Effect of recombinant human macrophage colony-stimulating factor 1 on immunopathology of experimental brucellosis in mice.

Brucella abortus injected into CBA mice replicated primarily in the spleen and liver, reaching a peak bacterial count in both organs about 7 days postinfection. The organism was eliminated from the liver but declined to a chronic phase in the spleen. The infection caused hepatosplenomegaly. An influx of macrophages into the two organs was monitored by quantitative Northern (RNA blot) analysis of the macrophage-specific marker lysozyme mRNA. Lysozyme mRNA was detectable in spleen and increased three- to fourfold during infection. In liver, lysozyme mRNA was initially undetectable, but at about the peak of infection it reached a level comparable to that in the spleen. Macrophage colony-stimulating factor 1 (CSF-1) has been reported to be elevated in the circulation of animals infected with B. abortus and is known to stimulate monocytopoiesis. To investigate the role of CSF-1 in pathogenesis, we studied the effect of further increasing the CSF-1 concentration by administration of recombinant human CSF-1. Since the infection is characterized by several distinct phases, recombinant human CSF-1 was administered at defined times relative to these phases. Pronounced effects were observed only when CSF-1 administration was begun during the developing acute phase. The consequences were decreased bacterial numbers in the spleen but an increase in the liver, reduced antibody generation, and increased hepatosplenomegaly. A feature of many chronic intracellular infections is immunosuppression. B. abortus caused a substantial diminution of responsiveness of spleen cells to T-cell mitogens, particularly concanavalin A. This action was mimicked by CSF-1 treatment of the animals prior to spleen cell isolation. The results suggest that CSF-1 plays a role in macrophage recruitment in brucellosis and that recruited macrophages contribute to the immunopathology and immunosuppression.

Suppressor cells and soluble factors from the spleens of UV-irradiated mice.

Several indices of splenic reactivity were measured daily after ultraviolet (UV) irradiation of mice. Spleen weight increased to a maximum after 5 days. Suppressor cell activity, manifested in various assays, peaked slightly earlier. The ability of passively transferred spleen cells to suppress the induction of contact hypersensitivity (CHS) was maximal after 4 days, as was the production of soluble suppressor factors in culture. The factors were detected by suppression of CHS in vivo and leucocyte adherence inhibition (LAI) in vitro. These assays appeared to detect different factors: an I-J- factor active in CHS assays and an I-J+ factor active in LAI assays. The latter factor required CD4-, CD8+ lymphocytes for its production.

The role of macrophages in experimental arthritis induced by Streptococcus agalactiae sonicate: actions of macrophage colony-stimulating factor (CSF-1) and other macrophage-modulating agents.

Intraperitoneal injection of Streptococcus agalactiae sonicated cells into Wistar rats causes a chronic relapsing polyarthritis resembling human rheumatoid arthritis. We report evidence favoring a role for macrophages in the pathology of this disease. S. agalactiae injected ip induced a high level of tumor necrosis factor release by peritoneal macrophages isolated subsequently, and had a similar effect when added to control peritoneal macrophages in culture. Ia antigen was induced on macrophages in both the peritoneum and affected joints following S. agalactiae injection. The role of macrophages in the disease process was studied by treating animals prior to S. agalactiae injection with varying concentrations of bacterial lipopolysaccharide (LPS), silica, and carrageenan, agents known to have a biphasic effect on macrophage function. They aggravated the pathology at low doses but prevented the disease at high doses. The most specific alteration of macrophage levels was achieved by injection of recombinant human macrophage colony-stimulating factor (CSF-1). Treatment with CSF-1 early in the disease lead to significant worsening of the pathology. Administration of CSF-1 after 2 weeks reactivated the disease and extended the chronic phase. These data in combination with previous findings are consistent with nonimmune, macrophage-mediated pathology for this model of arthritis. The results have implications for therapeutic application of CSF-1.

Distinctive molecular markers and biological activities in two tumour-specific murine T suppressor factors.

Murine suppressor factors (SF) in tumour-bearer serum (TBS) and tumour-bearer spleen cell culture supernatants (SCCS) were compared with regard to their molecular markers, cellular requirements and suppressive activities. Suppressor factors in SCCS bear the idiotype I-J, and markers recognized by B16G and anti-lipomodulin antibodies require Ly2+ I-J+ cells for their production and cells of the same phenotypes, for suppression to be manifested in leucocyte adherence inhibition (LAI) assays, are not immunoglobulin (Igh)-restricted and are active in both the afferent and efferent phases of the immune response. Suppressor factors from TBS resemble those from SCCS with regard to idiotype, I-J and lipomodulin-like markers, but in contrast, do not bind to B16G antibody, require Ly2+ but not I-J+ cells for suppression to be manifested in LAI assays, are Igh-restricted, and suppress delayed-type hypersensitivity (DTH) reactions only when injected in the afferent phase. These results show that the SF from the different sources in tumour-bearing mice are not identical; they may be different parts of a suppressor cascade, or they may belong to entirely separate suppressor cascades.

Effect of the novel triazinium zwitterion 1-methyl-3-phenyl-1,2,4-triazinium-5-olate on immunological responses and experimental brucellosis in mice.

The immunopharmacology of a novel triazinium zwitterion, designated JR-6, was studied in mice. Experiments show dose-response inhibition of antibody and delayed-type hypersensitivity (DTH) responses to sheep red blood cell antigens. Suppression of DTH was confirmed using trinitrochlorobenzene as a contact-sensitizing antigen. Using a model of experimental brucellosis in mice, it was found that JR-6 caused suppression of specific antibody and DTH reaction, as well as spleen weight, but a statistically significant increase in viable counts of Brucella abortus was not observed. Extensive short-term toxicology studies showed reduction in lymphocyte and neutrophil counts, and slow weight gain of treated mice. However, organ histology, liver function tests and biochemical profiles were normal.

Comparative immunopharmacology and toxicology of the bisbenzylisoquinoline alkaloids tetrandrine and berbamine.

Comparison of the bisbenzylisoquinolines tetrandrine and berbamine shows that both drugs are equipotent in terms of enhancement of antibody responses and suppression of delayed-type hypersensitivity (DTH) responses to sheep red blood cell antigens. Both compounds are also equally active when given to mice during the induction and expression phases of DTH. Using a model of experimental brucellosis in mice, it was found that both compounds did not affect antibody responses, while they caused equipotent suppression of DTH. By contrast, berbamine but not tetrandrine caused significant suppression of spleen weight. Also, berbamine caused a significantly greater enhancement of spleen colony counts of Brucella abortus than tetrandrine. Short-term toxicology studies showed no toxic effects at bioactive doses.

Effects of anti-idiotype vaccine on tumour growth and on production of soluble factors modulating cell-mediated immunity in vitro.

The previous observation, that single i.p. doses of a monoclonal anti-idiotypic antibody (MAIA) injected into BALB/c mice induced suppressor factors, was extended to multiple i.v. doses. These induced enhancing factors, which were produced in spleen cell cultures, required L3T4+ cells for their formation, lacked the IJ marker, and bound to anti-immunoglobulin, showing them to be antibodies. Selective immunoabsorption demonstrated two separate enhancing antibodies; both bound to MAIA but they had different affinities for specific and non-specific tumour antigens. Subsequently, single and multiple MAIA doses were tested in vivo for their effects on tumour growth. The single doses had variable effects depending on time of administration, and these effects were tumour-specific; the multiple doses strongly inhibited tumour growth when given before tumour challenge, but also had non-specific effects on another tumour as anticipated from the in vitro results.

Alkaptonuric ochronosis and multiple intracranial aneurysms.

Alkaptonuric ochronosis is a heritable disorder of tyrosine metabolism, with various systemic abnormalities related to pigment deposition and degeneration of collagen and other tissues, including the heart and aorta, though no cerebrovascular abnormalities have been reported. The authors report a patient with alkaptonuric ochronosis and multiple intracranial aneurysms presenting with subarachnoid hemorrhage. The ruptured aneurysm was surgically treated, with a satisfactory outcome. In view of the well-known association of other connective tissue disorders with intracranial aneurysms, a potentially causal relationship is suggested between cerebral aneurysms and alkaptonuric ochronosis.

Immunopathological investigations during the course of arthritis induced in rats by Streptococcus agalactiae.

A large single intraperitoneal injection of sonicated cells of Streptococcus agalactiae O90R induced polyarthritis in Wistar rats. The arthritis reaction score was monitored according to redness, edema, severity and deformity of rat ankle and wrist joints. The inflammation of joints was confirmed by radiology and histology. Acute arthritis was initiated within 48 h and the chronic form continued for more than 30 days. Although serum immunoglobulin was elevated within 48 h, anti-streptococcal antibody was detected only at later times (by enzyme-linked immunosorbent assay and Arthus-type hypersensitivity reactions) and neither serum nor splenocytes of arthritic rats were able to transfer disease to susceptible, normal rats. From these observations and the finding of streptococcal antigen in joint macrophages (by immunogold labelling) we conclude that arthritis is related to persistent streptococcal fragments rather than to antibody or immune complexes.

Comparison of T suppressor factors from tumour-bearing mice and mice immunized with a monoclonal anti-idiotypic antibody.

Certain dosage schedules of a monoclonal anti-idiotypic antibody (related to a murine bladder carcinoma) were found to induce suppressor factor production by syngeneic mice. This suppressor factor resembled the factor from tumour-bearing mice with respect to idiotype specificity, possession of molecular markers (reactive with anti-IJ and B16G antibodies) and production by Lyt2+IJ+ T cells in spleen cell cultures. The two factors differed with respect to Igh restriction in an in vitro assay (leucocyte adherence inhibition) and ability to suppress the induction of delayed-type hypersensitivity to tumour antigen.

Production of suppressor factors induced by ultraviolet irradiation or cis-urocanic acid requires Lyt-2+ lymphocytes.

Both ultraviolet (UV) irradiation and cis-urocanic acid (UCA) are reported to be associated with the suppression of contact hypersensitivity (CHS) and the induction of a soluble factor which suppresses leukocyte adherence inhibition (LAI) assays in vitro. The cellular origin of the suppressor factor (SF) has now been investigated in vivo and in vitro, using monoclonal antibodies against lymphocyte surface markers to deplete certain cell populations, namely T lymphocytes bearing the L3T4 (helper) and Lyt-2 (suppressor) markers. Depletion of Lyt-2+ cells from irradiated mice in vivo and from spleen cell cultures in vitro led to the elimination of detectable levels of SF. Depletion of L3T4+ cells had no such effect. Similarly, Lyt-2+ cells (but not L3T4+ cells) were shown to be necessary for the production of SF by normal spleen cells cultured with cis-UCA. These data suggest that the production of SF following UV irradiation may be related to the action of cis-UCA on Lyg-2+ lymphocytes.

Suppression of contact hypersensitivity by short-term ultraviolet irradiation: II. The role of urocanic acid.

Cis-urocanic acid (cis-UCA), produced from trans-UCA (a normal component of epidermis) by UV irradiation, suppressed cell-mediated immunological reactions in vivo and in vitro. It suppressed the development of contact hypersensitivity (CHS) when injected into mice, and it suppressed leucocyte adherence inhibition (LAI) reactions of previously sensitized lymphocytes exposed to antigen. Serum from mice injected with cis-UCA was also immunosuppressive in vitro. Normal murine spleen cells cultured with cis-UCA produced a non-dialysable factor which suppressed LAI reactivity. Trans-UCA was ineffectual in all of these systems. Both the ability of cis-UCA to induce an immunosuppressive serum factor and its ability to suppress CHS were abrogated by prior administration of cyclophosphamide, indicating that cis-UCA (normally from irradiated epidermis) stimulates T suppressor cells to produce the previously described suppressor factor in serum and the immunosuppression associated with short-term irradiation.

Auto-anti-idiotypic antibodies in mice hyperimmunized with a chemically induced bladder carcinoma.

Purified immunoglobulin from the serum of mice hyperimmunized with syngeneic tumour cells was compared with a monoclonal anti-idiotope antibody, obtained by immunizing mice with antibody to the same tumour. Both the immunoglobulin and the monoclonal antibody were specifically immunogenic in syngeneic (BALB/c) mice as tested by in vitro and in vivo assays of cell-mediated immunity. In both cases the reactivity was absent in immunoglobulin heavy chain gene complex-1-(Igh) allotype congenic (CB.20) mice, indicating Igh-restriction of immunogenicity. The active material in hyperimmune serum thus has the properties of auto-anti-idiotypic antibody, complementary to T cell idiotopes.

Suppression of contact hypersensitivity by short-term ultraviolet irradiation: I. Immunosuppression by serum from irradiated mice.

Serum from UV-irradiated mice was shown to be immunosuppressive in vitro and in vivo. It suppressed leucocyte adherence inhibition reactions of cells from sensitized syngeneic and allogeneic mice, and suppressed the development of contact hypersensitivity after passive transfer to mice. Supernatants of cultures of spleen cells from irradiated mice were also suppressive. The suppressive factors in sera and culture supernatants were non-dialysable. The suppressive effect of UV irradiation was abrogated by cyclophosphamide, but this restored reactivity was still inhibited by serum from irradiated donors; this suggests that the serum factor requires T suppressor cells for its production but not for its action. The level of interleukin 1 (IL-1) was not raised in serum from UV-irradiated mice; thus the serum factor appears not to be IL-1.

Tumor-specific idiotopes on suppressor factors and suppressor cells revealed by monoclonal anti-idiotope antibodies.

Two monoclonal anti-idiotope antibodies, previously found to induce tumor-specific cell-mediated immunity in mice, were examined for their relationship to tumor-associated suppressor factors (SF), produced in culture by spleen cells from tumor-bearing mice or present in sera from such mice. A leukocyte adherence inhibition assay was used to detect cellular immunoreactivity to tumor antigens and its inhibition by SF, using peritoneal cells from mice bearing tumor or sensitized with anti-idiotope antibody. The SF were specifically absorbed by the corresponding anti-idiotope antibodies coupled to a solid phase and were recovered by elution. They were also specifically neutralized by the addition of the respective antibodies to the assay system. Anti-idiotope antibody, used with complement to pretreat spleen cells from tumor-bearing mice, prevented these cells from producing SF in culture. Tumor antigen-reactive effector cells, suppressor cells, and SF thus share similar idiotopes, permitting their respective functions to be modulated by appropriate anti-idiotopes.

Cell-mediated immunoreactivity of tumor-bearing mice with myelin basic protein and related peptides.

Spleen cells (SC) from tumor-bearing mice and mice immunized with porcine myelin basic protein (MBP) reacted in vitro in E-rosette augmentation assays with MBP and certain of its constituent peptides. Peptides 1-115, 43-169, 64-83, 113-121, and 153-161 reacted significantly with both types of SC, while peptide 1-19 reacted only with SC from MBP-immunized mice. The phenomenon of specific inhibition of peptide reactivity by a moderate excess of a related protein was used to identify peptides as accessible epitopes of that protein. Peptide 113-121 was specifically inhibited by excess MBP when reacted with both types of SC, whereas peptide 64-83 was inhibited by excess MBP only when reacted with SC from MBP-immunized mice. These reactions suggest that the immunizing antigen in tumor-bearing mice is related to MBP but differs in epitopes associated with peptides 1-19 and 64-83.

Cellular requirements for the suppression of leucocyte adherence inhibition reactions by serum factors from tumour-bearing mice.

Tumour-bearing mice produce circulating serum factors that block cell-mediated immunological reactions in vitro. The mechanism by which these specific suppressor factors (SF) block leucocyte adherence inhibition (LAI) was studied. It had previously been shown that the antigen-reactive effector cells in the LAI assay are Ly-1+ T cells. We have now found that Ly-2+, I-J+ cells are required in the reactive cell population to observe the blocking action of SF from serum. Tumour-bearer spleen cells (containing Ly-2+, I-J+ lymphocytes) reacted only with the specific tumour-related serum factor (SF1) and relevant tumour antigen, to produce a non-specific suppressor factor (SF2). Specificity studies were conducted with contact-sensitized mice: hapten-specific spleen cells reacted only with hapten-related SF1 and the relevant hapten, to produce a similar SF2. SF2 differed from SF1 in suppressing allogeneic as well as syngeneic cells, in suppressing populations depleted of Ly-2+, I-J+ cells, and in being unaffected by absorption with immobilized anti-I-J antibody. Gel-filtration of SF2 revealed two forms of differing MW (greater than 190,000 and 20,000-50,000).

Circulating suppressor factors in mice subjected to ultraviolet irradiation and contact sensitization.

BALB/c mice subjected to a single dose of UV-B radiation showed suppressed contact hypersensitivity to trinitrochlorobenzene. The in vitro antigen reactivity of peritoneal cells from these mice was investigated using the leucocyte-adherence inhibition assay. These cells showed a high degree of reactivity with specific antigen. However, this reactivity, but not the reactivity of cells from non-irradiated sensitized mice, could be suppressed by serum from UV-treated sensitized mice. The suppressive effect of this serum could also be demonstrated on other syngeneic systems with unrelated antigens and was partially effective with allogeneic cells, indicating a lack of antigen specificity and genetic restriction. Suppressive properties were also found in serum taken from mice 3-5 days (but not at other times) after irradiation without subsequent sensitization.

Leukocyte adherence inhibition to myelin basic protein by cancer patients' T-lymphocytes in association with class II major histocompatibility antigens on monocytes.

Patients' leukocytes were shown to react consistently in tube leukocyte adherence inhibition (LAI) assays with myelin basic protein (MBP) at optimal concentration, whereas control leukocytes were nonreactive. Mononuclear cells from patients with cancer gave positive LAI reactions with MBP, but separated T-lymphocytes, monocytes, and neutrophils did not. The mononuclear cell LAI responses were blocked by monoclonal antibody (MAb) to monomorphic determinant of class II major histocompatibility complex (MHC) antigens and to T4+ (Leu-3a+) and T3+ (Leu-4+) T-cell differentiation antigens but not by antibody to class I MHC antigens or T8+ (Leu-2a+) antigens. MBP was thus recognized by helper T-cells, requiring presentation in association with class II MHC determinants on monocytes. MAb to class I and class II MHC antigens and to T8+ (Leu-2a+), T4+ (Leu-3a+), and T3+ (Leu-4+) differentiation antigens did not negate LAI mediated by peripheral blood lymphocytes to organ-specific cancer neoantigens (OSN) of crude extracts of allogeneic cancer, which had previously been shown to react with cytophilic antibody on allogeneic monocytes. When membrane OSN and leukocytes were autologous, T8+ (Leu-2a+) phenotypic T-cells also mediated LAI that was blocked by anti-T8 (Leu-2a) and anti-T3 (Leu-4). LAI induced by MBP was also negated by drugs that antagonize thromboxane-leukotriene biosynthesis, indicating that, in common with other LAI reactions, the terminal mediators of nonadherence are oxidative metabolites of arachidonic acid. In addition to clarifying the role of MBP in the cellular in vitro immunoreactivity of cancer patients, the present observations have important implications for theories of LAI. Sensitized leukocytes have different mechanisms for the recognition of antigens in different forms, and the antigen-stimulated leukocytes produce mediators that in a final common pathway induce nonadherence of surrounding cells through leukotriene-like metabolites.

Monoclonal antiidiotypic antibodies related to a murine oncofetal bladder tumor antigen induce specific cell-mediated tumor immunity.

Rat monoclonal antibody 6.10 recognizes a 175-kDa protein expressed in all BALB/c mouse transitional cell bladder carcinomas tested, in epithelial cells of the mouse embryo, and in a few epithelial cells of adult mice. The antibody was used as an immunogen to generate two mouse monoclonal antibodies, 21D9 and 43A10, which bind to idiotopes on antibody 6.10 associated with the binding site for the 175-kDa antigen. The antiidiotypic antibodies induced bladder tumor-specific, cell-mediated immunity when injected into syngeneic mice, as shown by delayed-type hypersensitivity reactions in vivo and leukocyte adherence inhibition reactions in vitro. Tumor specificity was demonstrated by employing as controls a chemically induced BALB/c fibrosarcoma, MCA-1511 (MCA, 3-methylcholanthrene), and its corresponding antiidiotypic antibody, 5.96. Lymphocytes from mice sensitized with antibody 21D9 or 5.96 specifically recognized antigens in extracts of BALB/c bladder carcinoma BTCC-1660 (BTCC, bladder transitional cell carcinoma) and sarcoma MCA-1511, respectively, as shown by leukocyte adherence inhibition reactivity. This reactivity was selectively abrogated by prior treatment of the sensitized cells with the appropriate antiidiotypic antibodies and complement. An antigen recognized in vitro by antibody 21D9-sensitized lymphocytes could be separated from BTCC-1660 extract by immunoabsorption with antibody 6.10 and elution with acidic buffer. Our findings indicate that the oncofetal antigen defined by antibody 6.10 is recognized by the immune system of syngeneic mice and suggest that antiidiotypic antibodies related to certain oncofetal antigens can be used to immunize against syngeneic tumors.