Assessment of cell-mediated hypersensitivity against coxsackievirus B3 viral-induced myocarditis utilizing hypertonic salt extracts of cardiac tissue.

Hypertonic KCl extracts prepared from heart tissues of adolescent CD-1 mice inoculated with coxsackievirus B3 (CVB3) were tested for antigenicity in evaluating cell-mediated sensitivity to CVB3 virus utilizing the agarose droplet cell-migration-inhibition assay. Immune mouse peritoneal exudate cells (IMPEC) from mice immunized against CVB3 virus and Freund's complete adjuvant were specifically inhibited in the cell-migration-inhibition assay with graded doses of KCl-extracted antigen and purified protein derivative (PPD). Unimmunized for "normal" mouse peritoneal exudate cells (NMPEC) were not inhibited in the presence of the CVB3 KCl extracts. KCl heart extracts from mice inoculated with a cardiotropic strain of antigenically distinct mengovirus failed to inhibit CVB3 IMPEC, and noncardiac KCl extracts of liver and spleen from CVB3-inoculated mice also failed to inhibit cellular migration of CVB3 IMPEC. Reciprocal specificity experiments utilizing KCl-extracted antigens from mice infected with antigenically distinct cardiotropic mengovirus failed to inhibit cellular migration of IMPEC from mice immunized against the mengovirus. Serum-blocking power experiments indicate the antigenic KCl extracts failed to bind virus-neutralizing antibodies, indicating absence of detectable quantities of virion antigens. The results indicate that inoculation of mice with CVB3 virus results in the appearance of a new antigen(s) in cardiac tissue reacting with CVB3-IMPEC, but not with mengovirus IMPEC.     

RNA extracts with polyadenylic acid sequences transfer specific sensitivity for a low molecular weight antigen (MW 486).

RNA prepared from the lymphoid tissues of guinea pigs specifically sensitized to mono(p-azobenzene-arsonate)-N-chloroacetyl-l-tyrosine (ARSNAT) (MW = 486) was fractionated by oligo(dT)-cellulose affinity chromatography. Two fractions designated as I and II were eluted from the column. Fraction II, binding to the column and containing polyadenylic acid sequences, transferred ARSNAT or keyhole-limpet-hemocyanin (KLH) sensitivity to nonsensitized guinea pig peritoneal exudate cells (GP-PEC) in 14 experiments. Fraction I was unable to transfer KLH or ARSNAT sensitivity to GP-PEC. The amount of Fraction II needed to transfer ARSNAT sensitivity was 10 times less than previously reported. Synthetic nonlymphoid cell poly(A) tested in this system failed to transfer ARSNAT or KLH sensitivity to GP-PEC. Both Fractions I and II were inactivated by ribonuclease. The results suggest a possible messenger function for the poly (A)-containing RNA fractions.     

Involvement of natural killer cells in coxsackievirus B3-induced murine myocarditis

The role of natural killer cells in the temporal development of coxsackievirus B3-induced myocarditis in adolescent CD-1 male mice was examined. Inoculation of purified CVB3m induced maximum NK cell activity in the splenic populations at 3 days postinoculation (p.i.) as assessed by lysis of YAC-1 cells; maximum virus titers in heart tissues were also found at day 3 p.i. Mice depleted of NK cells after injection of anti-asialo GM1 antiserum i.v. had decreased NK cell activity, increased CVB3m titers in heart tissues, and exacerbated myocarditis. Although lesion number was not increased in heart tissues of the latter mice, lesions in these mice exhibited increased myocyte degeneration and dystrophic calcification above that found in lesions of mice inoculated with CVB3m only. No alteration in interferon titers were observed in CVB3m-infected mice treated with anti-asialo GM1 antiserum as compared with normal CVB3m-infected mice. Measurements of splenic NK cell activity in mice inoculated with doses of 10(2) to 10(8) PFU of CVB3m per mouse or UV-irradiated virus suggest that replication of CVB3m is required for NK cell activation. An amyocarditic variant of CVB3m (ts5R) was shown to replicate in heart tissues and to elicit NK cell activity comparable to that elicited by CVB3m. Therefore, the data suggest that NK cell activation depends on virus replication and that these cells provide some protection against CVB3m-induced myocarditis by limiting virus replication in heart tissues.     

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.     

Ontogenic development of the reactivity of macrophages to antigenic stimulation

Experiments were performed to test the ability of macrophages from newborn mice to participate in immune reactions. It was found that peritoneal cells from 4-day-old mice injected at birth with thioglycollate did not reconstitute reactivity to Shigella in adult, irradiated mice, while normal adult macrophages did.The total yield of peritoneal exudate cells (PEC) was relatively low, yet the number of precursor cells of macrophages in the newborn spleen was found in significantly higher concentrations than in the adult. The number of precursor cells in the spleens of 0–3-day-old mice did not increase in response to antigenic stimulation, indicating that they too are unable at this stage to develop reactivity to immunological signals.     

Plasminogen activator and MIF-like activities in Kirsten virus transformed mouse NIH culture fluids.

Kirsten virus transformed mouse NIH cells produce both a macrophage migration inhibition activity for guinea pig and mouse peritoneal exudate cells and a plasminogen activator. The migration inhibition factor activity exhibited thermal stability up to 80°C while the plasminogen activator was inactivated after 15 minutes at 70°C. Separation of these activities was achieved by absorption of the migration inhibition activity on agarose-fucosamine or high speed centrifugation.     

Effect of injection of adult mouse peritoneal macrophages on suppressor cell activity in neonatal mice

Injection of adult mouse peritoneal exudate cells into newborn mice results in a premature decrease of splenic suppressor cell activity in the neonates. The effect becomes apparent 4–5 days after ip injection of 10–15 × 10⁶ thioglycollate-induced peritoneal exudate cells into mice on the day of birth. The macrophage in the peritoneal exudate is the responsible cell type. The effect is not H-2 restricted or strain limited. Heat-killed peritoneal exudate cells or peritoneal cells from unstimulated donors can also decrease neonatal suppressor cell activity prematurely. Adult spleen cells, injected into neonatal mice, do not affect suppressor cell activity. The data are discussed in light of the hypothesis that macrophages control suppressor activity in neonatal mice and that an increase in the number and/or function of macrophages shortly after birth results in a decrease in the number and/or function of suppressor cells, allowing for immunological competence to emerge.     

Activity and Properties of Macrophage Migration Inhibitory Factor produced by Mixed Lymphocyte Cultures

THE macrophage migration test is an in vitro demonstration of delayed hypersensitivity. Supernatant fluids of sensitive lymphocytes cultured for 24 h in the presence of specific antigen contain migration inhibitory factor (MIF) that arrests the migration of macrophages of unsensitized animals in vitro¹,². In vivo, it induces delayed skin reactions³. The use of the macrophage migration test, based on differences of transplantation antigens in donor and recipient, to show histocompatibility has been suggested⁴. The test was also recommended as an indicator of immunological reactivity after organ transplantation, to demonstrate impending rejection⁵. It can demonstrate homograft sensitivity, for migration of peritoneal exudate cells (containing lymphocytes and macrophages) of CBA mice previously sensitized by grafts from A/Jax donors was inhibited when they were mixed with peritoneal exudate cells of the donor strain. However, histocompatibility was not demonstrated, for mixtures of peritoneal exudate cells of ungrafted CBA mice and A/Jax mice migrated regularly during the 24 h test⁶.     

Refractoriness to migration inhibitory factor of macrophages of LPS nonresponder mouse strains.

The responsiveness to macrophage migration inhibitory factor (MIF) of peritoneal exudate cells (PEC) from the LPS unresponsive C3H/HeJ and C57BL/10ScCR mice was assessed by the indirect agarose microdroplet macrophage migration inhibition assay. No migration inhibition with PEC from C3H/HeJ nor C57BL/10ScCR mice was detected, whereas PEC from both C3H/HeN and C57BL/10Sn mice were significantly inhibited by even a 1/32 dilution of MIF-containing supernatants. Responsiveness to MIF of C3H/HeJ PEC could, however, be induced. In vivo inoculations of Mycobacterium bovis, strain BCG, 7 days before in vitro assay rendered C3H/HeJ PEC responsive to MIF. The lack of responsiveness to MIF by C3H/HeJ PEC appeared related to some form of suppression, since a mixture of PEC from C3H/HeN mice with 10 to 15% PEC from C3H/HeJ mice resulted in undetectable migration inhibition at any MIF dilution. In contrast to the usual lack of responsiveness of their macrophage to MIF, C3H/HeJ mice were able to produce MIK in response to PPD as well as their counterpart C3H/HeN mice after BCG sensitization. These results demonstrate that macrophages from C3H/HeJ and C57BL/10ScCR mice are unable to be inhibited in their in vitro migration of MIF (possibly being directly or indirectly influenced by a suppressor cell), whereas lymphoid cells from at least one of these strains, the C3H/HeJ mice, can produce MIF in response to antigenic stimulation.     

Generation of cytolytic T lymphocytes in vitro. VIII. failure of anti-RS antisera to inhibit the generation of cytolytic T lymphocytes or cytolytic T lymphocyte activity.

Antibody reactive with "recognition structures" (RS) of mouse lymphoid cells for alloantigens (anti-RS) was prepared by immunization of F1 hybrid mice with parentalstrain lymphoid cells or with antibody produced in one parental strain against alloantigens of the other parental strain. Such antisera prevented generation of the "product of antigenic recognition" (PAR) that is produced within a few hours in cultures prepared with a mixture of lymphoid cells from genetically disparate mice. However, treatment of responding lymphoid cells with anti-RS sera and complement did not inhibit generation of cytolytic T lymphocytes (CTL) in mixed lymphocyte cultures (MLC). Treatment of cells obtained from MLC with anti-RS sera and complement failed to inhibit cytolytic activity of such cells for specific alloantigens.     

Augmentation of mouse natural killer cell activity by LS 2616, a new immunomodulator

The quinoline-3-carboxamide LS 2616 administered to mice in drinking water increased spontaneous cytotoxicity against YAC-1 cells in a dose-dependent manner. The enhancement of spontaneous cytotoxicity was found to be mediated by NK cells, as judged by their lack of adherence to nylon wool columns, relative resistance to treatment with antibodies to Thy-1.2 and complement, and almost total abrogation after depletion of asialo-GM1+ cells. Enhancement of NK activity was evident after 2 days of treatment, was maximal after 4 days, and remained elevated during the 14-day exposure period studied. NK activity returned to control levels 4 days after cessation of treatment. NK activity was significantly increased in spleen, peripheral blood, lymph nodes, and bone marrow of LS 2616-treated mice, while activity in peritoneal exudate cells and thymus remained low. LS 2616 was able to elevate NK activity in several mouse strains studied, including mice homozygous for the beige gene. Serum interferon levels were not increased during treatment with LS 2616. Combined injection of the interferon inducer Poly I:C and LS 2616 did not increase NK activity above that of animals injected with Poly I:C alone. However, Poly I:C, in contrast to LS 2616, increased NK activity in peritoneal exudate cells. Studies at the single cell level revealed that LS 2616 increased NK activity by increasing the number of lytically active cells via recruitment of new target-binding cells and not by increasing the lytic activity of pre-existing binders.     

Migration inhibition by an agarose microdroplet assay: monitoring of tumor-associated antigens on a simian virus 40-induced sarcoma.

Macrophage migration inhibition assays, with a direct agarose microdroplet method, were used to monitor TAA activity of preparations of SV-40-induced mKSA cells. These preparations included cell-free crude membranes, papain-solubilized and NP40 detergent-solubilized membrane extracts from mKSA tumor cells. The assay was extremely sensitive and could detect migration inhibition reactivity with all three types of antigenic preparations with concentrations as low at 250 ng protein/ml. The reactivities were quite reproducible from experiment to experiment using the same or different lots of these antigen preparations, and the reactivities were specific in that peritoneal exudate cells from BALB/c mice, immunized with antigenically unrelated but syngeneic plasmacytomas, were not inhibited by these antigens. The results demonstrated the usefulness of this assay in rapidly detecting small concentrations of partially purified TAA preparations by using small number of immune cells.     

Antiviral effect of lymphokine-activated killer cells: chemotaxis and homing to sites of virus infection.

Lymphokine-activated killer (LAK) cells generated from C57BL/6 mouse spleen cells cultured with interleukin-2 are effective prophylactically against virus infection when inoculated at the site of virus injection. To predict the therapeutic efficacy of LAK cells, we determined whether LAK cells would home to sites of virus infection. In vitro, LAK cells responded chemotactically to cell-free peritoneal exudate fluids collected from virus-infected mice and to preparations of purified beta interferon. In vivo, radiolabeled LAK cells injected intravenously accumulated in the peritoneal cavities of intraperitoneally infected mice in amounts three to eight times greater than in uninfected mice. This ability to respond to chemotactic agents and migrate into sites of virus infection may make LAK cells useful as antiviral therapeutic agents.     

Mouse peritoneal cells confer an antiviral state on mouse cell monolayers: role of interferon.

Vesicular stomatitis virus and encephalomyocarditis virus do not multiply in the majority of peritoneal macrophages freshly explanted from 4- to 8-week-old male or female mice. However, when peritoneal macrophages were cultivated in vitro for 3 to 5 days, these cells became permissive for both viruses. The loss of antiviral state in "aged" macrophages paralleled a significant decrease in the intracellular levels of (2'-5')oligo-adenylate synthetase activity. Although biologically active interferon was not detected in the nutrient medium of macrophage cultures, freshly harvested peritoneal cells could confer an antiviral state on monolayer cultures of mouse cells (aged macrophages, embryonic fibroblasts, and L cells) but not on heterologous chicken embryo, rabbit kidney, or human cells infected with vesicular stomatitis virus or encephalomyocarditis virus. The conferred antiviral state required at least 7 h to develop in target cells and was totally inhibited by the presence of antibody to mouse interferon alpha/beta but not to interferon gamma in the cocultures. Heterologous guinea pig and rabbit peritoneal cells could not transfer an antiviral state to target mouse cells. Donor peritoneal cells from mice preinjected with antibody to interferon alpha/beta could not transfer an antiviral state to target mouse cells. This ensemble of results indicating that freshly harvested peritoneal cells transfer interferon (which is responsible for inducing an antiviral state in susceptible mouse target cells) adds further experimental evidence that interferon is spontaneously expressed in normal mice and plays an important role in maintaining some host cells in an antiviral state.     

Variation among mouse strains in responsiveness to migration inhibitory factor.

Mineral oil-induced peritoneal exudate cells (PEC) from 10 different inbred mouse strains were tested for their responses to macrophage migration inhibitory factor (MIF). PEC from 5 out of 10 mouse strains responded to MIF, PEC from BALB/c mice showed an intermediate responsiveness, and PEC from A/J, C3H/HeJ, CBA/N, and C57BL/10ScCR mice were refractory to MIF. MIF responsiveness was not linked to the H-2 complex. However, a possible link between responsiveness to LPS and MIF was suggested, since the mouse strains not responding to MIF were previously reported to be deficient for responses to LPS.     

gamma delta+ T cells regulate major histocompatibility complex class II(IA and IE)-dependent susceptibility to coxsackievirus B3-induced autoimmune myocarditis.

Coxsackievirus B3 (CVB3) infection induces myocardial inflammation and myocyte necrosis in some, but not all, strains of mice. C57BL/6 mice, which inherently lack major histocompatibility complex (MHC) class II IE antigen, develop minimal cardiac lesions despite high levels of virus in the heart. The present experiments evaluate the relative roles of class II IA and IE expression on myocarditis susceptibility in four transgenic C57BL/6 mouse strains differing in MHC class II antigen expression. Animals lacking MHC class II IE antigen (C57BL/6 [IA+ IE-] and ABo [IA- IE-]) developed minimal cardiac lesions subsequent to infection despite high concentrations of virus in the heart. In contrast, strains expressing IE (ABo Ealpha [IA- IE+] and Bl.Tg.Ealpha [IA+ IE+]) had substantial cardiac injury. Myocarditis susceptibility correlated to a Th1 (gamma interferon-positive) cell response in the spleen, while disease resistance correlated to a preferential Th2 (interleukin-4-positive) phenotype. Vgamma/Vdelta analysis indicates that distinct subpopulations of gamma delta+ T cells are activated after CVB3 infection of C57BL/6 and Bl.Tg.Ealpha mice. Depletion of gamma delta+ T cells abrogated myocarditis susceptibility in IE+ animals and resulted in a Th1-->Th2 phenotype shift. These studies indicate that the MHC class II antigen haplotype controls myocarditis susceptibility, that this control is most likely mediated through the type of gamma delta T cells activated during CVB3 infection, and finally that different subpopulations of gamma delta+ T cells may either promote or inhibit Th1 cell responses.     

The suppressive effect of peritoneal exudate macrophages on production of antibody to sheep erythrocytes in vitro

The effects of peritoneal exudate macrophages on antibody response to sheep erythrocytes (SRBC) were investigated in mice. Peritoneal exudate macrophages obtained from mice injected intraperitoneally with proteose peptone or Corynebacterium parvum 4 days earlier had stronger ability to phagocytize and degrade SRBC than normal resident macrophages. These macrophages suppressed antibody formation to SRBC in vitro as well as in vivo. This suppression was overridden by increasing the amount of SRBC and diminished completely by pretreatment of the macrophages with iodoacetate and partly by pretreatment with 2-deoxyglucose, both known to be inhibitors of phagocytosis, but not by addition of indomethacin to the in vitro culture. These results suggest that the suppression of antibody response by peritoneal exudate macrophages was due to the increased activity of these cells as scavenger cells, resulting in a reduced amount of effective antigenic stimulation, and that it was not mediated by a prostaglandin-dependent mechanism. The scavenger function of these macrophages may be due to Ia-negative macrophages.     

B2 but not B1 cells can contribute to CD4+ T-cell-mediated clearance of rotavirus in SCID mice

Studies utilizing various immunodeficient mouse models of rotavirus (RV) infection demonstrated significant roles of RV-specific secretory immunoglobulin A (IgA), CD4+ T cells, and CD8+ T cells in the clearance of RV and protection from secondary infection. Secretion of small but detectable amounts of IgA in RV-infected alphabeta T-cell receptor knockout mice (11) and distinctive anatomical localization and physiology of B1 cells suggested that B1 cells might be capable of producing RV-specific intestinal IgA in a T-cell-independent fashion and, therefore, be responsible for ablation of RV shedding. We investigated the role of B1 cells in the resolution of primary RV infection using a SCID mouse model. We found that the adoptive transfer of unseparated peritoneal exudate cells ablates RV shedding and leads to the production of high levels of RV-specific intestinal IgA. In contrast, purified B1 cells do not ablate RV shedding and do not induce a T-cell-independent or T-cell-dependent, RV-specific IgA response but do secrete large amounts of polyclonal (total) intestinal IgA. Cotransfer of mixtures of purified B1 cells and B1-cell-depleted peritoneal exudate cells differing in IgA allotypic markers also demonstrated that B2 cells (B1-cell-depleted peritoneal exudate cells) and not B1 cells produced RV-specific IgA. To our knowledge, this is the first observation that B1 cells are unable to cooperate with CD4+ T cells and produce virus-specific intestinal IgA antibody. We also observed that transferred CD4+ T cells alone are capable of resolving RV shedding, although no IgA is secreted. These data suggest that RV-specific IgA may not be obligatory for RV clearance but may protect from reinfection and that effector CD4+ T cells alone can mediate the resolution of primary RV infection. Reconstitution of RV-infected SCID mice with B1 cells results in the outgrowth of contaminating, donor CD4+ T cells that are unable to clear RV, possibly because their oligoclonal specificities may be ineffective against RV antigens.     

Cytotoxic T cells in the peritoneal cavity of mice infected with ectromelia virus.

Peritoneal exudate cells from mice infected with ectromelia virus were cytotoxic for virus-infected target cells as measured in a 51Cr release assay. Cytotoxic activity seemed to be T cell-dependent as it was largely abolished by treatment with anti-theta serum and complement but was not impaired by macrophage depletion. The kinetics of development of cytotoxicity in the peritoneal cavity lagged behind spleen cytotoxicity by 1-2 days. Peak activity in peritoneal cells was present about 6 days after intravenous infection with virus. These studies suggest that macrophages present in the free peritoneal cell populations of ectromelia-infected mice are not cytotoxic for virus-infected target cells. The effect of macrophages in virus clearance is therefore likely to be due to phagocytic rather than cytotoxic effects.