The use of the PCR to quantitate gene expression.
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Autoradiography↗
Biomedical subjects
W C Gause
Publications and source records attributed to W C Gause.
Germline and productive C epsilon gene expression during in vivo IgE responses.
In vitro studies have established that Ig isotype switching typically involves deletion of CH genes that are located between VDJ and the CH gene that will be expressed, and is preceded by transcription of a germline (g) form of that CH gene. Increases in g epsilon transcript levels are induced by the cytokine IL-4, and always precede switching to IgE. To evaluate whether a similar relationship occurs in vivo, we examined IL-4 mRNA, g epsilon RNA, productive (p) epsilon mRNA, and serum IgE levels in two in vivo systems: one in which the injection of anti-IgD antibody induces mIgD+ B cells to switch to the expression of IgE and to secrete this isotype, and a second in which the injection of anti-IgE antibody stimulates IgE secretion by B cells that had been induced to express membrane IgE by earlier treatment with anti-IgD antibody. Increases in IL-4 transcript levels in anti-IgD-injected mice were followed within 24 h by increases in g epsilon RNA, and, one to two days later, by increased p epsilon mRNA and serum IgE levels. IL-4 antagonists blocked the g epsilon and p epsilon RNA and serum IgE responses in these mice, whereas the injection of otherwise untreated mice with IL-4 stimulated, within 24 h, a large increase in g epsilon RNA levels, followed 1-2 days later by a small increase in p epsilon mRNA. Injection of anti-IgD-primed mice with anti-IgE antibody also stimulated increases in IL-4, g epsilon and p epsilon RNA levels; however, the increases in IL-4 and g epsilon RNA were considerably smaller, and the increases in p epsilon mRNA and serum IgE considerably larger, than those observed in anti-IgD antibody-injected mice. IL-4 antagonists blocked the anti-IgE antibody-induced g epsilon RNA response, but not the p epsilon mRNA or serum IgE responses. Thus, IL-4 is required for the induction of g epsilon RNA in at least two in vivo systems, increased g epsilon RNA levels precede increases in p epsilon RNA levels in vivo as in vitro, and neither IL-4 nor g epsilon RNA is required to induce B cells that have already switched to IgE expression to differentiate into IgE-secreting cells.
Animals↗
Analysis of cytokine mRNA expression during primary granuloma formation induced by eggs of Schistosoma mansoni.
Granulomas induced by parasite eggs are the primary lesions in mice infected with Schistosoma mansoni. Temporal analysis of cytokine mRNA expression in lung tissue containing synchronized granulomas demonstrated a Th0-like pattern of lymphokine expression. IFN-gamma, IL-1 beta, and IL-6 were the primary cytokines induced by day 1 in developing lung granulomas initiated by i.v. egg injection. These changes were followed by increases in expression of IL-2, IL-4, and IL-10 mRNA on day 3 and TNF-alpha and IL-5 mRNA on day 6. Nearly all cytokine mRNA reached maximal levels by day 6, which preceded the peak in granuloma size seen on day 14. In vivo treatment of egg-injected mice with either anti-IL-2 or anti-IL-4 antibodies significantly diminished the size of circumoval granulomas in the lungs. Both groups of antibody-treated animals displayed a marked reduction in IL-4 as well as IL-5 mRNA expression, although IFN-gamma and IL-2 mRNA levels were unchanged or slightly increased. These findings confirm previous observations suggesting a role for IL-2 in egg-induced pathology via the generation of Th2-associated responses, and also indicate a primary function for IL-4 in granuloma formation. Analysis of responses after injection of eggs into nude mice demonstrated that only the Th2 cytokines IL-4 and IL-5 are exclusively dependent on T cells for their induction. Taken together, these data suggest that Th2 cells producing IL-4 play a major role in egg granuloma formation, and that the induction and ultimate down-modulation of Th2-like responses may be influenced by non-T-cell-derived cytokines.
Animals↗
A primary intestinal helminthic infection rapidly induces a gut-associated elevation of Th2-associated cytokines and IL-3.
The immune response that is characteristic of parasitic helminth infections includes components associated with immediate-type hypersensitivity: elevated serum IgE, eosinophilia, and intestinal mast cell hyperplasia. In infection with the parasitic nematode, Heligmosomoides polygyrus, IL-4 mediates protective immunity, suggesting the presence of a host-protective Th2 response. In this investigation, we examined early stages of immune responsiveness to H. polygyrus infection to determine whether and at what stage a specific Th2-like pattern first appears. Using a quantitative reverse transcriptase-polymerase chain reaction assay, we analyzed changes in IL-2, IFN-gamma, IL-3, IL-4, IL-5, IL-6, IL-9, and IL-10 gene expression in the spleen, mesenteric lymph node, and Peyer's patch at various time points after infection. Our results demonstrate a highly specific and reproducible pattern of cytokine gene expression that remains localized to the enteric region. By 6 h after infection, IL-5 and IL-9 mRNA were elevated in the Peyer's patch and IL-3 was elevated by 12 to 24 h after infection. IL-4 RNA became elevated by 4 to 6 days after infection, but little change was observed in IFN-gamma, IL-2, or IL-10 mRNA levels. The early increases in IL-3, IL-5, and IL-9 gene expression after infection were probably T cell-independent, inasmuch as they were observed in Peyer's patches of congenitally athymic mice and anti-CD4, anti-CD8 mAb-treated conventional mice. However, treatment with these mAb considerably decreased cytokine gene expression 6 days after infection, and 8 days after infection, increased IL-4 gene expression in mesenteric lymph node cells was restricted to the CD4+ population. Thus, H. polygyrus infection induces cytokine gene expression that is restricted to some Th2-associated cytokines, is initiated by a T-independent response, and culminates in a T-dependent response.
Animals↗
Brucella abortus induces a novel cytokine gene expression pattern characterized by elevated IL-10 and IFN-gamma in CD4+ T cells.
Immunization of BALB/c mice with killed Brucella abortus (BA) has previously been shown to increase serum IgG2a levels and long-term T cell clones from these mice secrete Th1-associated cytokines: IFN-gamma and IL-2 but not IL-4 or IL-5. We analyzed cytokine gene expression following primary immunization with BA to determine when CD4+ T cells first express cytokine genes and whether specific hypothesized cytokine patterns (e.g. Th precursor, Th0) could be identified prior to a Th1-like pattern. Our results demonstrated a highly consistent and novel pattern of Th1/Th2 cytokine gene expression characterized by elevated IL-10 and IFN-gamma in CD4+ T cells which rapidly manifests itself and is sustained for at least 10 days after immunization. No elevation in IL-2 cytokine gene expression was observed and treatment of BA-immunized mice with blocking anti-IL-2 antibodies had no effect on the cytokine gene expression pattern, although treatment with anti-IFN antibodies resulted in increased IL-4, IL-5, and IL-9 cytokine gene expression, in the absence of any change in IFN-gamma or IL-10 as early as 4 days after immunization. These results suggest that a whole pathogen may trigger sufficient costimulatory signals to rapidly induce effector T cells in the absence of elevated IL-2 and that IL-10 is specifically elevated in certain Th1-like responses.
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Murine models of autoimmune disease and Sjögren's syndrome.
Murine models of autoimmune disease exhibit heterogenous features that are now being understood in terms of their basic molecular defects. Defects in apoptosis genes or genes that contribute to B-cell activation, T-cell tolerance loss, and development of autoimmune disease, including glomerulonephritis, have been identified. The interaction of primary and secondary predisposing genes leads to a diverse spectrum of autoimmune features that is different in different strains of mice. The challenge in the future will be to correlate these primary and secondary autoimmune genes with specific environmental triggers to explain the diverse spectrum of autoimmune disease. The ability to correct the apoptosis defect and autoimmune disease in CD2-Fas transgenic MRL-lpr/lpr mice brings hope that genetic therapy will eventually be possible in humans.
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The importance of Th2 cytokines in protective immunity to nematodes.
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Animals↗
Regulation by interferon alpha of immunoglobulin isotype selection and lymphokine production in mice.
Antigens and infectious agents that stimulate interferon alpha(IFN-alpha) production in mice induce antibody responses that are predominantly of the immunoglobulin (Ig)G2a isotype and contain little or no IgE. This suggested the possibility that IFN-alpha might have a role in directing Ig isotype selection. Consistent with this possibility, we have found that injection of mice with recombinant mouse IFN-alpha suppresses IgE secretion, enhances IgG2a secretion, and has no independent effect on IgG1 secretion in mice stimulated with a foreign anti-IgD antibody. Injection of mice with polyinosinic acid.polycytidylic acid (poly I.C), an inducer of macrophage IFN-alpha production, also suppresses the anti-IgD antibody-induced IgE response and stimulates the IgG2a response; these effects are blocked by a sheep antibody that neutralizes mouse IFN-alpha/beta. Both recombinant IFN-alpha and poly I.C have maximum IgE suppressive and IgG2a stimulatory effects when injected early in the anti-IgD antibody-induced immune response. Addition of IFN-alpha to mouse B cells cultured with lipopolysaccharide (LPS) + interleukin 4 (IL-4) suppresses both IgG1 and IgE production, but much less potently than IFN-gamma. IFN-alpha suppresses anti-IgD antibody-induced increases in the level of splenic IL-4 mRNA, but enhances the anti-IgD antibody-induced increase in the splenic level of IFN-gamma mRNA. These results are consistent with the effect of IFN-alpha on Ig isotype expression in mice, as IL-4 stimulates IgE and suppresses IgG2a secretion while IFN-gamma exerts opposite effects. These observations suggest that antigen presenting cells, by secreting IFN-alpha early in the course of an immune response, can influence the nature of that response both through direct effects on B cells and by influencing the differentiation of T cells.
Animals↗
Cytokine gene expression after in vivo primary immunization with goat antibody to mouse IgD antibody.
Cytokines are important mediators of effector lymphoid cell function during an immune response, but their expression during an in vivo immune response has not been well documented. We analyzed the kinetics of cytokine gene expression during the course of an in vivo primary immune response to goat antibody to mouse IgD antibody. Total RNA was purified from spleens taken from freshly killed BALB/c mice 1 to 7 days after immunization. The reverse transcriptase polymerase chain reaction was used to evaluate the expression of seven cytokine genes, all of which encode cytokines that are secreted by T cells and are important in T and/or B cell activation and differentiation. These were IFN-gamma, IL-2, IL-4, IL-5, IL-6, IL-9, and IL-10. IL-2 and IL-9 exhibited an early elevated expression at days 2 to 3, and declined as the expression of IL-4, IL-6, IL-10, and IFN-gamma increased. In contrast, IL-5 gene expression showed little change, exhibiting a similar pattern to the housekeeping gene, hypoxanthine-guanine phosphoribosyl transferase. Cell sorting of CD4+ and CD4- cells at day 3 and day 5 after immunization revealed that CD4+ cells were the predominant source of the elevated cytokines (with the exception of IL-6). Our results demonstrate a specific and highly reproducible cytokine gene expression pattern during the course of a primary in vivo immune response that is marked by an absence of a clear-cut Th1/Th2 dichotomy.
Animals↗
Interleukin 1-induced maturation of progenitor thymocytes.
Regulation of thymocyte development was assessed by culturing purified CD4-CD8- thymocytes with cytokines. Sorted CD3-CD4-CD8- adult thymocytes responded to the combination of interleukin (IL) 1 plus IL 2 without additional mitogens or co-mitogens with both cellular proliferation and cell surface expression of the T cell receptor (TcR)/CD3 complex. IL2 alone induced neither proliferation nor cell surface TcR/CD3 expression. IL1 alone was sufficient to induce cell surface TcR/CD3 without proliferation. Prior to stimulation with cytokines, the progenitor CD4-CD8- thymocytes accumulated TcR beta and CD3 gamma, delta, epsilon and zeta mRNA but TcR alpha mRNA was not detectable. Stimulation with IL 1 led to a dramatic induction of TcR alpha mRNA without an increase in the other transcripts. These studies suggest that IL 1 regulates the differentiation status of immature adult thymocytes. Nuclear run-on studies suggested that the increase in TcR alpha mRNA accumulation induced by cytokines might result from post-transcriptional accumulation.
Animals↗
Murine models for systemic lupus erythematosus and Sjögren's syndrome.
Of the types of defects that have been observed in autoimmune mice, two are common to most strains and appear to be of critical importance in the development of autoimmune disease. The first of these is a deficiency in the maintenance of self-tolerance by B or T lymphocytes. This is an intrinsic defect due to genetic abnormalities that presumably affect an early developmental signaling mechanism. The disruption of early B- and T-cell development leads to the appearance of lymphocytes that are predisposed to loss of tolerance. In certain strains of autoimmune mice, it also leads to overproduction of Ly1+ (CD5+) B cells or CD4-CD8-B220+ T cells, which apparently do not contribute to autoimmunity. The second type of defect is a strain-specific, antigen-driven autoimmune disease syndrome. This defect is limited by the affinities of the available T-cell receptor and immunoglobulin variable regions and the efficiency of antigen presentation. Current evidence suggests that the major histocompatibility complex molecules play the most important role in determining which autoantigens lead to a pathogenic antigen-driven immune response. For example, the type and magnitude of the autoimmune response may be directed by the production of specific cytokines. The type of antigen and the context of its presentation can bias the production of cytokines that favor the production of the Th2 subset of CD4+ T helper cells. These cells secrete predominantly interleukin-4, interleukin-5, and interleukin-10, and may contribute to a chronic response and autoimmune disease.
Animals↗
Anti-CD3 antibody induces rapid expression of cytokine genes in vivo.
Anti-CD3 antibody was administered to mice i.v. and the kinetics of spleen cell cytokine mRNA expression studied by Northern analysis. Untreated mice and mice receiving control antibody had low or undetectable amounts of mRNA for c-fos, c-myc, IL-2, IL-4, and IFN-gamma. After injection of anti-CD3 antibody, substantial increases in all were found. Induction of c-fos was detected at 10 min and of c-myc at 30 min after injection. IL-2, IL-4, and IFN-gamma mRNA were induced by 30 min and reached peak levels at 60 min. Thereafter, IL-2 and IL-4 mRNA declined, whereas IFN-gamma mRNA persisted. The induced cytokine mRNA was not observed in athymic nu/nu mice nor in normal spleen cells from which T cells had been depleted in vitro. The early in vivo induction of IL-4 mRNA contrasts with prior in vitro studies in which IL-4 production was difficult to detect after primary stimulation. To assess the possibility that many T cells had been preprimed in vivo, germfree mice were compared with conventional mice and no differences in cytokine mRNA were found. These data show that T cell-dependent IL message production can be induced rapidly in vivo without prepriming and that the cytokine messages induced after anti-CD3 antibody administration do not suggest a predominance of either Th1 or Th2 type cells.
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The cDNA sequence of mouse Pgp-1 and homology to human CD44 cell surface antigen and proteoglycan core/link proteins.
We describe the isolation and sequencing of a cDNA encoding mouse Pgp-1. An oligonucleotide probe corresponding to the NH2-terminal sequence of the purified protein was synthesized by the polymerase chain reaction and used to screen a mouse macrophage lambda gt11 library. A cDNA clone with an insert of 1.2 kilobases was selected and sequenced. In Northern blot analysis, only cells expressing Pgp-1 contained mRNA species that hybridized with this Pgp-1 cDNA. The nucleotide sequence of the cDNA has a single open reading frame that yields a protein-coding sequence of 1076 base pairs followed by a 132-base pair 3'-untranslated sequence that includes a putative polyadenylation signal but no poly(A) tail. The translated sequence comprises a 13-amino acid signal peptide followed by a polypeptide core of 345 residues corresponding to an Mr of 37,800. Portions of the deduced amino acid sequence were identical to those obtained by amino acid sequence analysis from the purified glycoprotein, confirming that the cDNA encodes Pgp-1. The predicted structure of Pgp-1 includes an NH2-terminal extracellular domain (residues 14-265), a transmembrane domain (residues 266-286), and a cytoplasmic tail (residues 287-358). Portions of the mouse Pgp-1 sequence are highly similar to that of the human CD44 cell surface glycoprotein implicated in cell adhesion. The protein also shows sequence similarity to the proteoglycan tandem repeat sequences found in cartilage link protein and cartilage proteoglycan core protein which are thought to be involved in binding to hyaluronic acid.
Amino Acid Sequence↗
A role for endogenous retroviral sequences in the regulation of lymphocyte activation.
The genomes of most vertebrates contain numerous retroviral sequences, the great majority of which are non-infectious. These endogenous retroviral sequences are transcribed and translated in many host tissues, and are induced by mitogens. The function, if any, of endogenous retroviruses has been unclear. The transmembrane envelope proteins of some infectious type C retroviruses suppress lymphocyte activation, but it is unknown whether any endogenous type C retroviruses share this suppressive activity. To study the possible effects of murine endogenous retroviral expression, specific antisense oligonucleotides were synthesized complementary to type C retroviral sequences, and were cultured with murine spleen cells. If any of these endogenous retroviruses are suppressing lymphocyte activation, then inhibiting such endogenous retroviral-mediated suppression with antisense might result in lymphocyte stimulation. Three classes of endogenous type C retroviral sequences may be distinguished by antisense oligonucleotides (based on their homology to infectious retroviruses): ecotropic, xenotropic, and mink cell focus-forming (MCF). Antisense oligonucleotides to endogenous MCF envelope gene (env) initiation regions caused: i) doubling or tripling of spleen cell RNA synthesis, and ii) marked increases in lymphocyte surface Ia and Ig expression relative to control oligonucleotides. Antisense oligos to xenotropic or ecotropic env sequences or to endogenous MCF non-envelope sequences had no effect. These data suggest that endogenous MCF sequences exert an inhibitory influence on the murine immune system. Because endogenous MCF expression is inducible by immune stimuli, such expression could constitute an inhibitory feedback circuit that participates in the regulation of immune homeostasis.
Animals↗
Responsiveness of fetal and adult CD4-, CD8- thymocytes to T cell activation.
Day-14 fetal CD4-, CD8- thymocytes showed a greater proliferative response to PMA + IL-4 than did adult double-negative thymocytes. In contrast, adult double-negative thymocytes were more responsive to PMA + IL-1 + IL-2 or to IL-1 + IL-2 alone. The adult double-negative thymocytes showed significantly greater proliferation than fetal thymocytes after stimulation via anti-CD3 or anti-Thy-1 in the presence or absence of interleukins (IL-1 + IL-2 or IL-4). Adult CD4-, CD8- thymocytes also exhibited greater calcium mobilization following anti-CD3 stimulation IL-2-dependent activation with anti-Thy-1 or IL-1 + IL-2 in the absence of PMA resulted in marked expansion of CD 3+, F23.1+, CD4-, CD8- thymocytes, a population absent in fetal thymocytes but constituting 4% of pre-cultured CD4-, CD8- adult thymocytes. IL-4 + PMA failed to expand this CD 3+ population. It is hypothesized that before expression of functional TCR, T cell development may be more dependent on activation pathways not using IL-2; after TCR expression, IL-2-dependent pathways, including Thy-1-mediated stimulation, become functional.
Animals↗
Activation of CD 4-, CD 8- thymocytes with IL 4 vs IL 1 + IL 2.
Thymocytes from C57BL/6 mice were highly purified to obtain the CD 4-, CD 8- subpopulation which constitutes only 5% of all thymocytes. Substantial proliferation was induced in vitro with either IL-1 + IL-2 or with IL-4 in the presence of PMA. IL-1 and IL-2 synergized in inducing proliferation of these purified CD 4-, CD 8- thymocytes whereas neither synergized with IL-4. In order to determine whether stimulation with IL-1 + IL-2 acted via IL-4 or vice versa, cultures were treated reciprocally with affinity-purified anti-IL-2 or anti-IL-4 antibodies. Cultures with IL-4 were inhibited by anti-IL-4 but were unaffected by anti-IL-2. The CD 4-, CD 8- thymocytes cultured with IL-1 + IL-2 + anti-IL-2 were inhibited to baseline IL-1 stimulation. At low concentrations of IL-1 (1 U/ml) and IL-2 (100 U/ml), anti-IL-4 had no effect, whereas at higher levels of IL-1 (2 U/ml IL-1), and 100 or 200 U/ml IL-2, anti-IL-4 significantly reduced DNA synthesis. This result suggests that at higher concentrations the combination of IL-1 + IL-2 can induce cells to produce IL-4 which then contributes to overall proliferation. When CD 4-, CD 8- thymocytes were cultured with the low doses of IL-1 + IL-2 for 72 h, 62% expressed cell surface T3 complex (vs 11% at initiation) and 27% were F23.1+ (vs 5% at initiation). In contrast, culture with IL-4 led to no increase in numbers of T3+ cells and none were F23.1+; however, there was coexpression of Thy1 and 6B2 on 20% of cells at the end of culture (vs 4% at initiation). Thus, IL-1 + IL-2 causes expansion of a CD 4-, CD 8- thymocyte population expressing the alpha, beta-T cell receptor, whereas IL-4 induces cells to express a phenotype present in small numbers in the periphery of normal mice and in larger numbers in mice bearing the lpr gene.
Animals↗
Prevention of lymphadenopathy in MRL-lpr/lpr mice by blocking peripheral lymph node homing with Mel-14 in vivo.
MRL-lpr/lpr mice develop massive lymphadenopathy and autoimmunity. There is evidence that both migration and local proliferation contribute to the accumulation of Ly-2-, L3T4-, 6B2+ T cells in the peripheral lymph node (PLN). Mel-14 is an antibody which binds to the lymphocyte lymph node homing receptor (gp90Mel-14) and can block migration of lymphocytes to the PLN. Treatment of mice from birth to 11 wk of age with Mel-14 and another rat IgG2a mAb, 6B2, resulted in reduction (10- to 20-fold) in lymphadenopathy. Mel-14, but not 6B2, preferentially reduced the percentages of Thy-1+, 6B2+ lymphocytes in the lymph node. Treatment with a third antibody, anti-Ly-1, had no effect on lymphadenopathy. Mel-14 treatment resulted in diversion of the Ly-2-, L3T4-, 6B2+, gp90Mel-14 cells to the spleen and consequently induced marked splenomegaly. Thymocytes from MRL-lpr/lpr and MRL-+/+ mice were analyzed by two-color flow cytometry analysis after depletion of Ly-2+ and L3T4+ T cells. There was no difference in the percent of Ly-2-, L3T4-, 6B2+, gp90Mel-14 positive thymocytes comparing these two strains. Mel-14 treatment did not alter Ig levels or autoantibody production. These studies suggest Mel-14 reduced lymphadenopathy by interfering with homing to PLN, whereas 6B2 may have interfered with marrow production of precursor cells or killed 6B2+ cells after they exited the marrow. The data are consistent with the idea that lymphadenopathy occurs in MRL-lpr/lpr mice due to increased homing gp90-Mel-14 T cells to the PLN and that gp90Mel-14 is a necessary receptor for the abnormal 6B2+ T cells.
Animals↗
Characterization and differentiation of CD4-, CD8- thymocytes sorted with the Ly-24 marker.
Heterogeneity within the CD4-, CD8- thymocyte population was explored by flow microfluorometry on thymocytes from 6-wk-old female C57BL/6 mice. Double negative cells were obtained by twice killing thymocytes with anti-CD4 and anti-CD8 antibodies. The resultant population lacked CD4, CD8, and Ig on cell surfaces; it contained cells bearing Ly-24 (27%), Ly-6C (6%), and Ly-5 (B220) detected by 6B2 (6%). These markers are the same as those characteristic of lpr/lpr T cells; they also are found on bone marrow cells. In order to investigate the maturational pathway of CD4-, CD8- thymocytes, such cells were cultured in vitro for 6 days with phorbol myristate acetate + calcium ionophore. There was a marked increase in cells bearing Ly-24 with time; by 6 days essentially all bore Ly-24. A lesser increase (to 15%) in 6B2 + Thy-1 positive cells was observed. Small numbers of cells bearing CD4 and/or CD8 also were found after 6 days in vitro. In additional studies, CD4-, CD8- cells were first sorted with respect to Ly-24 and then cultured with phorbol myristate acetate + calcium ionophore. Ly-24+ cells proliferated vigorously and formed clusters whereas Ly-24- cells did not. The former gave rise to large numbers of CD4+, CD8+ cells; the latter exhibited little differentiation. These studies demonstrate substantial heterogeneity within the CD4-, CD8- thymocyte population. Use of the markers Ly-24, Ly6C, and 6B2 allows a subdivision of such progenitor thymocytes. Different stages of maturation as well as possible lineages of cells may be investigated by combining such hemopoietic cell surface markers with in vitro culture.
Animals↗