Radiation leukemia virus-transformed immunocompetent T cells. II. Antigen-induced macrophage migration inhibition factor and leukocyte migration inhibition factor production

OVA-specific T cells were immortalized by infection with radiation leukemia virus (RadLV). Some clones derived from such population were shown to exhibit helper activity. We then tested clones without such function and found among them some that secreted macrophage migration inhibition factor (MIF) and leukocyte migration inhibition factor (LIF) upon exposure to the antigen in vitro. The lymphokine-producing clones, which were Thy-1+, Ly-1+ and Ly-2-, did not secrete MIF and LIF constitutively. Like other antigen-specific T cells, the immortalized clones could not be stimulated by free soluble antigen but required macrophages for presentation and for triggering the lymphokine production. The antigen-activated clones exclusively produced MIF and LIF, but not interleukin 2 or colony-stimulating factor. They neither provided helper activity nor induced delayed-type hypersensitivity. The data suggest that the T-cell clones carry the antigen receptors and that their antigen-inducible biological function is restricted to the migration inhibitory factor production.     

Reactivation of herpes simplex virus is associated with production of a low molecular weight factor that inhibits lymphokine activity in vitro

Peripheral blood mononuclear cells obtained during recrudescent Herpes simplex virus (HSV) infection and stimulated with UV-inactivated viral antigen (UV-HSV) for 24 hr produced a low molecular weight (dialyzable) factor that inhibited lymphokine activity. This factor prevented the expression of leukocyte inhibitory factor (LIF) activity, but not its production. It was not made in UV-HSV-stimulated cultures grown in presence of 2 X 10(-6) M indomethacin nor in cultures of peripheral blood mononuclear cells obtained during convalescence or quiescence (greater than 4 days from onset of clinical symptoms) or from seropositive controls without a history of recurrent HSV disease. Dialyzable inhibitory factor production required OKM1+, OKT8+, and OKIa+ cells as determined by complement-mediated lysis with monoclonal antibody. Dialyzable inhibitory factor activity was associated with a trypsin-sensitive 8.2 K fraction as determined by Sephadex chromatography followed by sodium dodecyl sulfate-acrylamide gel electrophoresis.     

A suppressive lymphokine of platelet cytotoxic functions

The in vitro stimulation of mononuclear cells from human peripheral blood with mitogens is known to induce the release of factors (monokines and lymphokines) that possess distinct biologic activities. The present data describe the presence in Con A- and antigen-stimulated T cell supernatants (of man or rat) of a factor able to inhibit, in a dose-dependent manner, the platelet cytotoxicity toward the young larvae of Schistosoma mansoni. The production of oxygen metabolites by IgE-coated platelets, stimulated by anti-IgE or the specific antigen, was, likewise, strongly inhibited by this lymphokine. The producing T lymphocyte subpopulation was identified as OKT 8+. This suppressive lymphokine of platelet functions had an m.w. of 15,000 to 20,000 and a pI of 4.6. It was heat- and acid-stable and sensitive to trypsin and proteinase K, but neuraminidase had no effect on its activity. This platelet suppressive activity was specifically absorbed by platelet membrane, suggesting its action through the binding to a receptor.     

Enhancement of DTH response by cholera toxin B subunit inoculated intranasally together with influenza HA vaccine

The effects of B subunit of cholera toxin (CTB) on delayed-type hypersensitivity (DTH) response to influenza vaccine derived from influenza virus A/PR/8/34 (PR-8, HlNl) virus were investigated in B10 mice that were immunized intranasally with both influenza vaccine and CTB. The result showed that intranasal inoculation of this combination augmented DTH response to influenza vaccine, which reached its peak 6 days after inoculation, and also induced accelerated DTH response upon a second inoculation of influenza vaccine alone 4 weeks later, that the cross-reactive DTH response to PR-8 vaccine was elicited by the injection of the different influenza A-type virus vaccine into the footpad of the vaccinated mice, but was not by influenza B-type virus vaccine, that the DTH-mediating T cells were detected selectively in the lungs of mice that received the nasal inoculation of the vaccine and CTB together, but that subcutaneous inoculation of this combination failed to induce DTH-mediating T cells in the lungs. These results, together with the previous papers (Tamura et al, Vaccine 7: 257-262; 314-320, 1989), suggest that CTB could augment both humoral and DTH responses against influenza vaccine in the respiratory mucosal tract.     

O antigen-dependent mutant of bacteriophage T5.

The administration of cyclophosphamide (50 to 100 mg/kg) at 48 to 72 h before removal of murine lung or spleen mononuclear cells for culture rendered DBA/2 mice incapable of generating an effective cytotoxic T-lymphocyte response to influenza A virus-infected cells. The cytotoxic T-lymphocyte precursor frequency to influenza A virus in lung and spleen cells from cyclophosphamide-treated mice was significantly decreased when compared with that of normal littermate controls. The low cytotoxic T-lymphocyte activity in the lungs and spleens of cyclophosphamide-treated mice could be partially restored in vitro by human interleukin 2.     

Systemic suppression of delayed-type hypersensitivity by supernatants from UV-irradiated keratinocytes. An essential role for keratinocyte-derived IL-10.

Exposing murine keratinocyte cultures to UV radiation causes the release of a suppressive cytokine that mimics the immunosuppressive effects of total-body UV exposure. Injecting supernatants from UV-irradiated keratinocyte cultures into mice inhibits their ability to generate a delayed-type hypersensitivity reaction against allogeneic histocompatibility Ag, and spleen cells from mice injected with supernatant do not respond to alloantigen in the in vitro MLR. A unique feature of the immunosuppression induced by either total-body UV-exposure or injecting the suppressive cytokine from UV-irradiated keratinocytes is the selectivity of suppression. Although cellular immune reactions such as delayed-type hypersensitivity are suppressed antibody production is unaffected. Because the selective nature to the UV-induced immunosuppression is similar to the biologic activity of IL-10, we examined the hypothesis that UV exposure of keratinocytes causes the release of IL-10. Keratinocyte monolayers were exposed to UV radiation and at specific times after exposure mRNA was isolated or the culture supernatant from the cells was collected. IL-10 mRNA expression was enhanced in UV-irradiated keratinocytes. The secretion of IL-10 by the irradiated keratinocytes was determined by Western blot analysis. A band reactive with anti-IL-10 mAb was found in supernatants from the UV-irradiated but not the mock-irradiated cells. IL-10 biologic activity was determined by the ability of the supernatants from the UV-irradiated keratinocytes to suppress IFN-gamma production by Ag-activated Th 1 cell clones. Anti-IL-10 mAb neutralized the ability of supernatants from UV-irradiated keratinocytes to suppress the induction of delayed-type hypersensitivity in vivo. Furthermore, injecting UV-irradiated mice with antibodies against IL-10 partially inhibited in vivo immunosuppression. These data indicate that activated keratinocytes are capable of secreting IL-10 and suggest that the release of IL-10 by UV-irradiated keratinocytes plays an essential role in the induction of systemic immunosuppression after total-body UV exposure.     

In vivo effects of cyclosporine on influenza A virus-infected mice

Cyclosporine (cyclosporin A, CsA) administered to mice substantially affects their immune response to an influenza A virus infection. If treated with CsA for 21 days, the mouse lungs contain high titers of virus which are cleared more slowly than in controls. Indicators of pathological damage--lung weight, extent of consolidation, fine morphology, and the extent of infiltration of dividing cells into the lung--showed that administration of CsA greatly decreased the level of inflammation. The production of hemagglutination-inhibiting (HI) antibody was delayed but reached almost control levels and NK cell activity in the lung was also comparable to control levels. In contrast, a delayed-type hypersensitivity (DTH) response to the virus could not be elicited in the CsA-treated, infected mice at 6 or 12 days after infection. Cytotoxic-T-cell (Tc-cell) activity was present in the lungs of such mice though its appearance was delayed and the activity recovered was less than that of the control infected mice. If administered with a dose of virus lethal for normal mice. CSA-treated mice survived, probably due to the greatly reduced level of immunopathological damage in the infected lung.     

Interleukin 1 activity in normal human urine

Human leukocyte dialysates contain components capable of amplifying cutaneous delayed-type hypersensitivity (DTH) reactions. In the present study, two such amplifiers, both less than 3500 m.w., were partially purified from human leukocyte dialysates by gel filtration on Sephadex G-10 followed by high pressure reverse-phase liquid chromatography. These amplifiers of DTH were examined for their effects on production of the migration inhibitory lymphokines leukocyte migration inhibition factor (LIF) and macrophage migration inhibition factor (MIF). The amplifiers were found to increase LIF and MIF production by antigen- or alloantigen-stimulated human peripheral blood lymphocytes in a dose-dependent fashion. Further analysis demonstrated that although antigen-stimulated T4 and T8 cell subpopulations could produce LIF activity under the assay conditions employed, amplification of lymphokine production by modulator was only observed with the T4 subset.     

Amplification of the activity of human leukocyte inhibitory factor (LIF) by the generation of a low molecular weight inhibitor of PMN leukocyte chemotaxis

Leukocyte inhibitory factor (LIF), which was derived from human peripheral blood lymphocytes by stimulation with concanavalin A ad partially purified by Sephadex G-100 gel filtration, inhibited the in vitro spontaneous migration and chemotaxis of human PMN leukocytes as assessed in a Boyden chamber micropore filter assay. The inhibitory activity was attributed to LIF, a principle defined in terms of its inhibition of PMN leukocyte migration from glass capillary tubes since it was preferentially directed to PMN leukocytes as compared to mononuclear leukocytes, exhibited a size comparable to LIF by gel filtration, and was inactivated by diisopropyl fluorophosphate in parallel with LIF. Incubation of PMN leukocytes with LIF released additional inhibitory activity, distinct from LIF, which resembled the neutrophil-immobilizing factor (NIF) by virtue of its approximate m.w. of 4000 by filtration on Sephadex G-25, inactivation by trypsin digestion, and preferential noncytotoxic inhibition of spontaneous migration and chemotaxis of PMN leukocytes as compared to mononuclear leukocytes. Thus LIF inhibits PMN leukocyte migration both by a direct action on the cells and by an amplification pathway that is mediated by low m.w. chemotactic inhibitors similar to NIF.     

Selective regulation of chemotactic lymphokine production. I. Selective potentiation of eosinophil chemotactic lymphokine production in alum hydroxy gel- and Bordetella pertussis vaccine-treated guinea pigs

Delayed tissue eosinophilia in DNP-ovalbumin-induced allergic inflammatory skin lesions of guinea pigs was markedly enhanced by previous treatment with alum hydroxy gel (Alum) or Bordetella pertussis vaccine. This enhancement seemed due to increased production of a lymphocyte-derived eosinophil chemotactic factor (ECF) at the skin site. Treatment of animals with Alum potentiated antigen-induced in vitro ECF production by lymphoid cells from spleen and mesenteric lymph node of sensitized animals. The co-culture supernatants of lymphoid cells from Alum-treated animals also potentiated concanavalin A (Con A)-induced in vitro ECF production. The potentiating effect of Alum on ECF production seemed to be ascribed to the release of soluble factors from macrophages of the Alum-treated animals. The macrophage-derived soluble factor ECF-potentiating factor (ECF-PF) selectively potentiated ECF production but not macrophage chemotactic lymphokine production by Con A-stimulated lymphoid cells from normal animals. ECF-PF activity was associated with two separate m.w. fractions: one was 50,000 to 70,000 and the other was 10,000 to 20,000. The present study provides one of the explanations for enhanced ECF production by adjuvants, such as Alum and Bordetella pertussis vaccine.     

Attenuation of inducible respiratory immune responses by oseltamivir treatment in mice infected with influenza A virus

The antiviral neuraminidase inhibitor oseltamivir (OSV) is widely used to suppress viral replication in the treatment of influenza. Here, we report that OSV administration significantly suppressed respiratory mucosal secretory IgA responses with respect to antigen (Ag)-specific antibody (Ab) production and also the induction of Ag-specific IgA Ab-forming cells, but not systemic IgG responses, in weanling mice as a model of pediatric influenza. Neutralizing activities of the airway fluids in oral OSV-treated mice were significantly less than those of sham-treated mice. Our findings suggest the risk of re-infection in patients showing a low mucosal response following OSV treatment.     

An antigen-specific hypersensitivity which does not fit into traditional classification of hypersensitivity

A unique type of Ag-specific hypersensitivity was induced by challenging the Ag-sensitized mice at the ear. It was elicited within 1 h after the Ag challenge, and thus was distinct from either the delayed-type hypersensitivity (DTH) which developed in 24 h or the immune complex-mediated hypersensitivity which evolved in 4 to 6 h. This hypersensitivity was referred to as early-type hypersensitivity (ETH). The time required for these types of hypersensitivity to develop after immunization was also different; DTH required 4 to 6 days, ETH 9 to 11 days, whereas plasma protein-induced immune complex-mediated hypersensitivity needed 18 to 21 days. The ETH could be induced by a smaller amount of Ag than DTH, and unlike DTH could be transferred by either immune sera or T cell-derived culture factor which was small m.w. Although the ETH developed later than DTH after sensitization, it lasted longer once developed and the pattern of response was inversely related to DTH. Furthermore, the denatured hepatitis B surface Ag induced DTH but not ETH, in contrast to native hepatitis B surface Ag that induced both, suggesting that the epitopes recognized by TETH cells were distinct from those recognized by TDTH cells. The ETH could be induced by most Ag tested including poly(Glu60Ala10Tyr10, L-lactic dehydrogenase, insulin, chicken egg white lysozyme, polymerized human serum albumin, horse gamma-globulin, transferrin, fibrinogen, and plasminogen, but not by purified protein derivative. Because poly(Glu60Ala10Tyr10, L-lactic dehydrogenase, egg white lysozyme and insulin were under the Ir gene control and the inducibility of ETH was Ag dependent and was closely correlated with that of DTH, the expression of ETH also must be regulated by Ir gene. The histopathologic changes in ETH consisted of capillary congestion and edema. The vasopermeability was increased and there was the leakage of plasma proteins into the tissue. Based on these data, we concluded that the ETH reported in this study was a novel type of Ag-specific hypersensitivity.     

Antigen-specific augmentation of delayed-type hypersensitivity by immune serum factor in mice: augmentation of anti-tumor cytostatic activity

A humoral factor capable of augmenting delayed-type hypersensitivity antigen specificity (DAF) is present in the serum of mice sensitized with heterologous erythrocytes to induce a delayed footpad reaction (DFR). In the present study, a similar factor was identified when xenogeneic tumor cells were used as antigens. This factor also could augment the in vitro anti-tumor cytostatic activity against homologous tumor cells, which correlated with in vivo DFR to the same tumor cells. The cytostatic activity augmented by the transfer of this factor had the following characteristics: The activity appeared in the whole peritoneal exudate cells (PEC) from serum recipients at 4 days after the antigenic challenge. Such an activity was revealed in the collaboration of plastic dish-nonadherent and -adherent PEC as the primary and final effectors, respectively. The appearance of primary effector cells for such an activity was also accelerated in spleen and lymph node cells. However, a sufficient number of macrophages were always required as the final effectors in their functional expression. These primary effectors were sensitized T lymphocytes which produced lymphokine(s) such as macrophage-activating factor(s) and which contributed to this augmented cytostatic activity through the activation of macrophages. Thus, this immune serum factor seems to exert functional expression by accelerating the generation of lymphokine-producing delayed-type T lymphocytes, which is also responsible for cytostatic anti-tumor immunity.     

Virus-specific delayed-type hypersensitivity (DTH). Cells mediating lymphocytic choriomeningitis virus-specific DTH reaction in mice

We had previously shown that the local lymphocytic choriomeningitis virus-induced delayed-type hypersensitivity (DTH) reaction in mice consists of two well delineated phases that are mediated by CD8+ and CD4+ T lymphocytes, respectively. These findings have been confirmed and extended by showing that the first CD8+ cell-dependent part of the response was enhanced by either the presence of CD4+ cells or systemic treatment with IL-2 and that it developed in the absence of detectable numbers of mononuclear phagocytes, whereas the later CD4+ cell part required monocytes or related elements. Furthermore, in the DTH reaction that was elicited with noninfectious viral Ag in mice previously immunized by infection, only the CD4+ cells participated. Thus, the two phases of the lymphocytic choriomeningitis-viral DTH reaction are principally different, which has to be taken into account when trying to assess the relevance of DTH during this virus infection.     

Mechanisms of suppressed cell-mediated immunity and impaired antigen-induced interleukin 2 production in granuloma-bearing mice

Pulmonary granulomas were induced in BALB/c mice immunized with methylated bovine serum albumin in complete Freund's adjuvant by the intratracheal injection of plain agarose beads or beads conjugated to specific antigen. Large hypersensitivity granulomas developed around antigen-coupled beads in immunized animals. Smaller but still prominent granulomatous reactions developed around plain beads in immunized mice. In nonimmunized animals, both plain and antigen conjugated beads produced very small granulomas. Granuloma formation in sensitized animals was associated with suppressed delayed-type hypersensitivity reactions induced by the footpad injection of specific and nonspecific antigens. Lymph node cells from sensitized granuloma-bearing mice with cutaneous anergy showed suppressed specific and nonspecific antigen-induced proliferative responses in vitro. These cells also showed suppressed interleukin 2 production in response to specific antigen. Although no soluble suppressive factor was detected in granuloma extracts, suppressor cells were found in lymph nodes of granuloma-bearing mice, which could inhibit antigen-induced production of interleukin 2 by lymph node cells from immunized mice. Antigen-specific immunoglobulin G antibody production was not suppressed in immunized granuloma-bearing mice. Previous studies from our laboratory have demonstrated migration inhibition factor and interleukin 1 activities in aqueous extracts prepared from granuloma-bearing lungs of immunized mice. These results and the findings reported here indicate that granuloma formation and the associated anergy observed in this system are primarily expressions of cell-mediated immunity; selective suppression of in vivo and in vitro expressions of cell-mediated immunity in granuloma-bearing mice may be due to impaired antigen-induced interleukin 2 production; and such impairment is caused by suppressor cells.     

Characterization of human chemotactic lymphokine production induced by mitogens and mixed leukocyte reactions using a new microassay.

Quantification of lymphokine production in vitro can be a useful tool in the evaluation of delayed hypersensitivity in various disease states. A micro-method for the measurement of chemotactic lymphokine production by human mononuclear leukocytes (MNL's) has been developed. MNL's are isolated on Ficoll-Hypaque gradients and cultured without plasma in microtiter plates. Culture supernatants are harvested through glass fibre filter paper under vacuum in a semi-automatic harvester. Chemotactic lymphokine activity in the supernatants is quantified in miniaturized Boyden chambers using human monocytes as responder cells. The production of chemotactic activity can be initiated by mixed leukocyte reactions as well as by soluble antigens or mitogens, and therefore may be a useful adjunct in tissue typing. Studies of lymphokine production in normal individuals indicate that these methods are quantitative, reproducible, and readily applicable to the study of this parameter of immune function in human disease.     

Production of a fibronectin-associated lymphokine by cloned mouse T cells

Azobenzenearsonate-specific cloned mouse T cells able to transfer delayed hypersensitivity reactions in vivo produced macrophage agglutination factor (MaggF) after stimulation with mitogen or antigen in vitro. Mitogen (Con A) elicited MAggF production directly from T cells. Responses to Ag were Ag-specific, required syngeneic accessory cells in addition to T cells, and were independent of T cell fine specificity for azobenzenearsonate. Mouse MAggF shared a number of biochemical and immunochemical properties with the fibronectins (FN): 1) high Mr similar to that of plasma FN; 2) binding to gelatin, heparin, and polyclonal antibodies and mAb specific for cellular and plasma FN; 3) inhibition of activity in solution by monoclonal anti-human FN directed against plasma FN gelatin-binding domain; and 4) action on peritoneal exudate macrophages mediated through a FN-receptor cross reactive with one on human monocytes. MAggF production required active protein synthesis and was associated with significant increases in gelatin-binding immunoreactive FN (Mr 440 kDa on immunoblotting) in culture supernatants and T cell lysates. Metabolically labeled peptides could be precipitated by anti-FN from culture supernatants of activated T cells. Stimulated cultures contained significantly more cells with immunohistologically demonstrable cytoplasmic FN than unstimulated control cultures. We suggest that T cell FN is a distinct species of cellular FN which may play an important role in mediating delayed hypersensitivity inflammatory reactions in vivo.     

Low dose ultraviolet B-irradiated Langerhans cells preferentially activate CD4+ cells of the T helper 2 subset

UVB radiation distorts the Ag-presenting function of epidermal Langerhans cells (LC); this has been shown for the presentation of soluble Ag to primed T cells in vitro and for the initiation of delayed-type hypersensitivity in vivo, such as contact hypersensitivity (CH). Previous work has also demonstrated UVB-induced suppression of CH to be mediated ultimately by T cells. Two subsets of CD4+ Th cells, Th1 and Th2, have been identified, based on their cytokine production and functional activities. In particular, Th1 mediate delayed-type hypersensitivity, whereas Th2 do not. To investigate whether the perturbation of LC function induced by UVB radiation leads to a differential activation of these subsets of CD4+ cells, we examined the capacity of unirradiated and irradiated (200 J/m2) APC from adult BALB/c mice to present keyhole limpet hemocyanin to Ag-specific, H2d-restricted Th1 and Th2 cell lines. Four sources of APC were utilized: epidermal cells (EC), flow microfluorometry-purified Ia+ EC (LC), flow microfluorometry-purified Ia- EC, and splenic adherent cells (SAC). Unirradiated EC, LC, and SAC, but not Ia-EC, presented keyhole limpet hemocyanin to both Th1 and Th2. Irradiated EC and LC lost their ability to stimulate Th1, but retained fully their capacity to stimulate Th2. On the other hand, irradiated SAC were unable to induce proliferation of either Th1 or Th2. These findings indicate that suppression of CH mediated by UVB-irradiated LC may result from an alteration of the ratio and/or activity of Th1 and Th2 cells normally generated during the induction of such responses.     

The role of mast cells in virus-induced inflammation in the murine central nervous system

The mononuclear inflammatory response to Sindbis virus infection of the central nervous system is analogous to the cutaneous delayed-type hypersensitivity reaction. It is dependent on sensitized T cells for initiation, but many of the cells present are nonsensitized bone marrow-derived cells. Tissue mast cells have been shown to be important for the development of the delayed-type hypersensitivity reaction in the skin where capillary endothelial cells are joined by tight junctions. To determine whether mast cells are also important for the development of an immune-mediated inflammatory response across the endothelial tight junctions of the blood-brain barrier, the development of mononuclear inflammation in the central nervous system of reserpine-treated mice and mast cell-deficient mice (WBB6F1-W/Wv) was studied after infection with Sindbis virus. Three central nervous system compartments, the cerebrospinal fluid, the meninges, and the brain parenchyma, were evaluated for inflammation by counting the number of cells present, by grading the histopathologic lesions, and by labeling infiltrating cells with 125IUDR. By all parameters inflammation was reduced when mice were treated with reserpine or were deficient in mast cells. Antigen-specific humoral and cellular immune responses were depressed and virus clearance delayed in reserpine-treated mice, but not in mast cell deficient mice. It is concluded that the vasoactive amines released by mast cells in the central nervous system play a facilitating role in the development of the inflammatory response to Sindbis virus.