Immune responses in mice infected with lactic dehydrogenase virus. IV. Functional status of the macrophage during acute LDV infection.

Macrophages from uninfected and lactic dehydrogenase virus (LDV)-infected mice were compared with respect to the affinity and number of their Fc receptors for IgG2a; no differences were found regarding these parameters. When the uptake of DNP-BGG by macrophages from uninfected and acutely LDV-infected mice was compared, again no differences were observed. However, when the per cent membrane-bound DNP-BGG was determined as a function of time after antigen uptake in these two groups, more DNP-BGG was found membrane-bound on the macrophages from the LDV-infected mice, than on uninfected macrophages. In view of the fact that humoral immunity is enhanced during acute LDV infection, these data provide a positive correlation between increased retention of membrane-bound antigen and enhanced humoral immune responses.     

Suppression of development of diabetes in NOD mice by lactate dehydrogenase virus infection

It has been reported that lactate dehydrogenase virus (LDV) selectively infects a subpopulation of macrophages, thereby affecting the immune system. We studied the effects of LDV infection on the development of diabetes in non-obese diabetic (NOD) mice. Five-week-old female NOD mice were infected with LDV (10(8) ID50/mouse) and observed until 23 weeks of age. None of the 21-LDV-infected mice developed diabetes, whereas 10/14 (71.4%) uninfected mice did. Although the subpopulations of T cells and the percentage of Mac1-positive cells in the NOD murine spleen and the number of harvested peritoneal macrophages were unaffected by LDV infection, the proportions of Ia-positive peritoneal macrophages were significantly decreased in LDV-infected compared with uninfected mice (1.1 +/- 0.2%, 6.5 +/- 2.9%; P < 0.01). In LDV-infected NOD mice, insulitis of the same grade as that seen in uninfected NOD mice was observed. In another experiment, 3, 5, 10 or 16-week-old female NOD mice were infected with LDV. None of the mice infected with LDV at 3, 5 or 10 weeks of age developed diabetes and only one of six infected at 16 weeks of age did. These findings indicate that LDV infection suppresses the development of diabetes in female NOD mice by reducing the capacity of Ia-positive macrophages, and suggest that the development of human type 1 diabetes may be suppressed by certain viral infections.     

The peritoneal antigen-presenting macrophage: control and immunogenic properties of distinct subpopulations

The control of the immunogenic antigen-presenting capacity of different subpopulations of thioglycollate-induced peritoneal macrophages has been investigated. The experiments revealed the existence of two major subpopulations of macrophages, only one of which was highly efficient in educating antigen-specific T cells. The other subpopulation, while highly phagocytic, was devoid of antigen-presenting capacity. Further analysis, using specific antisera directed at H-2I region gene products, revealed that the immunogenic antigen-presenting population expressed H-2I region-controlled membrane antigens. Searching for cellular elements which control the differentiation of this antigen-presenting macrophage subpopulation, it was found that its function was strictly controlled by T cells. T cell-deficient mice (nu/nu) failed to generate a functional antigen-presenting macrophage subpopulation. Transplantation of mature T lymphocytes to T cell-deprived mice restored the immunogenic function of their antigen-presenting macrophages. The results obtained suggest the existence of heterogeneity of functions among macrophage subpopulations and add a new regulatory function for T cells.     

A tumor-associated lactic dehydrogenase virus suppresses the host resistance to infection with listeria monocytogenes

Infection of mice with lactic dehydrogenase virus (LDV) causes a lifelong chronic infection which is followed by alterations in immune responses during the acute phase of the infection. LDV was found to impair many functions of the reticuloendothelial system and to suppress macrophage-dependent immune responses. We tested the effect of acute infection with LDV in mice on the macrophage-mediated resistance to infection with a virulent bacterium. We found that LDV reduces the host's capacity to resist infection with Listeria monocytogenes.Many tumor lines which are transferred in mice are infected with LDV, and their growth rate is affected by the presence of the virus. It is therefore important to distinguish between immune alterations in tumor-bearing mice which are caused by the progressive growth of the tumor and those which are secondary to the viral infection.We tested whether LDV and a circulatory factor from tumor-bearing mice with similar suppressive effects on anti-Listeria immunity are two different entities or whether they are similar. We found that the factor is associated with LDV-infected tumor cells and is absent in LDV-free tumor cells. Other biological and physical characteristics supported the assumption that the tumor-associated factor is the LDV.     

Extensive cytocidal replication of lactate dehydrogenase-elevating virus in cultured peritoneal macrophages from 1-2-week-old mice

Indirect fluorescent antibody staining was used to examine the replication of lactate dehydrogenase-elevating virus (LDV) in primary cultures of peritoneal macrophages from BALB/c mice of different ages. Up to 80% of the total peritoneal macrophages from 1-2-week-old mice were susceptible to productive infection by LDV, though only 1-2% of the cells expressed detectable levels of IA antigen. The proportion of LDV-permissive peritoneal macrophages progressively decreased to 5-15% between 2 and 5 weeks of age of the mice. Macrophages from 9-day-old mice, when cultured in the presence of L cell conditioned medium, retained undiminished LDV permissiveness for at least 10 days in culture. The maximum proportion of LDV antigen-positive cells was detected between 8-10 h post infection of macrophages cultured from both 1-2-week-old and adult mice, concomitant with maximum LDV RNA synthesis. The LDV antigen positive macrophages disappeared between 12 and 48 h post infection. In cultures of macrophages from 9-10-day-old mice, the loss of infected cells was clearly due to cell killing, proving unequivocally that LDV replication is cytocidal. Disintegration of LDV-infected macrophages or phagocytosis of killed macrophages by surviving macrophages must be very sudden and complete since infected cells disappeared without the appearance of trypan blue-stainable cells in the culture. Ten cell lines established from macrophages of 2, 9, and 10-day-old mice all contained a small proportion of LDV-permissive cells (1-4%). Individual clones of one of the lines contained a similar small proportion of LDV-permissive cells.     

Lactic dehydrogenase virus infection prevents development of anti-nuclear antibody in (NZB x NZW)F1 mice; role of prostaglandin E2 and macrophage Ia antigen expression.

Persistent lactic dehydrogenase virus (LDV) infection prevents the development of antinuclear antibody (ANA) in (NZB x NZW)F1 mice. To assess the suppressive mechanisms, we focused on the role of the E series of prostaglandin(PGE), since previously we have shown enhanced production of PGE by macrophages from chronically LDV-infected mice. Treatment with PGE2 suppressed ANA titres more markedly in non-infected mice than in LDV-infected mice. Indomethacin enhanced ANA titres more markedly in LDV-infected mice than in non-infected mice. The number of Ia antigen positive(Ia+) macrophages was less in LDV-infected mice than in uninfected mice. The number of Ia+ macrophages was decreased in non-infected mice by PGE2 treatment and increased in LDV-infected mice by indomethacin treatment. These results suggest that the low ANA production in LDV-infected (NZB x NZW)F1 mice may be related to the decreased number of Ia+ macrophages and that one of the factors responsible for suppression of Ia+ macrophages may be the enhanced PGE2 production in the LDV-infected mice.     

Polyclonal B cell activation of IgG2a and IgG2b production by infection of mice with lactate dehydrogenase-elevating virus is partly dependent on CD4+ lymphocytes

Concentrations of IgM and IgG isotypes were determined by capture ELISA in plasma of Swiss, BALB/c and C58/M mice. Plasma IgG isotype concentrations, especially of IgM, IgG1 and IgG2a, varied considerably between mouse strains, batches of mice of the same strain and individual mice and as a function of age. Infection of the mice with LDV, which is known to replicate primarily in a subpopulation of macrophages, consistently resulted in a rapid elevation of plasma IgG2a (or of IgG2b in some Swiss nu/+ mice), but no plasma IgG increases were observed in mice immunized with inactivated LDV. Plasma IgG2a elevation after LDV infection was greatly delayed and reduced by depletion of the mice of CD4+, but not of CD8+, T cells by administration of protein-G-purified anti-CD4 or anti-CD8 mAbs, and completely inhibited by repeated treatment of the mice with cyclophosphamide. Treatment with anti-CD4 mAbs, or cyclophosphamide also greatly reduced the production of anti-LDV antibodies, while not significantly affecting the replication of LDV in these mice. Nude Swiss mice also failed to produce anti-LDV antibodies, though supporting normal LDV replication. Plasma IgM, IgG1, IgG2a and IgG2b levels increased in LDV-infected nu/nu mice, but similar changes were observed in uninfected mice. The results indicate that the LDV-induced polyclonal activation of B cells requires productive LDV infection of mice and is, at least partly, dependent on functioning CD4+ cells. They suggest that productive infection of the LDV-permissive subpopulation of macrophages leads to the activation of CD4+ T lymphocytes of subset 1 and their Spleen cells from 5-day LDV-infected BALB/c mice incorporated [3H]thymidine 2-3 times more rapidly in vitro than spleen cells from companion uninfected mice, whereas their responses to concanavalin A and lipopolysaccharide were reduced 60-70%.     

Altered macrophage antigen-presenting cell function following Friend leukemia virus infection.

To investigate the mechanism by which Friend leukemia virus (FV) causes immunosuppression, the ability of peritoneal macrophages to mediate antigen-specific T-cell activation following FV infection was examined. Decreased IL-2 production was observed when antigen-primed T cells were cultured with antigen-pulsed macrophages from mice infected with FV, compared to T cells cultured with macrophages from control mice. Macrophages from FV-infected mice demonstrated decreased phagocytic and pinocytic activity, suggesting that antigen uptake may be impaired in these cells. In addition, FV-infected mice had decreased numbers of MHC class II positive macrophages compared to uninfected controls, as measured by immunofluorescence. The alterations in antigen uptake and class II expression observed in macrophages from FV-infected mice may be the result of infection of these cells by FV, which was demonstrated by in situ hybridization using a FV-specific probe. The ability of FV to infect and modulate the functions of macrophages may account, at least in part, for the immunosuppression observed in FV-infected mice.     

Involvement of the spleen in the control of the immunogenic and phagocytic function of thioglycollate-induced macrophages

The immunogenic capacity of thioglycollate-induced peritoneal macrophages of adult splenectomized animals was compared to that of macrophages of sham-operated controls. Macrophages from splenectomized animals were found to be impaired in their function as antigen-presenting cells, both in the education of virgin initiator T lymphocytes and in the stimulation of antigen-specific T memory cells. Macrophages from splenectomized animals were also severely impaired in their phagocytic capacity, as assessed in an opsonin-dependent bacterial phagocytosis assay. However, they were not impaired in their ability to pinocytose soluble keyhole limpet hemocyanin. These results indicate that the spleen may play a decisive role in controlling the differentiation of peritoneal macrophages.     

Increased superoxide anion release by peritoneal macrophages in mice with a chronic infection of lactic dehydrogenase virus.

The function of macrophages in mice chronically infected by lactic dehydrogenase virus (LDV) was studied. Superoxide anion (O2-) release was examined by using peritoneal macrophages. O2- release increased markedly from 3 weeks to 12 months, but not at 1 week post infection. O2- release was 1.2 to 1.5 times greater than in uninfected mice. Increased O2- release from macrophages in LDV-infected mice may explain, at least in part, suppressive effects on tumour growth seen in the chronic phase of infection.     

Lactic dehydrogenase virus infection inhibits allergic eosinophil reaction and IL-5 gene expression in vivo.

The effects of lactic dehydrogenase virus (LDV) infection on allergic eosinophil reaction and IL-5 gene expression were studied. LDV infection suppressed antigen-induced eosinophil recruitment into the peritoneal cavity in sensitized mice. The elevation of IL-5 gene expression in the spleen and mesenteric lymph nodes 6 h after ovalbumin challenge was significantly suppressed in LDV-infected mice compared with uninfected (control) mice. The expression of the interferon-gamma and IL-2 genes in the spleen, but not in mesenteric lymph nodes, was significantly suppressed in LDV-infected mice compared with control mice. The present results suggest, that suppression of IL-5 gene expression by LDV infection may not be mediated by a mutual inhibitory mechanism between Th1 and Th2 cells.     

Lactic dehydrogenase virus replicates in somatic cell hybrids of mouse peritoneal macrophages and SV40-transformed human fibroblasts

Hybrid clones obtained from mouse peritoneal macrophages and SV40-transformed human Lesch-Nyhan fibroblasts are permissive for the replication of lactic dehydrogenase virus. Immunofluorescent assays show that all of the cells are infected. These hybrid cells produce interferon in response to LDV infection as had previously been shown for LDV-infected macrophages.     

Defective induction of antigen-reactive proliferating T cells in B cell-deprived mice. II. Anti-mu treatment affects the initiation and recruitment of T cells

Mice injected from day of birth onwards with rabbit anti-mouse IgM (antim-mu) antibodies were found to be B cell-deficient and defective for the induction of antigen-reactive proliferating T cells (TPRLF). This defective induction was not due to the absence of circulating antigen-specific antibodies since the daily injections of such antibodies during exposure to antigen did not restore the ability of anti-IgM treated animals to generate TPRLF. Analyzing the cellular events implicated in the induction of virgin antigen-reactive T cells, anti-mu-treated mice manifested impairment of the three interacting cell types involved in the induction of TPRLF. Thus, peritoneal and splenic antigen-presenting cells from such animals were impaired in their capacity to signal a primary antigen-specific T cell reaction. Their splenic lymphocytes could not function as initiator cells in transferring immunogenic signals to recruit TPRLF in normal recipients. Potent antigen-specific splenic initiator cells failed to induce the recruitment of specific TPRLF in anti-mu-treated mice. The defective induction of TPRLF in anti-mu-treated mice may be due to a functional impairment of cells expressing membrane-bound IgM molecules which seemingly play a central role in the transfer of immunogenic signals for the recruitment of antigen-specific circulating T cells. We suggest that splenic B cells function as initiators in the transfer of antigen-induced signals from peritoneal antigen-presenting cells to T cells. These seems to be the primary targets of anti-mu treatment.     

Effect of Anesthesia on the Immune System: Suppression of the Immunogenic Capacity of Macrophages and of Lymphocyte Transformation

Experiments were carried out aimed at investigating the effects of anesthesia without surgery on certain functional components of the immune system. We observed that the immunogenic antigen-presenting capacity of mouse peritoneal macrophages was significantly reduced one week post-anesthesia, with either injected or inhaled anesthetic drugs. On the other hand, an augmentation of the phagocytic activities of these macrophages was manifested. Hence, the impaired immunogenic potency could not be attributed to a reduced antigen uptake. Lymphocyte transformations, i.e., response to PHA and Con A were suppressed, whereas the response to LPS was augmented.     

Effect of Anesthesia on the Immune System: Suppression of the Immunogenic Capacity of Macrophages and of Lymphocyte Transformation

Experiments were carried out aimed at investigating the effects of anesthesia without surgery on certain functional components of the immune system. We observed that the immunogenic antigen-presenting capacity of mouse peritoneal macrophages was significantly reduced one week post-anesthesia, with either injected or inhaled anesthetic drugs. On the other hand, an augmentation of the phagocytic activities of these macrophages was manifested. Hence, the impaired immunogenic potency could not be attributed to a reduced antigen uptake. Lymphocyte transformations, i.e., response to PHA and Con A were suppressed, whereas the response to LPS was augmented.     

The IA antigen is not the major receptor for lactate dehydrogenase-elevating virus on macrophages from CBA and BALB/c mice

Double staining and labeling procedures were employed to simultaneously identify IA+ cells and cells permissive for the replication of the lactate dehydrogenase-elevating virus (LDV) in populations of peritoneal and spleen macrophages from BALB/c and CBA/J mice. No correlation between the expression of IA antigen and LDV permissiveness was observed. Only a low proportion of resident peritoneal macrophages expressed IA antigen and the antigen was lost within 1-2 days in culture whether or not L cell-conditioned medium was present, whereas the cells retained undiminished LDV permissiveness for 4 days and longer. Induction of IA expression on macrophages by injection of mice with concanavalin A, starch or indomethacin (up to 50% of the total macrophages became IA+), or elimination of IA+ macrophages by treatment with anti-IA monoclonal antibodies plus complement had little or no effect on the ability of the cells to support LDV replication in vivo or in vitro. LDV infection of untreated or concanavalin A-treated or starch-treated mice caused a drastic decline in IA+ peritoneal macrophages within 1 day, but the number of IA+ macrophages returned to pre-infection levels by 7 days post-infection without rendering the cells LDV permissive. Treatment of macrophages with trypsin destroyed the LDV receptor on macrophages with minimal loss of IA antigen from the cells. We conclude that the IA antigen is not the major receptor for infection of macrophages from BALB/c or CBA mice by LDV.     

The potentiating, mitogenic and inhibitory effects on lymphocytes in vitro, of macrophages in the lymph nodes of mice 'overloaded' with mycobacterial products.

The lymph nodes of mice overloaded with mycobacterial products, either by the injection of whole or ultrasonicated organisms, or as a consequence of severe infection with Mycobacterium ulcerans, contain phagocytic cells which cause spontaneous transformation of the lymph node cells in a low volume, high cell density culture system. This spontaneous mitosis is unaffected by trypsinization but is inhibited by specific antigen and by PHA, and eliminated by treatment with carbonyl iron. Replacement of the macrophages removed with carbonyl iron by a critical number of peritoneal cells, restores the spontaneous transformation. Normal lymph node, thymus or peritoneal lymphocytes will also undergo mitosis if small numbers of peritoneal cells are added to them. This phenomenon therefore appears not to be antigen-dependent, but is probably due to a mediator released from macrophages. The possible role of this phenomenon in the pathogenesis of mycobacterial disease and the 'overloading' of T lymphocytes in vivo is discussed, with reference to similar macrophage-dependent mechanisms reported in other systems.     

Antigen-presenting function of human peritoneum mesothelial cells

Mesothelial cells (MC) from human peritoneal omentum fragments obtained during surgical insertion of peritoneal catheters for continuous peritoneal dialysis in end stage renal failure (ESRF) patients were cultured in vitro. MC exhibited a phenotype different from macrophages, but MHC class II molecules were well expressed. Therefore MC lines were tested for antigen-presenting capacity by pulsing with soluble antigens (tetanus toxoid and purified protein derivative (PPD)) or with a corpusculate antigen (Candida albicans bodies). Autologous peripheral blood mononuclear cells (PBMC) depleted of adherent monocytes and cloned T cells generated from an individual matched for the MHC class II antigen DR2 were used to test antigen-presenting function. MC effectively presented the soluble and corpusculate antigens to autologous and MHC-compatible allogeneic lymphocytes, indicating that they are endowed with both endocytic/phagocytic activity and with processing/presenting capacity. Preincubation of MC with human recombinant interferon-gamma (IFN-γ) up-regulated MHC class II and intercellular adhesion molecule-I (ICAM-I) expression, but the effect on antigen-presenting function was not consistent. Since MC are an important component of the peritoneal environment, they may participate, along with macrophages, in activation of specific T cells and in the generation of local cell-mediated immunity to various pathogens.     

Antigen-presenting capacity of macrophages and dendritic cells in the peritoneal cavity of patients treated with peritoneal dialysis

In this study the antigen-presenting capacity of human peritoneal cells and the influence of continuous ambulant peritoneal dialysis (CAPD) were studied. On average 6% of the peritoneal cells were dendritic cells (DC), with no difference between CAPD and control peritoneal cells. DC were enriched by selecting for non-adherent, Fc receptor-negative, low density cells. A typical spot-like CD68 positivity was seen in DC, in contrast to the pancytoplasmic staining pattern in macrophages. Peritoneal DC morphologically and functionally showed features of cells belonging to the DC lineage. Peritoneal DC were superior antigen-presenting cells for both allo-antigen, and Candida albicans antigen or purified protein derivative. CAPD peritoneal macrophages were two- to threefold better stimulator cells for allogeneic T cells compared with control macrophages. The level of integrins/adhesins or MHC class I or II, as measured semi-quantitatively on the FACS, could not account for this phenomenon. In addition, a double chamber system showed that dialysate-activated macrophages produced soluble factors that could enhance DC-induced allogeneic T cell proliferation. In conclusion, human peritoneal cells contain a relatively high percentage of classical DC. CAPD treatment does not impair the antigen-presenting capacity of peritoneal cells, but instead up-regulates the allo-antigcn-presenting capacity of peritoneal macrophages.     

Presentation of mycobacterial antigens by human dendritic cells: lack of transfer from infected macrophages.

When exposed to a challenge of 10 Mycobacterium bovis BCG cells per antigen-presenting cell, most human monocytes engulf several organisms. In contrast, blood dendritic cells which are potent antigen-presenting cells for several antigens are not detectably phagocytic for mycobacteria. We investigated the possibility that infected macrophages might regurgitate antigens for presentation by populations of human blood dendritic cells. Macrophages were infected with M. bovis BCG, mixed with uninfected dendritic cells, and added to immune T cells, either bulk T cells or cloned populations from BCG vaccinees or patients recovering from tuberculosis. The macrophages were from donors who were mismatched to the T cells so that transfer of antigen to major histocompatibility complex-matched dendritic cells could be evaluated. As we describe, there was no evidence for the transfer of mycobacterial antigens from macrophages to dendritic cells in a form that was stimulatory for the T cells.