Suppressive effect of nonviable Mycobacterium paratuberculosis on the delayed-type hypersensitivity reaction to sheep erythrocytes in mice.

The effect of nonviable Mycobacterium paratuberculosis on the delayed-type hypersensitivity reaction to sheep erythrocytes (SRBC) in mice was evaluated by means of delayed-type footpad swelling. Intraperitoneal (i.p.) injection with nonviable M. paratuberculosis into mice from 28 days before to 1 day after immunization with SRBC resulted in a significant suppression of foot-pad swelling to SRBC. The suppressive effect could be transferred by i.p. injection of spleen cells or peritoneal exudate cells from mice which had been pre-treated with nonviable M. paratuberculosis into non-treated recipient mice. The suppressive effect of spleen cells was retained even after passing them through a nylon wool column. The suppressive effect of spleen cells was abolished by treatment with anti-Thy 1.2 monoclonal antibody plus complement or anti-Lyt 2.2 monoclonal antibody plus complement. However, treatment of spleen cells with anti-mouse gamma globulin antiserum plus complement or anti-Lyt 1.2 monoclonal antibody plus complement did not affect the suppressive effect of spleen cells. The suppression of footpad swelling to SRBC induced by pre-treatment with nonviable M. paratuberculosis could be reversed by i.p. administration of cyclophosphamide. Serum antibody response to SRBC in mice was not affected by pre-treatment with nonviable M. paratuberculosis. These findings indicate that T cells appear to be involved in the suppression of delayed-type hypersensitivity reaction to SRBC in mice by pre-treatment with nonviable M. paratuberculosis.     

Site of action of a soluble immune response suppressor (SIRS) produced by concanavalin A-activated spleen cells.

The cellular site of action of SIRS, a soluble immune response suppressor released by Con A-activated spleen cells which suppresses antibody responses to heterologous erythrocytes by murine spleen cells in vitro, was investigated. Exposure of spleen cells to SIRS for 2 hr at 37 degrees C or 1 hr at 4 degrees C was sufficient to suppress 5-day antibody responses in vitro. Similar exposure of splenic or peritoneal exudate macrophages to SIRS also suppressed antibody responses by untreated splenic lymphoid cells; exposure of splenic lymphoid cells to SIRS was without effect. SIRS did not act via T cells which might have contaminated the macrophage preparations. SIRS-mediated suppression could be partially overcome by an excess of normal peritoneal exudate macrophages, but not by an excess of T or B cells. These data indicate that the target cell of SIRS activity is the macrophage. The results are discussed in the context of macrophage functions that could be affected by SIRS.     

Prostaglandin-mediated suppression of delayed-type hypersensitivity to infected erythrocytes during Babesia microti infection in mice

The mechanism of suppression of delayed-type hypersensitivity (DTH) to intraerythrocytic Babesia microti which occurs during infection in mice was examined. The suppression was not specific for anti-parasite DTH; infected mice immunized and challenged with sheep red blood cells had a similar depression of anti-sheep red blood cell DTH. Sublethal or lethal irradiation did not significantly alter the suppression of the DTH response, and cyclophosphamide pretreatment of infected mice also had no effect on suppression. Multiple passive transfer experiments using serum or regional lymph node cells from immunized or infected and immunized (suppressed) donor animals failed to demonstrate any ability to transfer suppression of DTH. Adherent cells from the spleens or peritoneal exudates of suppressed mice, however, did significantly depress the ability of immunized mice to express a DTH response. The cells responsible for this suppression were Thy 1- and nonspecific esterase+. Treatment of suppressive cell populations with 10 micrograms/ml indomethacin for 24 hr in vitro abrogated their suppressive ability, and in vivo administration of indomethacin to suppressed mice also restored DTH to normal levels. By examining levels of prostaglandin E2 (PGE2) in supernates of cultured peritoneal exudate cells from immune or suppressed mice, it was shown that infected mice had peritoneal exudate cells which produced significantly more PGE2 than similar cells from immune mice. These data suggest that B. microti infection elicits synthesis of PGE2 by macrophage-like cells which results in suppression of DTH to parasite as well as heterologous antigens.     

Immune responsiveness and phagocytic activity of macrophages in streptozotocin (SZ)-induced diabetic mice

We succeeded in inducing different severities of diabetic state in C3H male mice by repeated intraperitoneal injections of various doses of SZ. SZ-induced diabetic mice were divided into four groups as follows: Group A, B, C and D. SZ, respectively, 3, 5 doses of 45 mg/kg, 5 doses of 60 mg/kg on consecutive days and one of a dose of 200 mg/kg BW. The degree of hyperglycemia and glycosuria were mild in group A and D. Group B was moderate and group C severe with ketonuria and loss of body weight. We investigated the immune response to anti-sheep red blood cells (SRBC) and the phagocytic activity of macrophages in the above mentioned various SZ-induced mice. Antibody forming activities (values of anti-SRBC plaque-forming cells (PFC) and serum agglutinin) were markedly depressed in all of SZ-diabetic groups. The degree of the suppression of antibody response to SRBC in SZ-diabetic mice corresponded with the severity of the diabetic state (C greater than B greater than A = D). However, the phagocytic activity of peritoneal macrophages in SZ-diabetic mice was as high as or higher than that in normal controls, using both latex beads and immune complex as test particles. Moreover, we observed that insulin treatment reversed the defect in the immune response in SZ-diabetic mice. These results indicate that the phagocytic activity of peritoneal macrophages was retained but the antibody response was impaired in the SZ-diabetic mice, and this suggested that the impaired antibody response may be a contributing cause of increased susceptibility to infections in a diabetic state.     

Target cells for the activity of a synthetic adjuvant: muramyl dipeptide.

Muramyl dipeptide (MDP), a synthetic adjuvant, increased the primary response of CBA mice to sheep red blood cells (SRBC). In reconstituted irradiated recipients, cooperation between T and B lymphocytes was required for the expression of adjuvant activity and MDP increased the efficiency of SRBC-educated T cells. The role of T-derived lymphocytes in mediating the MDP adjuvant activity was also demonstrated in irradiated mice and in mice reconstituted with various splenic cellular types of donors which had received SRBC and MDP 24 hr earlier. In our experiments, the macrophage did not seem to be involved, since MDP did not increase the phagocytic capacity of peritoneal exudate cells and MDP- and SRBC-pretreated macrophages had no increased ability to induce an anti-SRBC immune response. These results demonstrate the importance of T lymphocytes as mediators of the adjuvant activity of MDP.     

Inhibition of macrophage accessory cell function in casein-treated B6C3F1 mice

Humoral immunity of casein-treated B6C3F1 mice was evaluated. Splenocytes from casein-treated mice sensitized in vitro exhibited a marked suppression in their antibody response to the T-dependent antigen, SRBC (82%) and T-independent antigen, DNP-Ficoll (80%). In contrast, a control response to the polyclonal antigen, lipopolysaccharide (LPS), was observed. Cell fractionation and crossover reconstitution assays of adherent (ADH) and nonadherent (NAD) splenocyte populations from vehicle and casein-treated mice indicated that: 1) ADH splenocytes from casein-treated mice were responsible for suppression of humoral responses, 2) NAD splenocytes from casein-treated mice reconstituted with vehicle or naive ADH cells abrogated the immunosuppression, and 3) suppression of humoral responses in cultures containing casein ADH splenocytes was due to this cell populations inability to function as accessory cells in humoral responses rather than induction of suppressor macrophages. Results from in vivo studies with casein-treated mice sensitized with sheep red blood cells or dinitrophenyl-Ficoll paralleled the in vitro results.     

In vitro immune response to sheep erythrocytes in macrophage depleted cultures. Restoration with interleukine 1 or a monokine from resident macrophages and stimulation by N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP)

The involvement of macrophages in the adjuvanticity of N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP) has been examined. The stimulation of the in vitro primary immune response to sheep red blood cells (SRBC) has been studied, because it is known that macrophages cooperate through the mediation of soluble compounds for the induction of the anti-SRBC response. The cultures depleted of macrophages by passing spleen cells on Sephadex G-10 were unable to give any response to SRBC. Their immune responsiveness was fully restored by the addition of either Interleukine 1 (IL 1) obtained from P388D1 cells or a factor able to replace macrophages (FRM) obtained from resident peritoneal macrophages. MDP alone, at any dose, was unable to induce any response in such macrophage depleted cultures, but it was able to enhance the antibody response of these cultures reconstituted with monokines, with the same characteristics in dose effect and timing dependence than in whole spleen cells.     

Effect of Bacillus Calmette-Guérin infection on delayed footpad reaction to Listeria monocytogenes

The role of peritoneal macrophages induced by Bacillus Calmette-Guérin (BCG) in the induction of immune responses to Listeria monocytogenes was studied in mice. The peritoneal macrophages from mice treated with BCG 14 days previously contained a high proportion of Ia-bearing macrophages (approximately 56%) and the cells showed not only a high level of listericidal activity but also a strong ability for presentation of listerial antigen to Listeria-immune T cells. An intraperitoneal inoculation with a low dose of Listeria, which can induce the maximal level of delayed footpad reaction (DFR) and positive migration inhibitory activity of macrophages in untreated mice, did not induce a detectable level of such responses in BCG-treated mice. The bacterial growth at an early stage of infection was suppressed by scavenger macrophages in these mice. On the other hand, BCG-treated mice showed the early development of DFR and macrophage migration inhibitory activity after an inoculation with a high dose of Listeria. It is revealed in transfer experiments that Listeria-pulsed peritoneal exudate cells induced by BCG elicited the highest level of DFR and positive migration inhibition of macrophages in normal mice at the earlier period of injection compared with Listeria-pulsed resident peritoneal cells. These results suggested that the increased activities of macrophages acting as scavenger cells and as antigen-presenting cells play important roles in the modification of immune responses to Listeria in BCG-treated mice.     

Higher ADCC of murine peritoneal cells after immunization with allogenic tumor cells as compared with stimulation by adriamycin, BCG, and thioglycolate

Normal peritoneal cells or spleen cells from C57BL mice could not lyse SRBC in an ADCC assay. After intraperitoneal injection of Adriamycin, BCG or thioglycolate the ADCC of peritoneal cells toward antibody-coated SRBC was elevated to 30% in contrast to the ADCC of spleen cells. However, peritoneal cells but not spleen cells of mice immunized with allogenic tumor cells (DBA SL2) showed ADCC levels at least two times higher than the levels observed after stimulation by other agents. Maximal ADCC levels (55.8%) were observed 10 to 15 days after immunization. Direct cytotoxicity towards SRBC increased to a maximum of 17.7% at 9 days after immunization. The effector cells in this system are thought to be macrophages, for ADCC activity was only present in the plastic-adherent cell fraction. Cell to cell contact was necessary for ADCC to occur; nonsensitized erythrocytes were not lysed when added to a mixture of effector cells and sensitized erythrocytes. Concentrations of antibody of 1 pg/ml were sufficient to induce ADCC, and effector cell to target cell ratios could be as low as 0.05. The finding that macrophages of mice immunized with allogenic tumor cells exhibit higher ADCC levels than macrophages elicited in other ways can contribute to the investigation of combined cancer therapy with antibodies and biological response modifiers.     

A novel rat monoclonal antibody directed against a population of activated mouse peritoneal macrophages

A hybridoma clone secreting rat monoclonal antibody (MAB) designated as 3F3.5F and which reacted with a population of activated tumoricidal mouse peritoneal macrophage (M phi) was produced by the fusion of mouse myeloma cells with rat spleen cells immunized against adherent BCG-activated mouse peritoneal exudate cells (adherent BCG-PEC). The antibody was cytotoxic and of the rat IgM class. The specific reactivity of the antibody with mouse primary cells and cell lines was examined by complement-dependent cytotoxicity and indirect immunofluorescence flow cytometry analysis. The antibody was found to bind to about 40% of the adherent BCG-PEC activated in vivo and elicited peritoneal macrophages activated in vitro by lymphokine and lipopolysaccharide (LPS), to about 35% of polymorphonuclear neutrophils (PMN) 15 hr after intraperitoneal injection of BCG, to about 30% of bone marrow cells from BCG-infected mice, to about 10% of P815 mastocytoma cells and to thioglycollate-induced PEC to some degree. It did not bind to other cells tested including BCG-induced peritoneal lymphocytes, non-tumoricidal PEC, thymocytes, spleen cells, resting bone marrow cells from normal mice, lymphomas, myelomas, fibroblasts, or macrophage-cell lines. Pretreatment of adherent BCG-PEC with MAB 3F3.5F and rabbit complement caused a considerable decrease in tumor cytotoxicity toward P815 cells, but the same pretreatment of non-adherent BCG-PEC had no inhibitory effect on natural killer activity for YAC-1 cells.     

Chemotactic effect of human recombinant interleukin 2 on mouse activated large granular lymphocytes

Human recombinant interleukin 2 (hrIL-2) was demonstrated in vitro to be chemotactic for mouse large granular lymphocytes (LGL) activated in vivo by virus infection. Peritoneal exudate cells harvested from virus-infected mice were used as a source of LGL. LGL collected from mouse hepatitis virus-infected mice at 3 days postinfection were a source for NK 1.1 positive natural killer (NK)/LGL. LGL collected from mice treated with antiserum to gangliotetraosylceramide and infected with lymphocytic choriomeningitis virus for 7 days were used as a source for Lyt-2 positive cytotoxic T lymphocytes (CTL)/LGL. Both NK/LGL and CTL/LGL responded chemotactically to hrIL-2, purified IFN-beta, and to crude cell-free washout fluids collected from the peritoneal cavity of virus-infected mice. hrIL-2 had chemotactic activity for virus-elicited granular and agranular lymphocytes but did not attract the contaminating macrophages, in contrast to IFN-beta, which displayed chemotactic activity for virus-elicited granular and agranular lymphocytes as well as macrophages. The migration to hrIL-2 was inhibited by a monoclonal antibody (7D4) to the IL-2 receptor, but treatment with 7D4 did not affect migration in response to IFN-beta. Microscopic examination of Wright's-Giemsa-stained migrated NK/LGL and CTL/LGL revealed that the majority of migrated LGL in either LGL population had a blast cell morphology (enlarged cells with rich basophilic cytoplasm). The frequency of cells bearing the LGL morphology within the virus-elicited nonadherent peritoneal exudate cell population was on incubation in vitro, stabilized by either hrIL-2 or IFN-beta. These data suggest that another important immunomodulating function of IL-2 may be to attract activated NK/LGL and CTL/LGL to sites of inflammation.     

Effect of modified low density lipoproteins on humoral immune response and functional activity of macrophages in mice

Intravenous injection of acetylated low density lipoproteins (acLDL) in mice in a dose of 0.5 mg per mouse decreased the intensity of humoral immune response to sheep red blood cells (SRBC) by 35%. The addition of acLDL to mouse peritoneal macrophages in vitro resulted in inhibition of Fc-dependent phagocytosis of SRBC and fourfold increased secretion of prostaglandins E2 by macrophages. Fc-dependent phagocytosis of SRBC was also found to be inhibited by oxysterols (25-hydroxycholesterol and 7-ketocholesterol), added to the incubation medium of macrophages in vitro in doses of 0.5-5 mg/ml. The conclusion was made that oxidative metabolism of cholesterol and arachidonic acid, contained in LDL, may mediate the immunomodulating effects of modified LDL.     

Enhanced antibody response in retrovirus-infected mice treated with endotoxin or nontoxic polysaccharide derivative

Friend leukemia virus (FLV) is a retrovirus which causes marked suppression of the immune response of genetically susceptible mice. In the present study the depressed antibody response to sheep erythrocytes by spleen cells from FLV-infected mice was partially reversed by injection of either a bacterial endotoxin or a nontoxic polysaccharide derivative directly into infected mice or by addition to spleen cell cultures from these mice immunized in vitro with sheep red blood cells (SRBC). The endotoxin and PS in a dose-related manner markedly increased the antibody responsiveness of the spleen cells to SRBC. Thus these results indicate that the nontoxic polysaccharide derivative has properties equivalent to the toxic endotoxin in enhancing the antibody responsiveness of FLV-suppressed spleen cells to a T-cell-dependent antigen like SRBC.     

Effects of increased major histocompatibility complex dosage on chicken monocyte-macrophage function

The influence of major histocompatibility complex (B complex) dosage on monocyte-macrophage function was examined using 4- to 6-week-old trisomic strain chickens. Di- (B15B15), tri- (B15B15B15), and tetrasomic (B15B15B15B15) progeny were produced from trisomic x trisomic crosses. Although mononuclear leukocytes from tetrasomics exhibited enhanced chemotactic activity in response to both f-met-leu-phe and Enterobacter cloacae culture supernatant as compared with that of cells from other groups, the ability to generate peritoneal exudate cells in response to intraperitoneal Sephadex stimulation was similar in all groups. Among peritoneal exudate cells, tetrasomic birds produced a significantly lower percentage of adherent macrophages with a higher proportion of Fc receptor-positive and CMTD-2-reactive macrophages than either disomic or trisomic chickens. Both tetrasomic and trisomic peritoneal macrophages exhibited a reduced phagocytic activity for unopsonized but not opsonized SRBC than was found with disomic macrophages. Thus, the number of major histocompatibility complex copies present in cells appears to influence monocyte-macrophage function.     

Recruitment of exogenous macrophages into metastases at different stages of tumor growth

Summary The endogenous tumor-associated macrophage content and recruitment of labeled peritoneal exudate cells into experimental murine B16 melanoma metastases has been examined at different stages in the progressive growth of metastatic lesions. The recruitment of thioglycollate-elicited peritoneal exudate cells and peritoneal exudate cells activated in vitro with muramyl dipeptide was studied. Tumor-associated macrophages and labeled peritoneal exudate cells were identified in paraffin sections by specific histochemical staining and their density in individual metastases measured morphometrically. The density of tumor-associated macrophages and exogenously recruited peritoneal exudate cells was high in very small lesions but decreased rapidly as a function of enlargement of metastases, MD:Aⁿ; where MD is macrophage density, A is the cross-sectional area of the lesion and n is a negative number. No significant difference was observed in the recruitment of activated and nonactivated peritoneal exudate cells. These results suggest that decreased recrutiment of macrophages from the circulation may explain the decrease in the density of tumor-associated macrophages as metastases grow and indicate that macrophage activation is not accompanied by enhanced localization and/or uptake of macrophages into metastases.     

Specific immunosuppression by passive antibody. V. Participation of macrophages in reversal of suppression by peritoneal exudate cells from immune animals

Thioglycollate-stimulated peritoneal exudate cells (PEC), harvested from mice immunized against sheep erythrocytes (SRBC) and transferred to normal syngeneic recipients, reverse the immunosuppression caused by passively administered anti-SRBC antibody. Macrophages purified from PEC on BSA gradients did not reverse immunosuppression; neither did suspensions of cells from mesenteric lymph nodes of immune mice. Mixtures of the purified macrophages and lymph node cells were fully capable of reversing immunosuppression. Thus, two types of cell, one a macrophage and one a lymphocyte, are required. Both must be compatible with the recipient mice at the H-2 complex. However, only the macrophages must necessarily be obtained from an immune donor. When “immune” macrophages were preincubated in vitro with “normal” lymph node cells before transfer to antibody-treated syngeneic recipients, a significant reversal of the immunosuppressive effect occurred. The ability of whole PEC or spleen cells to reverse the immunosuppressive effect of passive antibody is acquired rapidly after injection of a single low dose of antigen. Development of this ability precedes the appearance, in the circulation, of immunosuppressive antibody.     

Infection of macrophages with Friend virus: relationship to the spontaneous regression of viral erythroleukemia.

Resident peritoneal macrophages from normal mice were refractory to infection with the RFV or conventional strains of Friend virus (FV). Stimulation of DNA synthesis in the macrophage population by induction of an exudate in vivo or treatment in vitro with macrophage colony-stimulating factor resulted in productive infection following exposure to virus. Similarly, normal resident macrophages did not become infected in vivo following transfer to leukemic mice, while exudate macrophages did become infected. Bone marrow macrophage stem cells were stimulated to replicate and mature in clonal agar cultures in the presence of colony-stimulating factor. These replicating stem cells could be infected with RFV, as shown by virus production in the resultant progeny macrophages. Transfer of normal resident peritoneal macrophages to leukemic progressor mice caused regression of the disease. In contrast, transfer of normal bone marrow cells was ineffective in causing leukemia regression. During erythroleukemogenesis induced by RFV, macrophage precursor cells in all of the mice became infected with virus. In mice with a progressive and lethal leukemia, mature end-stage macrophages were produced which were also infected with virus. In mice in which the leukemia would later spontaneously regress, the infected stem cells were eliminated and the marrow became repopulated with uninfected cells. The resultant progeny macrophages which appeared in the peritoneal cavity were uninfected and thus capable of participating in or causing leukemia regression.     

Peritoneal exudate cells. I. Growth requirement of cells capable of forming colonies in soft agar

Mouse peritoneal exudate cells induced by thioglycollate medium can form colonies in soft agar with a plating efficiency of about 5% (0.6%–10%). Cells from an unstimulated peritoneal cavity form no colonies or have a plating efficiency of less than 0.001 %. These colony-forming cells from the peritoneal exudate are similar to bone marrow colony-forming cells in vitro in that they both require a substance(s) present in conditioned medium from L-cells or mouse embryo fibroblasts or the serum from endotoxin-treated mice for the initiation and the continuation of their growth. However, peritoneal exudate colony-forming cells have a much longer initial lag period (10–14 days) and can survive longer in the absence of L-cell conditioned medium than bone marrow colony-forming cells. Only mononuclear cells, presumably macrophages, are observed in peritoneal exudate colonies, whereas bone marrow cell colonies contain both polymorphonuclear cells and macrophages.     

Correlation between the lipopolysaccharide response of mice and the capacity of mouse peritoneal cells to transfer an antiviral state. Role of endogenous interferon

Freshly harvested mouse peritoneal cells, from normal lipopolysaccharide (LPS)-responsive (Lpsn) mice, were capable of transferring an antiviral state (to vesicular stomatitis virus) to "in vitro aged" mouse macrophages permissive for viral replication. The transfer of the antiviral state was completely abrogated by addition of antibody to interferon (IFN)-alpha/beta in the co-culture medium. In contrast, even large numbers of donor peritoneal cells from LPS-hyporesponsive (Lpsd) C3H/HeJ and C57BL/10ScCR mice did not transfer an antiviral state to target cells. Although peritoneal macrophages from Lpsd mice did not transfer an antiviral state to target cells, they were nevertheless found to be in an antiviral state when first placed in culture. Injection of mice with antibody to mouse IFN-alpha/beta rendered peritoneal macrophages from both Lpsn and Lpsd mice permissive for vesicular stomatitis virus. The decay of this initial antiviral state in peritoneal macrophages during in vitro culture was far more rapid for Lpsd mice than for normal mice. Addition of antibody to mouse IFN-alpha/beta markedly enhanced the in vitro decay of the antiviral state of peritoneal macrophages. Treatment of total peritoneal cells from Lpsn mice with LPS resulted in IFN production, whereas IFN was not detected in the cellfree medium of LPS-treated peritoneal cells from Lpsd C3H/HeJ and C57BL/10ScCR mice. Genetic studies with F1 hybrids between Lpsn and Lpsd mice and with Lpsn and Lpsd recombinant inbred strains revealed a striking correlation between the capacity of peritoneal cells to transfer an antiviral state and their capacity to produce IFN after stimulation with LPS, suggesting that closely linked, if not identical, genes are in some way involved in the transfer of antiviral state as well as in the LPS response by peritoneal cells of normal mice.     

Early production of a chemotactic factor to T lymphocytes by peritoneal macrophages

Supernatant fluids (SNF) were obtained from peritoneal exudate adherent cells stimulated in vitro with sheep red blood cells (SRBC) or BCG, and SNF collected at 6 and 24 hr were able to induce the migratory responses of rat leukocytes from the spleen and peripheral blood. The production of these SNF was dependent on protein active synthesis upon in vitro antigenic stimulation. The chemotactic activity from 6-hr SNF was inhibited by using several proteolytic enzymes and temperatures. We found the macrophages to be the producer cell of this activity, while the T cells were the target cells. The chemotactic activity from 6-hr SNF was found not to be due to IL-1. Six-hour chemotactic activity has not been reported previously.