Titers Of IFN-gamma Research Articles

Relative susceptibilities of inbred mouse strains C57BL/6 and A/J to infection with Histoplasma capsulatum

Differences in the 30-day survival of Histoplasma capsulatum after intravenous injection indicated that the A/J strain of inbred mouse was more resistant to experimental infection than was the C57BL/6 strain. CFU from the spleens of infected animals increased during the first week after injection but gradually declined over the next 3 weeks. The CFU per gram of tissue in the C57BL/6 animals were 10- to 100-fold higher than were those in the A/J mice during the time between 7 and 28 days after infection. The units of gamma interferon (IFN-gamma) in supernatants of spleen cells stimulated with heat-killed yeast cells of H. capsulatum reached a peak at the time of the largest number of CFU per gram of tissue. The titers of IFN-gamma at days 3 to 5 were higher in the A/J mice than they were in the C57BL/6 mice, but from days 7 to 28, the titers of IFN-gamma were not correlated with the more efficient clearance of the fungus from the spleens of A/J mice. The L3T4+ spleen cells were shown to be active IFN-gamma producers. Treatment of Histoplasma-infected mice with anti-IFN-gamma antibody resulted in much larger tissue burdens of the fungus in the lungs and spleens of treated animals than in untreated animals. There was no marked difference in the result of treatment with anti-IFN-gamma antibody between A/J and C57BL/6 mice. Treatment of Histoplasma-infected mice with recombinant murine IFN-gamma did not alter the course of infection in either inbred strain of mouse.

Dengue virus-specific human T cell clones. Serotype crossreactive proliferation, interferon gamma production, and cytotoxic activity.

The severe complications of dengue virus infections, hemorrhagic manifestation and shock, are much more commonly observed during secondary infections caused by a different serotype of dengue virus than that which caused the primary infections. It has been speculated, therefore, that dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) are caused by serotype crossreactive immunopathological mechanisms. We analyzed clones of dengue serotype crossreactive T lymphocytes derived from the PBMC of a donor who had been infected with dengue 3 virus. These PBMC responded best to dengue 3 antigen, but also responded to dengue 1, 2, and 4 antigens, in bulk culture proliferation assays. 12 dengue antigen-specific clones were established using a limiting dilution technique. All of the clones had CD3+ CD4+ CD8 phenotypes. Eight clones responded to dengue 1, 2, 3, and 4 antigens and are crossreactive, while four other clones responded predominantly to dengue 3 antigen. These results indicate that the serotype crossreactive dengue-specific T lymphocyte proliferation observed in bulk cultures reflects the crossreactive responses detected at the clonal level. Serotype crossreactive clones produced high titers of IFN-gamma after stimulation with dengue 3 antigens, and also produced IFN-gamma to lower levels after stimulation with dengue 1, 2, and 4 antigens. The crossreactive clones lysed autologous lymphoblastoid cell line (LCL) pulsed with dengue antigens, and the crossreactivity of CTL lysis by T cell clones was consistent with the crossreactivity observed in proliferation assays. Epidemiological studies have shown that secondary infections with dengue 2 virus cause DHF/DSS at a higher rate than the other serotypes. We hypothesized that the lysis of dengue virus-infected cells by CTL may lead to DHF/DSS; therefore, the clones were examined for cytotoxic activity against dengue 2 virus-infected LCL. All but one of the serotype crossreactive clones lysed dengue 2 virus-infected autologous LCL, and they did not lyse uninfected autologous LCL. The lysis of dengue antigen-pulsed or virus-infected LCL by the crossreactive CTL clones that we have examined is restricted by HLA DP or DQ antigens. These results indicate that primary dengue virus infections induce predominantly crossreactive memory CD4+ T lymphocytes. These crossreactive T lymphocytes proliferate and produce IFN-gamma after stimulation with a virus strain of another serotype, and demonstrate crossreactive cyotoxic activity against autologous cells infected with heterologous dengue viruses.(ABSTRACT TRUNCATED AT 400 WORDS)

Highly efficient action of autocrine mouse interferon-gamma expressed via a retroviral vector.

Following infection with a mouse interferon-gamma (IFN-gamma)-producing retrovirus, various types of mouse cell lines began to constitutively secrete IFN-gamma in the culture medium, but the IFN-gamma production level varied according to cell type. Effects of the IFN-gamma on the IFN-gamma-producing cell itself (autocrine effects) were examined. In the IFN-gamma-producing cells, the expression of the major histocompatibility complex class I genes was augmented; this augmentation was remarkable in T cell lines tested in this work, regardless of their poor IFN-gamma production. This autocrine effect was highly efficient and could not easily be abrogated by the exogenous addition of anti-IFN-gamma antibodies. Such antibody resistance was also observed for the antiviral effect of the autocrine IFN-gamma on an IFN-gamma gene-transferred fibroblast line. These results suggest that apparently low titers of physiological IFN-gamma might exert strong in vivo autocrine and possibly paracrine effects.

Supernatants of human leukocytes contain mediator, different from interferon gamma, which induces expression of MHC class II antigens.

In this report, data are presented on the regulation of MHC class II antigen expression by a mediator present in supernatants of human mixed leukocyte cultures (MLC-SN), and which is different from IFN-gamma. The capacity of supernatants to induce antigen expression did not correspond to titers of IFN-gamma. Removal of IFN-gamma using either dialysis against pH 2 or neutralizing mAb against human IFN-gamma did not abrogate the MHC class II antigen expression-inducing capacity of MLC-SN when tested on adenocarcinoma cell lines, kidney epithelial cells, and fibroblasts in vitro in an indirect immunofluorescence assay. Therefore, supernatants of human leukocytes contain a mediator, different from IFN-gamma, which induces expression of MHC class II antigens. Dose-response studies revealed that the mediator is produced after allogeneic and lectin stimulation of human leukocytes, and by unstimulated leukocytes. Activation of leukocytes resulted in increased titers of the mediator. The mediator markedly enhances expression of both HLA-DR and HLA-DQ antigens, whereas IFN-gamma had a similar effect on HLA-DR antigens, and only a minor effect on HLA-DQ antigens. Interaction of the mediator and IFN-gamma resulted in a potentiating effect of these two factors on MHC class II antigen expression. Biochemical analysis revealed a mediator, distinguishable by FPLC from IL-1, IL-2, and human IFN-gamma, and which has a molecular mass of 32 kD.

Interleukin 2-activated killer cells: generation in collaboration with interferon gamma and its suppression in cancer patients.

The generation of lymphokine activated killer (LAK) cells by recombinant IL2 (rIL2) in collaboration with interferon gamma (IFN gamma) was examined in peripheral blood mononuclear cells (PBMC) from patients with malignant tumors of the digestive organs and breast cancer. LAK cytotoxicity could be induced by rIL2 at 10 units/ml in 10 of 12 patients and 20 of 37 using fresh autologous tumor cells and PK-1, an established solid tumor cell line as a target, respectively. Among 34 patients, in which titers of IFN gamma produced were assayed, 12 showed no IFN gamma production. All of these 12 patients had no or extremely low LAK activity, suggesting the correlation of LAK generation with the production of IFN gamma in response to rIL2. LAK induction by rIL2 in PBMC of cancer patients was almost completely inhibited by addition of anti-IFN gamma serum. Depressed LAK generation, which was accompanied by no or low levels of IFN gamma production, was partially restored by addition of exogenous recombinant IFN gamma. These results indicate that LAK induction by rIL2 in cancer patients involves the production of IFN gamma and its interaction with rIL2. The results also suggested the presence of a factor(s) suppressing LAK induction by rIL2 in the serum of cancer patients. Based on these results, the cancer patients could be divided into the following three groups. Group 1, in which the serum suppressor activity was undetectable, had the same level of LAK cytotoxicity in PBMC as healthy controls. Group 2 showed the serum suppressor factor and had the lower level of cytotoxicity in PBMC when cultivated in autologous serum (AS) compared to healthy controls. The depressed LAK induction in AS medium was restored to a normal level in culture with fetal calf serum (FCS) plus rIL2, or by addition of rIFN gamma, or high concentrations of rIL2 in AS medium. The last group (group 3), in which the serum suppressor factor was also found, had the lowest level of cytotoxicity compared to healthy controls. The LAK induction in these patients could not be restored to a normal level by culture in FCS medium, addition of exogenous rIFN gamma or high concentrations of rIL2, suggesting the possibility that the deficit of LAK generation in this group might involve the dysfunction or the lack of IL2 responder cells, in addition to the presence of a serum suppressor factor(s).

A lymphokine distinct from interferon-gamma that activates human monocytes to kill Leishmania donovani in vitro.

Human interferon-gamma (IFN-gamma), a T cell lymphokine (LK), activates monocytes to kill many intra- and extracellular pathogens. In fact, previous reports assert that all activity in LK for macrophage activation is due to IFN-gamma. To test this assertion, we examined monocyte interactions with amastigotes of Leishmania donovani after treatment with recombinant DNA or affinity-purified leukocyte IFN-gamma and IFN-gamma containing LK. Cells treated with at least 200 IU/ml IFN-gamma were microbicidal for L. donovani. Analysis of IFN-gamma dose responses for induction of microbicidal activity by recombinant IFN-gamma (r-IFN-gamma) and LK, however, documented a striking difference: LK was 25-fold more efficient than r-IFN-gamma at equivalent IFN-gamma titers. This large difference suggested that monocyte activation factor(s) in LK may not be IFN-gamma. Rabbit anti-IFN-gamma completely inhibited antiviral activity in LK but did not abrogate the ability to induce monocyte cytotoxicity against leishmania. Furthermore, removal of IFN-gamma from LK by monoclonal anti-IFN-gamma affinity chromatography or by treatment with anti-IFN-gamma followed by staphylococcal protein A chromatography also did not inhibit LK activity. Fractionation of LK on Sephadex G-100 revealed two activity peaks: one in the 50,000 to 60,000 m.w. range coincident with IFN-gamma, and the other at 25,000 to 30,000 daltons with no IFN-gamma. These studies document LK physicochemically and antigenically distinct from IFN-gamma that activate monocytes to kill L. donovani. Such novel factors may have broad import for the study of macrophage-mediated host defenses and for development of immunotherapeutic regimens.

Cloning and expression of the chromosomal immune interferon gene of the rat.

The chromosomal immune interferon gene of the rat (IFN-gamma) was identified by screening a recombinant rat lambda phage library with a human IFN-gamma cDNA probe. In contrast to the genes of other rat IFNs, this rat IFN-gamma chromosomal gene contains introns and its structural organization closely resembles that of the human and murine IFN-gamma genes. The rat IFN-gamma gene encodes a signal sequence of 19 amino acids followed by the mature IFN-gamma protein of 137 amino acids. The gene was expressed under control of the simian virus 40 (SV40) early promoter in Chinese hamster ovary (CHO) cells deficient in dihydrofolate reductase (DHFR) after co-transformation with a plasmid containing the mouse DHFR gene. Initial transformants with a DHFR+ phenotype produced IFN-gamma titres ranging from 20 to 1600 units/ml. After stepwise increases in the concentration of methotrexate (MTX) in the growth medium of transformed CHO cells, MTX-resistant clones producing 80 000-100 000 units per ml were isolated. Protein analysis of supernatants of these MTX-resistant cells by polyacrylamide gel electrophoresis revealed a product with an apparent mol. wt. of 18 000 daltons which was not detectable in the growth medium of DHFR+ transformants that did not produce IFN. The product was identified as rat IFN-gamma and constituted approximately 5% of the proteins excreted from these cells.

Defective IFN-gamma production in the human neonate. I. Dysregulation rather than intrinsic abnormality.

Cord blood leukocytes (CBL) stimulated with PHA, Con A, or with the monoclonal antibody OKT3 proliferate normally but produce very low titers of IFN-gamma. This defect was not observed with maternal leukocytes collected at the time of delivery, indicating that the defective production of IFN-gamma in CBL is not a mere consequence of a hormonal change associated with labor. CBL produced large amounts of IFN-gamma (comparable to those observed in adult control and in mothers) after stimulation with staphylococcal enterotoxin A (SEA). Furthermore, gamma-irradiation with as little as 500 or 1000 rad, or incubation at 37 degrees C for 24 hr, reversed the defect in PHA-induced IFN-gamma secretion. This finding indicates that the defective secretion of IFN-gamma of CBL is not intrinsic, but rather is the consequence of a subtle dysregulation. We could not find evidence for a defective accessory function with cord blood monocytes, because the addition of adherent cells from adult donors did not reverse the defect. In co-cultures of adult leukocytes and CBL, PHA-induced IFN-gamma secretion was comparable to that of adult cultures tested alone. Nonirradiated CBL were not able to suppress IFN-gamma secretion by irradiated autologous leukocytes. Together, our results suggest that the defective PHA-induced IFN-gamma secretion of CBL is the result of an original type of dysregulation and is associated with an excessive sensitivity to suppressive signals rather than excessive suppressor function.

Production of high titers of interferon-gamma by prestimulated murine spleen cells.

Concanavalin A (Con A)-induced interferon-gamma (IFN-gamma) production by murine spleen cells prestimulated with Con A for different periods of time has been studied. Highest titers of antiviral activity were obtained by restimulation of cells that had been prestimulated with Con A for 3 days. These cells produced up to 30 times more IFN than freshly isolated spleen cells stimulated under the same conditions. In an effort to explain this rise in the capacity to produce IFN-gamma, the possibility that it was due to the inactivation of a suppressor cell was excluded. The addition of freshly isolated spleen cells at different concentrations to prestimulated cells did not affect subsequent Con A-induced IFN-gamma production. Separation of freshly isolated or prestimulated spleen cells by velocity sedimentation at unit gravity showed that in the latter case most of the IFN-gamma was produced by a population of large-sized cells not present in the former population. It was concluded from these experiments that prestimulation of spleen cells with Con A gives rise to a population of large-sized cells that produce, upon restimulation with the same mitogen, much higher titers of IFN-gamma than those obtained upon primary stimulation of small resting lymphocytes.

Heterogeneity of soluble T cell products. I. Precursor frequency and correlation analysis of cytotoxic and immune interferon (IFN-gamma)-producing spleen cells in the mouse.

In this study, the precursor frequency, relationship and activity of murine splenic cytotoxic and immune interferon (IFN-gamma)-producing cells has been determined. C57BL/6 spleen cells were activated by concanavalin A (Con A) and subsequently grown in microcultures under limiting dilution conditions. The progeny of cells plated in microcultures was divided and tested for (a) total cytotoxic activity on EL4 (H-2b) tumor target cells in the presence of Con A to nonspecifically attach the killer to the target cells, and (b) the quantity of IFN-gamma released by Con A stimulation. Cytotoxic T lymphocytes and IFN-gamma-producing cells were present in Con A-activated C57BL/6 spleen cells at a high frequency of approximately 1 out of 3 and 1 out of 2 cells, respectively. IFN-gamma could be released from both noncytotoxic and a large fraction of cytotoxic T cells and the cytotoxic activity was not necessarily associated with IFN-gamma release. In a few selected cultures with a progeny of cells plated at a low cell number (up to 10/well) very high IFN-gamma titers (greater than 10 000 U/ml) could be found. These results provide the first frequency estimate of IFN-producing cells and are discussed with respect to the physiological role of IFN-gamma release from T cells.

Mechanisms in in vivo release of lymphokines. III. Separation of gamma-interferon (IFN gamma) from cytotoxicity in inbred strains of mice.

Inbred strains of mice sensitized with BCG and challenged with old tuberculin (OT) vary in their capacity to release IFN gamma and MIF into circulation. Experiments were performed to correlate the presence of IFN gamma with that of the cytotoxic effect in an attempt to learn whether these two activities in the sera may be separated. Sera from eight inbred strains were obtained at times after challenge and assayed for IFN and for cytotoxic activity. A correlation did not exist between the titers of IFN gamma and the levels of cytotoxicity, i.e. sera from RF/J mice without detectable titers of IFN gamma had cytotoxic activity, while sera from C57BL/KsJ mice that had titers of IFN gamma were not cytotoxic. Regulation of IFN gamma titers could be achieved by administration of complete Freund's adjuvant to BCG sensitized mice. However, similar differences were not detected in cytotoxic activity. Therefore, IFN gamma may be released, or the activity of IFN gamma may be expressed in the circulation, in the absence of cytotoxicity.