Role of Corynebacterium parvum in the activation of peritoneal macrophages: I. Association between intracellular C. parvum and cytotoxic macrophages

We have investigated the association between intracellular C. parvum (CP) and macrophage (Mφ) cytotoxicity. Mouse peritoneal Mφs were activated by ip administration of CP and were subjected to a combination of fractionation techniques to study this. Velocity sedimentation demonstrated that only the largest cells were cytotoxic. These same cells contained CP and suggested an association between the two variables. Further separation of the largest Mφs using a BSA equilibrium buoyant density gradient demonstrated that cytotoxicity was due to Mφs and further substantiated the strong correlation between intracellular CP and cytotoxicity. Various fluorochrome tagged CP preparations were also used to activate Mφs and to isolate CP-containing Mφs using fluorescence-activated cell sorting. When velocity-enriched Mφs were sorted on the basis of the presence or absence of fluorescent CP, only the Mφ fractions which contained CP were cytotoxic. The results indicate that most cytolytic macrophages present at the peak of the response contain CP. Thus, a convenient probe with which to follow macrophage activation at the single cell level was provided.     

Functional heterogeneity among peritoneal macrophages. II. Enzyme content of macrophage subpopulations.

Rabbit peritoneal exudate (PE) macrophages were separated into subpopulations on discontinuous density gradients of bovine serum albumin. Four such macrophage subpopulations, referred to as bands A, B, C, and D (from lightest to heaviest buoyant density), were examined for differences in enzyme content. With regard to three acid hydrolases—acid phosphatases, β-glucuronidase, and cathepsin D—cells in bands A and B had greater enzyme activity than cells in bands C and D. A similar distribution of activities was observed for acid p-nitrophenylphosphatase. Peroxidase activity was present only in band D. Lysozyme activity was greatest in band D cells and least in band A cells. Only small differences in cytochrome c oxidase activity were observed among the subpopulations. Arginase activity was found to be greater in cells from band A than cells in bands B, C, and D. Macrophage subpopulations derived from PE macrophages placed in tissue culture for 7 days and macrophage subpopulation cells cultured for 2 days showed differences in acid phosphatase content similar to those seen with freshly obtained subpopulations. These results extend previous work demonstrating heterogeneity among PE macrophages.     

Functional heterogeneity in macrophages activated by Corynebacterium parvum: characterization of subpopulations with different activities in promoting immune responses and suppressing tumor cell growth.

Peritoneal cells (PEC) from mice injected i.p. with heat-killed Corynebacterium parvum (CP) showed enhanced immunostimulatory (accessor or A cell) activity as measured by their ability to restore the immune responsiveness of nonadherent spleen cells to sheep erythrocytes (SRBC) and polymeric flagellin (POL) of Salmonella adelaide in vitro. This was true whether the PEC and nonadherent spleen cells were in direct contact or separated by a cell-impermeable membrane which allowed the free passage of soluble mediators. CP-activated PEC also exhibited greatly increased cytostatic activity against the growth of syngeneic tumor cells in vitro. After fractionation of the PEC according to cell size by velocity sedimentation, a separation of A cell activity from anti-tumor activity was observed. Although both these functions were associated with phagocytic cells of the monocyte-macrophage series, the highest A cell activity was found in fractions containing small and medium-sized macrophages, whereas the anti-tumor activity increased with cell size to a maximum with the largest macrophages. Thus, there is a relative increase of suppressive activity over stimulatory activity with an increase in cell size. Cytochemical and morphologic evidence suggests that the A cell-rich fractions contained small and medium-sized macrophages which were derived from newly arrived monocytes, whereas the large tumor-suppressive macrophages were relatively more differentiated.     

Splenic suppressor macrophages induced in mice by injection of Corynebacterium parvum.

Spleen cells from C57BL/6N mice injected with killed Corynebacterium parvum (CP) had a marked growth inhibitory effect on the in vitro proliferation of RBL-5 murine lymphoma cells. It was most marked 12 to 14 days after injection and was usually no longer detectable later than 21 days. It could be demonstrated at effector cell to target ratios between 20:1 and 5:1 at which normal spleen cells had a growth-promoting effect. Addition of CP to an in vitro mixture of spleen cells and tumor cells augmented the inhibitory effect of spleen cells from CP-injected mice although it conferred no inhibitory potential on normal spleen cells. Growth inhibiton by CP spleen cells was not mediated by T cells and various depletion experiments suggested that the effector cells of the phenomenon were macrophages. Spleen cells of CP-injected mice also showed strongly depressed responses to the T cell mitogens PHA and Con A and suppressed the mitogen responses of syngeneic normal spleen cells. The characteristics of the suppressor cells mediating this effect appeared to be very similar to those inhibiting lymphoma cell growth. The responses to LPS were also strongly suppressed in mice injected with 2.1 mg of CP. However, after injection of one-tenth of the dose a relative sparing of the LPS response was noted, whereas the PHA response was still suppressed.     

Role of distinct subpopulations of peritoneal macrophages in the regulation of reactive oxygen species release.

It has been reported in vitro that during the respiratory burst of phagocytic cells the superoxide anion production per cell shows a negative relation with the cell density. This process has been described as autoregulation. The aim of this work was to analyze the superoxide anion production in thioglycollate-elicited peritoneal macrophage exudates to evaluate the importance of the peritoneal cavity environment in the autoregulation process. 12-O-tetradecanoylphorbol-13-acetate (PMA) was used to stimulate the respiratory burst and superoxide anion production was measured evaluating the intracellular formazan deposits that precipitate as a result of nitro blue tetrazolium (NBT) reduction. We have demonstrated a negative correlation between superoxide anion production and cell density in the peritoneal cavity in macrophages challenged with PMA. The response of individual cells was analyzed by means of an image analyzer, measuring the amount of formazan per cell and cell-size changes during the process of activation. The results revealed that the decrease in individual cell response as a function of higher cell densities were due to a significant increase in the amount of basal reaction macrophages. Concomitantly, the number of reactive cells remained unchanged irrespective of the cell density of the population. A direct correlation between cell size and superoxide anion production was observed. This phenomenon was demonstrated in SENCAR and Balb/c strains. However, macrophages from SENCAR mice showed greater superoxide anion production than those from Balb/c.The differences between strains could be associated to the increased sensitivity to PMA tumor promotion of SENCAR mice. Based on this property, macrophages from SENCAR mice were stimulated with opsonized zymosan, a particulate stimulus that reflects the interaction macrophage-microorganism during the phagocytic process. This data will contribute to the knowledge of infection control. We conclude that variations in basal reaction cells modulates the macrophage activation response when excess macrophages are recruited to the peritoneum. This is demonstrated using different stimuli, thus suggesting that this response may be applied to a wide variety of stimuli-macrophage interactions. The differences between strains may be associated to the increased sensitivity to PMA tumor promotion of SENCAR mice.     

Glycosaminoglycan profile of peritoneal and bone marrow-derived macrophages. Changes associated with macrophage activation.

1. We have analysed the glycosaminoglycan patterns of peritoneal and bone marrow-derived macrophages obtained from four different mouse strains which are resistant (A/J) or susceptible (BALB/c, DBA and C-57) to murine hepatitis virus type 3 (MHV3) infection. The glycosaminoglycans were biosynthetically labelled by exposing the macrophages to 35S-sulphate. The medium and cell fractions were collected and the 35S-glycosaminoglycans formed were identified by a combination of agarose gel electrophoresis and enzymatic degradation with bacterial mucopolysaccharidases. 2. Both peritoneal and bone marrow-derived macrophages synthesize and secrete a mixture of dermatan sulphate, heparan sulphate and chondroitin sulphate. Dermatan sulphate is the main glycosaminoglycan and most of the synthesized glycosaminoglycans are released to the culture medium. 3. The glycosaminoglycan patterns vary depending on the macrophage source. Bone marrow-derived cells synthesize glycosaminoglycans at lower rates, release a lower glycosaminoglycan percentage to the culture medium and express higher amounts of heparan sulphate in comparison with their peritoneal counterparts. Furthermore, LPS-induced activation leads to an increased glycosaminoglycan expression in bone marrow-derived macrophages and to a decrease in 35S-glycosaminoglycans of peritoneal macrophages from BALB/c, A/J and C-57 mice. 4. We have not established any correlation between macrophage glycosaminoglycans and resistance to MHV3 infection, since the glycosaminoglycan patterns of resistant (A/J) and susceptible (BALB/c, DBA and C-57) mouse macrophages are similar. Furthermore, the in vitro infection of both control and LPS-activated peritoneal macrophages with MHV3 did not cause any changes in the expression of glycosaminoglycans.     

Characteristics of macrophage activation by gamma interferon for tumor cytotoxicity in peritoneal macrophages and macrophage cell line J774.1

We investigated the characteristics of macrophage-mediated tumor cytotoxicity (MTC) against Meth A target, H2O2 generation and release of effector molecule(s) for MTC, by comparing with those of peritoneal macrophages (PMP) and macrophage cell line J774.1 during stimulation with recombinant gamma interferon (IFN-gamma). In PMP, MTC was demonstrated when they were stimulated with IFN-gamma for 12 hr (short-term stimulation) and was abrogated when they were stimulated for 48 hr (long-term stimulation). Enhanced H2O2 generation was observed in PMP activated by long-term stimulation followed by triggering with PMA, but not observed by triggering with Meth A cells. By contrast, whereas non-treated J774.1 cells have already attained a definite level of MTC, a higher MTC level was demonstrated both by short- and long-term stimulations. Conversely, J774.1 cells were unable to generate H2O2 at any stage of IFN-gamma stimulation followed by triggering both with PMA or Meth A cells. The time course for stimulation of PMP by IFN-gamma for release of cytotoxic factor (CF) corresponded to that for MTC by PMP, and activities of the CF released from both activated PMP and J774.1 cells also closely corresponded to those of MTC by both cells. The serological and physicochemical characteristics of CF released from both activated PMP and J774.1 cells were determined to be closely related to those of tumor necrosis factor (TNF). These results indicate that in contrast to PMP, the J774.1 cell line is free from suppression stage for MTC and CF release during stimulation with IFN-gamma. The results suggest that TNF-like CF plays a crucial role for MTC against Meth A target, and that H2O2 is irrelevant for MTC against Meth A.     

Kinetics and mechanisms of macrophage activation by Corynebacterium anaerobium

Kinetics and mechanisms of macrophage activation by heat-killed Corynebacterium anaerobium (CA) in mice were investigated. The carbon clearance test revealed that the function of the reticuloendothelial system rose markedly on the 4th day after a single intravenous injection of CA and continued in a highly enhanced state until the 14th day. This activity declined gradually and dropped to a normal level around the 21st day. On the other hand, both lysosomal enzymes, beta-glucuronidase and acic phosphatase, of peritoneal macrophages decreased after the CA injection and then recovered, taking an almost inverse course to the function of the reticuloendothelial system. These results might be attributable to possible extracellular secretion of the lysosomal enzymes in accordance with macrophage activation by CA. A remarkable cytotoxicity of peritoneal macrophages, examined in vitro against L 929 cells, was detected on the 4th day following intraperitoneal administration of CA. It was maintained up to the 14th day and then declined rapidly. The mechanisms of macrophage activation by CA were also examined in vitro. CA-homogenate, heat-killed CA disrupted with an ultrasonicator, directly activated thioglycollate-induced macrophages. The macrophages were aslo activated by simultaneous treatment with both CA-homogenate and CA-sensitized spleen cells. Furthermore, the supernatant obtained from the culture of CA-sensitized spleen cells with CA-homogenate was capable of inducing activation of the macrophages. Conversely, the culture supernatant of spleen cells from CA-immunized athymic nude mice with CA-homogenate was unable to activate them. It was ascertained from the above-mentioned results that macrophages are activated initially by direct action of CA in a nonspecific way and subsequently by a soluble factor elaborated by CA-sensitized lymphocytes in an immunological way.     

Role of the maleyl-albumin receptor in activation of murine peritoneal macrophages in vitro

It has been previously demonstrated that maley-lated-BSA (maleyl-albumin) induces functional activation in murine peritoneal macrophages. Furthermore, maleyl-albumin has been shown to interact with two distinct sites on human monocytes; one site is the scavenger receptor, a 260-kDa oligomeric protein which recognizes modified forms of low density lipoprotein (LDL), and the second is a lower affinity site which has yet to be structurally characterized. In the present study, we wished to quantitatively assess the number and character of maleyl-albumin-binding sites on murine peritoneal macrophages and to determine which site or sites are involved in signaling the macrophage to undergo extensive functional development. Binding studies. demonstrate at least two distinct receptors for maleyl-albumin on murine peritoneal macrophages. Scatchard analyses of the binding isotherms reveal two sites characterized by dissociation constants (Kd) of 17.6 nM and 4.9 microM and maximal binding of 1.2 x 10(5) and 1 x 10(6) sites/cell, respectively. The contribution of the scavenger receptor, determined by binding analyses of malondialdehyde-LDL, is described by two sites with Kd of 39.4 pM and 9.6 nM, and maximal binding of 2.7 x 10(3) and 1.9 x 10(4) sites/cell, respectively. Maleyl-albumin blocks binding of malondialdehyde-LDL, whereas modified LDL fails to inhibit binding of maleyl-albumin. Maleyl-albumin, at concentrations producing lower affinity binding, stimulates tumor cytolysis, expression of mRNA encoding TNF, and suppression of INF-gamma-induced expression of Ia Ag. Malondialdehyde-LDL fails to elicit these responses. We conclude that macrophage activation produced by maleyl-albumin is mediated by interaction with the low affinity, high capacity binding site for maleyl-albumin rather than the scavenger receptor.     

Cellular suppression of murine ADCC and NK activities induced by Corynebacterium parvum

Summary Administration of a single dose of C. parvum (CP) induces depression of splenic NK activity in mice after a lag period of 3–5 days and this depression lasts about 2 weeks. The depressed levels of NK activity noted in this study depended on time of CP administration and were associated with the induction of suppressor cell activity. Neonatally thymectomized or sublethally irradiated mice had unimpaired ability to generate suppressor cells following CP treatment. Depletion of adherent/phagocytic cells by carbonyl iron plus magnetism, Sephadex G-10 filtration, or both neither enriched NK activity nor removed suppressor activity from the spleens of CP-treated mice. Antibody-dependent cellular cytotoxicity (ADCC) against lymphoma targets was also depressed in CP-treated mice, accompanied by a concomitant appearance of suppressor cells that interfere with ADCC at the effector level.     

Biological effects of the adjuvant Corynebacterium parvum. II. Evidence for macrophage-T-cell interaction

The mechanism underlying the markedly reduced PHA responsiveness of spleen and peripheral blood lymphocytes from Corynebacterium parvum-treated mice is due to inhibition of the responsive T-lymphocytes by C. parvum-activated macrophages. Inhibition is a result of a qualitative rather than quantitative change in the macrophage population and GVH-activated macrophages behave similarly. Corynebacterium parvum-activated macrophages need to be viable and will inhibit normal lymphocytes. This inhibitory effect appears to be mediated through cell-cell contact.     

Role of culture supernatant of cytotoxic/cytostatic macrophages in activation of murine resident peritoneal macrophages

Summary Peritoneal macrophages elicited by Lactobacillus casei YIT9018 (LCEPM) were incubated in culture for 18 h with L. casei; the culture supernatant (LCM) was then harvested and tested for its ability to increase the cytostatic activity of resident peritoneal macrophages (RPM) and LCEPM. Treatment of RPM with LCM induced activation of macrophages to a cytostatic state against L929, Colon 26, P815, P388D1 and L1210 cells. A combination of recombinant human tumor necrosis factor (rhTNF), recombinant mouse TNF (rmTNF), recombinant human interleukin-1 (rhIL-1) or bacterial lipopolysaccharide with recombinant mouse interferon (rmIFN-) resulted in the synergistic induction of cytostatic activity in RPM. Recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) plus rhTNF increased the cytostatic activity of RPM a little but rmGM-CSF or rhTNF combined with rhIL-1 or alone had no effect. The effect of LCM on RPM was not inhibited by polymyxin B, anti-mTNF antiserum or below 20 U/ml monoclonal anti-rmIFN- antibody (anti-rmIFN-) but was inhibited by more than 40 U/ml anti-rmIFN-, and LCM did not have any interferon antiviral activity. These results suggest that the cytostatic activity of RPM was augmented by the LCM, and that the effect of the LCM may be not due to IFN-, TNF, GM-CSF, IL-1 or a small amount of contaminating lipopolysaccharide.     

Regulation of macrophage populations. II. Synthesis and expression of Ia antigens by peritoneal exudate macrophages is a transient event

We have evaluated the biosynthesis and surface expression of I-A antigens by peritoneal macrophages and found that both events terminated during the 1st day in culture, in contrast to the undiminished synthesis and expression of H-2K antigens. This pattern was observed regardless of the means by which the macrophages were elicited, but was subject to modulation for a limited period of time in vitro: phagocytic stimuli were able to augment both I-A synthesis and expression. The loss of I-A and the re-expression after phagocytosis were both reflected in the stimulatory capacity of these macrophages in the mixed leukocyte reaction. Moreover, we found that I-A-bearing macrophages were lost from the exudate in vivo after irradiation. Our data suggest that, as in vitro, this phenomenon is due to the transition of individual macrophages from I-A-positive to I-A-negative, and that constant renewal is required to maintain the I-A-bearing subset in vivo.     

Biological effects of Corynebacterium parvum. IV. Adjuvant and inhibitory activities on B lymphocytes

The PFC response to the thymus-independent antigen SIII (type 3 pneumococcal polysaccharide) was amplified in mice injected 4 days previously with killed Corynebacterium parvum. This adjuvant activity was demonstrable with high (2–50 μg) but not low (0.1–0.5 μg) doses of SIII. Induction of tolerance was unaffected. Depression of the response resulted from simultaneous injection of SIII with either C. parvum or Bordetella pertussis, while prior treatment with the latter was without effect. Responsiveness to SIII was transiently but potently suppressed in spleen cells transferred into lethally irradiated, C. parvum pretreated mice.Although C. parvum is an effective B cell adjuvant, other data imply that it acts indirectly on these lymphocytes. It is argued that both adjuvant and suppressive activities of C. parvum on the B cell response to SIII are most probably mediated by activated macrophages.     

Identification of the serum factor required for in vitro activation of macrophages. Role of vitamin D3-binding protein (group specific component, Gc) in lysophospholipid activation of mouse peritoneal macrophages

In vitro treatment of mouse peritoneal cells (mixture of adherent and nonadherent cells) with lysophosphatidylcholine (lyso-Pc) in 10% FCS supplemented medium RPMI 1640 results in a greatly enhanced FcR-mediated phagocytic activity of macrophages. This macrophage-activation process requires a serum factor. Fractionation studies with starch block electrophoresis of fetal calf and human sera revealed that alpha 2-globulin fraction contains a serum factor essential for macrophage activation. To identify the serum factor, human serum was precipitated with 50% saturated ammonium sulfate and fractionated on a Sephadex G-100 column. A protein fraction with a lower m.w. than albumin had the capacity to support activation of macrophages. The active serum factor in this protein fraction was analyzed by immunoabsorption by using rabbit antisera against three major proteins of human alpha 2-globulin. This active serum factor was shown to be a vitamin D3-binding protein (group specific component, Gc). By using a monoclonal anti-Gc-absorbed active column fraction of human serum, we observed no enhanced macrophage activation over the results with serum fraction-free cultivation of lyso-Pc-treated peritoneal cells. Cultivation of lyso-Pc-treated peritoneal cells in a medium containing a low concentration of purified human Gc protein (0.1 to 2.6 ng/ml) produced a greatly enhanced phagocytic activity of macrophages. When purified human Gc protein was used in a serum-free medium for stepwise cultivation of lyso-Pc-treated nonadherent cell types, a macrophage-activating factor was efficiently generated. Therefore, it is concluded that the vitamin D3-binding protein is the essential serum factor for the lyso-Pc-primed activation of macrophages.     

Identification of novel adhesion proteins in mouse peritoneal macrophages.

When mouse peritoneal macrophages were made to adhere firmly on glass surface and then removed by sequential treatment with hypotonic triethanolamine and Nonidet P-40, a set of proteins were found to be left behind at the sites of adherent cells. Such glass-adherent proteins were detected as round or ellipsoidal patches of autofluorescence under a confocal laser microscope, and visualized ultrastructurally as aggregates of narrow threads of unique loop structures which were composed of linearly aligned particles of 22 +/- 2 nm in diameter. Lithium dodecylsulfate-polyacrylamide gel electrophoresis of the glass-adherent proteins showed two major bands, 12 kDa and 14 kDa, which always co-existed in any different sample. The polyclonal antibody raised against these two proteins specifically stained the glass-adherent proteins in situ. The adhesion of macrophages to glass was significantly blocked with Fab fragments of the antibody. The in situ cross-linking experiment suggested that these two proteins might be closely associated with each other to form complexes. Hence, these proteins can be reasonably considered to be responsible for non-specific adhesion of macrophages to glass.     

Effect of Corynebacterium parvum on the immune response in guinea pigs. II. Passive transfer of enhanced anamnestic response and delayed hypersensitivity observed after treatment with Corynebacterium parvum]

After intradermal immunization with a mixture of Corynebacterium parvum (C. parvum) and ovalbumin guinea pigs show a markedly increased anamnestic response to an intradermal booster of ovalbumin as compared to controls treated with ovalbumin only. At the same time a reaction of delayed type hypersensitivity is observed in the treated animals, but not in controls. The enhanced anamnestic response as well as the posivitive skin reaction were transferred to strain 2 histocompatible guinea pigs by peripheral blood leukocytes as well as by peritoneal exudate cells. Passive transfer was not obtained after prior irradiation of donor animals.