoclonal antibodies (MAbs) to gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha). Intraperitoneal injection of mice with BCG increased urinary nitrate (NO3-) excretion coincident with development of activated macrophages capable of secreting nitrogen oxides and inhibiting F. tularensis growth in vitro. Eight days after BCG inoculation, mice survived a normally lethal intraperitoneal challenge with F. tularensis. Treatment of these BCG-infected mice with MAbs to IFN-gamma or TNF-alpha at the time of BCG inoculation reduced urinary NO3- levels to those found in normal uninfected mice for up to 14 days. The same anticytokine antibody treatment abolished BCG-mediated protection against F. tularensis: mice died within 4 to 6 days. Intraperitoneal administration of anti-IFN-gamma or anti-TNF-alpha antibody 8 days after BCG infection also reduced urinary NO3- and abolished protection against F. tularensis. Isotype control (immunoglobulin G) or anti-interleukin 4 MAbs had little effect on these parameters at any time of treatment. IFN-gamma and TNF-alpha were clearly involved in the regulation of macrophage activation by BCG in vivo. Protection against F. tularensis challenge by BCG depended upon the physiological generation of reactive nitrogen oxides induced by these cytokines.