Biomedical subjects
Publications and source records attributed to M S Meltzer.
Pigmentary demarcation lines are borders of abrupt transition between more deeply pigmented skin and that of lighter pigmentation. Two patients developed these lines on the lower extremities during pregnancy.
Macrophages from P/J mice demonstrated both quantitative and qualitative defects in lymphokine (LK)-induced activated macrophage antileishmanial effector reactions: a) these cells recognized the same LK signals that generated resistance to infection in responsive C3H/HeN macrophages, but more signal was required to observe maximal activity; b) LK-induced intracellular destruction of Leishmania tropica by P/J macrophages was minimal (less than 20%), and was induced by only one of three LK signals that regulate antimicrobial activities in C3H/HeN macrophages. The defective microbicidal activity of P/J macrophages observed with LK activation in vitro could also be demonstrated in vivo. Macrophages from P/J mice exposed to the macrophage-activating agent Mycobacterium bovis strain BCG in vivo were capable of restricting the intracellular replication of L. tropica but could not eliminate intracellular parasites, even with further incubation with LK during the 72-hr culture period. The defect of P/J macrophages for intracellular destruction of L. tropica, then, occurred in the activation sequence before the triggering stage that characterizes the macrophage defect of C3H/HeJ mice. Genetic regulation of the P/J macrophage defect appears to be by a single autosomal gene, with defective microbicidal activity as a recessive trait in these animals.
Amastigotes of Leishmania tropica and L. donovani were incubated with fresh or heat-inactivated normal human serum. Viability was estimated by amastigote conversion to promastigote forms and by the ability of serum-treated amastigotes to infect human monocytes. L. tropica, a parasite that causes local skin infection, was killed by fresh but not by heat-inactivated serum. The serum cytotoxic effect on L. tropica was inhibited by EDTA but not by Mg-EGTA. C2-deficient serum killed normally; C6-deficient serum was ineffective. These data indicate that L. tropica is killed by the complement membrane attack complex, in a sequence of reactions initiated by components of the alternate pathway. In contrast, L. donovani, a parasite that causes systemic visceral leishmaniasis, was 10-fold less susceptible to the cytotoxic effects of normal serum. Thus, a profound difference exists in the susceptibility of amastigotes of two species of Leishmania to a defense mechanism present in human serum. Serum complement factors may play an important role in limiting L. tropica to the skin. The resistance of L. donovani to such factors may be the primary reason for its ability to escape from the site of inoculation and cause catastrophic, disseminated disease.
Leishmania tropica infections of P/J mice are characterized by the development of progressive nonhealing cutaneous lesions, followed by visceral metastases to liver and spleen. To analyze the genetic control of this disease, we produced F1, backcross (BX), and F2 progeny by breeding susceptible P/J mice with L. tropica-resistant C3H/HeN mice. Infections in these hybrid animals suggested that genetic control of the cutaneous lesion was by a single, autosomal, dominant gene. Resistance was the dominant trait. Analysis of liver and spleen impression smears in these animals, however, indicated that development of the cutaneous lesion segregates independently of the second component of L. tropica infections, systemic disease.
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.
Conditioned medium from antigen- or mitogen-stimulated spleen cells, lymphokines, contained factors that induced formation of granulocyte and macrophage colonies in cultures of bone marrow cells (CSF). Lymphokines also contained factors that induced macrophage non-specific tumoricidal activity against fibrosarcoma 1023, antibody-dependent tumoricidal activity against lymphoma 18-8, and antimicrobial activities against amastigotes of the protozoan parasite, Leishmania tropica. The factors that regulated macrophage effector functions, however, were different from those that induced colony formation, and could be distinguished from CSF by Sephadex gel chromatography or heat sensitivity. To further analyze a role for CSF in induction of macrophage effector activities, conditioned medium from several nonlymphoid cell sources (L-929, WEHI-3, and endotoxin-treated lung cells) were assayed for CSF activities and capacity to induce tumoricidal and microbicidal activities. Conditioned medium that contained either macrophages CSF (CSF-1) or the factor that induced formation of both macrophage and granulocyte colonies failed to activate macrophages for effector activities against fibrosarcoma 1023, lymphoma 18-8, and L. tropica amastigotes (either resistance to infection or intracellular destruction). These data suggest that CSF has no direct role in activation of macrophages for tumoricidal and microbicidal activities against these targets.
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Peritoneal macrophages (M phi) from mice become cytotoxic after incubation in lymphokine (LK)-rich supernatants of antigen-stimulated spleen cell cultures. Tumoricidal activity is evident with M phi treated with LK for 4 hr, becomes maximal after 8-12 hr incubation and decreases to control levels by 24-36 hr. To gain insight into LK-induced functional changes, the lipid composition of M phi cultured with LK for 0-36 hr was analyzed by high pressure liquid chromatography. LK induced marked changes in M phi lipid composition: cellular content of cholesterol (CHOL) and polyunsaturated fatty acids increased 2- to 3-fold 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 further the role of CHOL and unsaturated fatty acids in M phi tumor cytotoxicity, M phi were enriched in CHOL or linolenic acid (18:3) and tested for their ability to kill 1023 tumor cells. Within 1 hr of culture, M phi showed a 3- to 4-fold increase in CHOL or 18:3 content. 18:3-enriched cells were markedly tumoricidal, whereas controls cultured in delipidized medium alone or enriched with saturated fatty acids were 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. These results suggest that UFA aids, whereas CHOL negates, expression of M phi tumor cytotoxicity.
Peritoneal macrophages (M phi) from C3H/HeN mice became cytotoxic for 1023 tumor cells after incubation with lymphokine (LK) for 8-12 hr and lost tumoricidal activity by 22 hr in the continued presence of LK; bacterial endotoxin (LPS) was ineffective in inducing tumoricidal activity. M phi from C3H/HeJ mice were not activated for tumor cytotoxicity after treatment with LK or LPS. C3H/HeN M phi acquisition of tumoricidal activity was accompanied by unique changes in M phi lipid composition: cellular content of cholesterol (CHOL) and polyunsaturated fatty acids (UFA) increased two- to threefold when the cells showed maximal tumoricidal activity and returned to control levels when the M phi lost tumoricidal activity. LPS treatment of C3H/HeN M phi and LK or LPS treatment of C3H/HeJ M phi did not cause characteristic M phi lipid alterations. To determine at what stage during M phi activation the correlative CHOL and UFA compositional changes were occurring, C3H/HeN M phi were primed with LPS or low concentrations of LK and triggered with LPS or Lk; M phi lipid and fatty acid composition was monitored at each stage. LK was shown to be able to prime and trigger whereas LPS could only trigger LK-primed M phi for tumor cytotoxicity. In all cases, the increase in M phi CHOL and UFA content occurred at the triggering step for tumor cytotoxicity rather than at the priming step. These data suggest that there is a correlation between the effects of endogenous and exogenous factors that control expression of M phi tumoricidal activity and their effects on M phi CHOL and UFA content; the establishment of these changes in M phi lipid composition occurs at a time when the cells are triggered for tumor cytotoxicity.
Macrophages treated with culture fluids from EL-4 cells, a continuous T cell line, were activated to kill mKSA-TU-5 fibrosarcoma cells, amastigotes of Leishmania tropica, and schistosomula of Schistosoma mansoni. Active EL-4 factors eluted from Sephadex G-100 in two distinct regions: molecular weight 45,000 (activities induced killing of unrelated intracellular and extracellular targets) and molecular weight 23,000 (activities induced killing of extracellular targets only). These results confirm heterogeneity among activation signals for the induction of macrophage microbicidal and tumoricidal activities. Factors that induced cytocidal activity against extracellular tumor cells and schistosomula were distinct from those that induced destruction of intracellular amastigotes.
Sera (BCG-lipopolysaccharide [LPS] serum) were obtained from mice infected with Mycobacterium bovis BCG 2 h after intravenous administration of bacterial endotoxin (LPS). Varying concentrations of sera were added to cultures of Plasmodium falciparum-infected human erythrocytes; parasite viability was assessed by hypoxanthine incorporation after 4 days in culture. At concentrations of 1 to 3%, cultures treated with BCG-LPS serum showed a two- to threefold increase in hypoxanthine incorporation; at higher concentrations (4 to 8%), hypoxanthine incorporation fell to 2 to 5% of that in control cultures. Concurrent assays with control sera (from untreated mice or mice treated with BCG or LPS alone) caused some stimulation but no inhibition at up to 8% concentration. Examination of cultures treated with BCG-LPS serum showed morphological, deterioration of parasites within erythrocytes. The presence of tumor necrosis factor in the BCG-LPS serum was confirmed by using a standard L-cell cytotoxicity assay. In addition, rabbit antiserum against partially purified tumor necrosis factor protected intraerythrocytic forms of P. falciparum from the toxic effects of BCG-LPS serum. These data suggest that the factor in BCG-LPS serum that is toxic to P. falciparum in human erythrocytes is antigenically similar or identical to tumor necrosis factor. This nonantibody mediator of killing may play a role in human malaria.
Mice of several inbred strains previously characterized as defective in various aspects of macrophage activation for tumor cell killing were utilized to compare the genetic control of induction of macrophage tumoricidal and schistosomulacidal activity and to examine the role of activated macrophages in concomitant immunity to Schistosoma mansoni infection. In all mouse strains tested, macrophage larvicidal activity developed concurrently with tumoricidal activity after in vivo bacillus Calmette Guérin or Corynebacterium parvum treatment. Lpsd strains, A/J, and P/N mice failed to develop macrophages capable of killing either tumor cells or helminth larvae in vitro, whereas C57BL/6J, C3H/HeN, and BALB/c mice all demonstrated strongly cytotoxic cells. In fact, analysis of the reactivity of peritoneal cells from C. parvum-treated F1 hybrids between H-2 congeneic high responder (B10.A) and low responder (A) strains of mice, as well as from F1 hybrid X parent backcross animals, suggested that macrophage activation for killing of these two unrelated extracellular targets is controlled by the same autosomal dominant gene. Strains of mice that failed to develop activated macrophages after in vivo treatment with bacterial stimuli also failed to develop tumoricidal and larvicidal macrophages as a result of S. mansoni infection. The addition of antischistosome sera to the cultures did not reverse the unresponsiveness of macrophages from S. mansoni-infected C3H/HeJ, A/J, or P/N mice in in vitro larvicidal assays. Furthermore, the pattern of concomitant immunity at 6 wk after primary S. mansoni infection correlated closely with the pattern of induction of activated macrophages in these strains of mice. Whereas animals with normal macrophage function developed highly significant levels of concomitant immunity, strains with the Lps defect demonstrated lower levels of acquired resistance, and A/J and P/N mice were not significantly resistant to S. mansoni challenge infection. These results indicate that the genes controlling macrophage activation for the killing of extracellular targets also influence the development of concomitant immunity in schistosomiasis, and suggest that macrophages activated as a consequence of primary S. mansoni infection may be involved in the in vivo effector mechanism of resistance to challenge infection.
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Activation of macrophages was assessed in strains of mice inoculated intraperitoneally with 1,000 times the 50% lethal dose of Rickettsia akari. Macrophages from mice resistant to R. akari infection (C3H/HeN, C57BL/10J, and BALB/cN) were nonspecifically tumoricidal 2 to 4 days after rickettsial inoculation. Moreover, these macrophages were microbial for R. akari in vitro; cells were resistant to infection with the bacterium and were capable of killing intracellular rickettsiae. In contrast, macrophages from strains of mice susceptible to R. akari (C3H/HeJ, C57BL/10SnCR, and A/J) failed to develop nonspecific tumoricidal activity over the course of lethal disease and became infected with R. akari in vivo within 2 days of rickettsial inoculation. Macrophages from uninfected mice of strains susceptible to R. akari also could not be activated for rickettsicidal or tumoricidal activities by treatment with macrophage-activating agents (Mycobacterium bovis BCG) in vivo or by treatment with lymphokines in vitro.
Susceptibility to lethal effects of Rickettsia akari varies among mouse strains. Although most strains are resistant, A/J mice are extremely sensitive (10,000-fold difference in LD50 between resistant and sensitive strains). In contrast to most strains of mice, A/J mice also fail to develop activated, tumoricidal macrophages after any of several in vivo or in vitro treatments. Are susceptibility to R. akari and inability to develop activated, cytotoxic macrophages in the A/J strain causally related traits? Genetic analysis of macrophage tumoricidal activity in responsive B10.A and nonresponsive A mice suggested this trait was controlled by a single, autosomal, dominant gene. Among (F1 X A) backcross mice, the traits for defective macrophage cytotoxicity and abnormal accumulation of macrophages during inflammation (a response controlled by a single, autosomal, dominant gene) segregated independently. Genetic analysis of resistance to R. akari in sensitive A and resistant B10.A mice suggested this trait was also controlled by a single, autosomal, dominant gene. Among (F1 X A) backcross mice, however, traits for defective macrophage cytotoxicity and R. akari resistance segregated independently. Thus, development of activated tumoricidal macrophages, accumulation of macrophages at sites of inflammation, and resistance to the lethal effects of R. akari in A and B10.A mice were each controlled by single, autosomal, dominant, non-H-2-linked genes. However, genes for control of macrophage activation for tumoricidal activity and for resistance to lethal effects of R. akari infection are distinct.
Supernatant culture fluids from a phorbol myristate acetate (PMA) stimulated variant of the murine EL-4 thymoma cell line activated inflammatory macrophages for nonspecific tumoricidal activity in vitro; active supernatants fluids were not directly toxic to tumor target cells in the absence of macrophages. Macrophages treated with culture fluids of unstimulated and concanavalin A-stimulated EL-4 or PMA-stimulated cells derived from any of ten other continuous T cell lines were not cytotoxic. The titer of activity in lymphokines from PMA-stimulated EL-4 was similar to that from antigen-stimulated spleen cell cultures. Spleen cell and EL-4 derived activities were both retained within a dialysis membrane and destroyed by heating at 100 degrees C for 15 min. However, unlike the activity in spleen cell-derived LK, activity in EL-4 culture fluids was stable to dialysis against pH 2 buffer. Further physicochemical characterization of the EL-4 activity showed two different factors that activated macrophages for cytotoxicity: one was indistinguishable from gamma interferon (IFN) (antiviral activity, 50,000 m.w., pI 4.2 to 6.0, pH 2 labile and neutralized by anti-gamma IFN serum); the other was clearly distinct (no antiviral activity, 23,000 m.w., pH 2 stable and unaffected by anti-gamma IFN serum). These results suggest that the EL-4 cell line may produce one or more of the macrophage activation factors present in spleen cell-derived lymphokines.