Multiple sclerosis: Fas signaling in oligodendrocyte cell death.

Fas is a cell surface receptor that transduces cell death signals when cross-linked by agonist antibodies or by fas ligand. In this study, we examined the potential of fas to contribute to oligodendrocyte (OL) injury and demyelination as they occur in the human demyelinating disease multiple sclerosis (MS). Immunohistochemical study of central nervous system (CNS) tissue from MS subjects demonstrated elevated fas expression on OLs in chronic active and chronic silent MS lesions compared with OLs in control tissue from subjects with or without other neurologic diseases. In such lesions, microglia and infiltrating lymphocytes displayed intense immunoreactivity to fas ligand. In dissociated glial cell cultures prepared from human adult CNS tissue, fas expression was restricted to OLs. Fas ligation with the anti-fas monoclonal antibody M3 or with the fas-ligand induced rapid OL cell membrane lysis, assessed by LDH release and trypan blue uptake and subsequent cell death. In contrast to the activity of fas in other cellular systems, dying OLs did not exhibit evidence of apoptosis, assessed morphologically and by terminal transferase-mediated d-uridine triphosphate-biotin nick-end-labeling staining for DNA fragmentation. Other stimuli such as C2-ceramide were capable of inducing rapid apoptosis in OLs. Antibodies directed at other surface molecules expressed on OLs or the M33 non-activating anti-fas monoclonal antibody did not induce cytolysis of OLs. Our results suggest that fas-mediated signaling might contribute in a novel cytolytic manner to immune-mediated OL injury in MS.

Sequences from sea urchin TU transposons are conserved among multiple eucaryotic species, including humans.

Sequences homologous to various structural domains of the Strongylocentrotus purpuratus TU family of transposons are present in sea urchin species closely related to S. purpuratus and were found in close proximity to each other in linkage patterns that differed for different species. Sequence homologs of the inverted repeat outer domain (IVR-OD) segment were, in addition, present in a sea urchin related only distantly to S. purpuratus and in all other eucaryotic organisms surveyed. In humans, a polymorphic hybridization pattern was seen for genomic DNA obtained from different individuals. Sequence comparisons revealed that repeated sequence motifs similar to those making up the 15-base-pair direct repeat unit of the IVR-OD domain of the TU elements exist in the IVRs of transposons identified in Drosophila melanogaster and maize and in the transcription control regions of certain eucaryotic viral and cellular genes. The remarkable evolutionary conservation of IVR-OD homologs may reflect a biological role for these sequences in DNA transposition, the regulation of gene expression, or both.

Interleukin 15 protects against toxicity and potentiates antitumor activity of 5-fluorouracil alone and in combination with leucovorin in rats bearing colorectal cancer.

5-Fluorouracil (FUra) modulated by leucovorin (LV) is active in the treatment of colorectal cancer. Diarrhea and stomatitis are the most common dose-limiting toxicities. We have developed a model system in rats bearing a transplantable colon carcinoma sensitive to FUra therapy with dose-limiting toxicity profiles similar to what is observed in patients treated with either daily or weekly schedules of FUra plus LV. Interleukin 15 (IL-15), a cytokine that shares many biological activities with IL-2, was used at different doses (25, 100, and 400 microg/kg) and schedules (three doses before a single dose of FUra, FUra/LV daily x 5, or before each week of FUra/LV weekly x 4, or three doses before a single dose of FUra or FUra/LV daily x 5, then twice daily x 5 for a total of 11 doses) to evaluate its role in the modulation of the therapeutic selectivity of FUra alone and modulated by LV. IL-15 induced a dramatic decrease in chemotherapy-induced gastrointestinal toxicities, significant potentiation of antitumor activity, and an increased therapeutic index of FUra administered on single dose, daily x 5 and weekly x 4 schedules. In contrast, IL-2 (400 microg/kg) significantly potentiated the toxicity of FUra administered as a single i.v. push, with minimal potentiation of the antitumor activity. Taken together, the results clearly demonstrated the ability of IL-15, but not IL-2, to provide significant improvement of the therapeutic index of FUra alone and in combination with LV. The clinical relevance of the results obtained in this model system needs to be confirmed.

Interleukin 15 offers selective protection from irinotecan-induced intestinal toxicity in a preclinical animal model.

Irinotecan (CPT-11) is a chemotherapeutic agent that is active in the treatment of a variety of solid tumor malignancies. Diarrhea represents the most common dose-limiting toxicity that is independent of the schedule of administration. A rat model with dose-limiting toxicity profiles that are similar to those observed in patients treated with CPT-11 was developed and used to evaluate the role of interleukin 15 (IL-15) in the modulation of the therapeutic selectivity of CPT-11 in normal rats and rats bearing advanced colorectal cancer. The maximum tolerated dose and lethal dose (LD) of CPT-11 by i.v. push daily x 3 were 150 and 200 mg/kg/day, respectively. CPT-11 at the LD induced a 93-100% incidence of severe diarrhea and an 86-100% incidence of lethality in treated animals. IL-15, a cytokine with multiple mechanisms of action, was used at a 100 or 400 microg/kg/dose with different schedules of administration (3, 8, and 11 doses, i.p.) to protect against CPT-11-induced toxicity. IL-15 offered complete and sustained selective protection against CPT-11-induced delayed diarrhea and lethality. IL-15 also moderately potentiated the antitumor activity of CPT-11 in rats bearing advanced colorectal cancer. Morphological examination of rat intestinal tissues after treatment with LD of CPT-11 revealed dramatic protection of duodenal and colonic tissue architecture by IL-15. CPT-11 alone produced serious damage to duodenal villi and colonic crypts. The results clearly demonstrated the ability of IL-15 to provide significant protection from CPT-11-induced intestinal toxicity with maintenance of antitumor activity, resulting in an increase in the therapeutic index of CPT-11. The clinical relevance of the results obtained in this model system needs to be confirmed.

Interleukin-17 antagonism inhibits acute but not chronic vascular rejection.

BACKGROUND: Blocking the action of interleukin (IL) 17 with an IL-17 receptor (R):Fc fusion protein inhibits T-cell proliferative responses to alloantigens and prolongs vascularized heart graft survival. In this study, we examined whether IL-17 antagonism could suppress the development of chronic rejection. METHODS: A 0.6-cm section of C57BL10 (H2b) thoracic aorta was transplanted to recipient C3H (H2k) abdominal aorta. IL-17R:Fc or control human immunoglobulin G was administered i.p. (500 microg/day) from days 0 to 6 or from days 0 to 29. Mice were killed on days 7 or 30. Grafts were examined histologically and stained for alpha-smooth muscle actin (alpha-smA). Antidonor mixed leukocyte reaction, cytotoxic T cell, and alloantibody responses were quantified. RESULTS: On day 7, control grafts showed mononuclear cell (MNC) infiltration, pronounced endothelial damage, and apoptosis of intimal and medial cell compartments. By day 30, there was concentric intimal thickening, accumulation of alpha-smA+ cells, and collagen deposition. Patchy destruction of the elastic membranes and loss of alpha-smA expression in media were evident. IL-17R:Fc for 6 days decreased MNC infiltration in the intimal and medial compartments at day 7. The endothelium was preserved (completely or partially) in all grafts. The medial compartment showed normal alpha-smA expression. Irrespective of IL-17R:Fc treatment for either 6 days or continuously, allografts harvested at day 30 showed circumferential intimal thickening, with accumulation of alpha-smA+ cells and collagen deposition. There was no effect on circulating alloantibody levels. CONCLUSIONS: These findings support a role for IL-17 in the immunopathogenesis of acute vascular rejection and demonstrate the potential of IL-17 antagonism for therapy. By contrast, IL-17 antagonism does not appear to prevent ensuing chronic graft vascular disease, in particular neointimal formation.

Fas expression on human fetal astrocytes without susceptibility to fas-mediated cytotoxicity.

Fas (APO-1/CD95) is a cell surface receptor, initially identified in lymphoid cells, but more recently detected in the central nervous system under pathologic conditions. Ligation of the fas receptor by fas ligand or by agonist antibodies induces apoptotic cell death in most fas-expressing cells. In the current study, using dissociated cultures of human fetal central nervous system-derived cells, we detected fas expression on astrocytes but not on neurons. Such expression differs from our previous results using cultures of human adult central nervous system-derived cells, which demonstrated fas expression on oligodendrocytes but not on astrocytes; the oligodendrocytes were susceptible to cell death via this pathway. Using multiple assays of cell death, including nuclear propidium iodide and TUNEL staining to detect nuclear-directed injury, cytofluorometric propidium iodide inclusion, and lactate dehydrogenase release to detect membrane-directed injury, we found that fas ligation, however, did not induce cell death in the cultured fetal astrocytes. Cytokines that augmented (gamma-interferon) or inhibited (interleukin-4) fetal astrocyte proliferation did not alter fas expression or resistance to fas ligation. Cells obtained immediately ex vivo from human fetal but not from adult central nervous system tissue expressed fas; such expression was restricted to astrocytes as assessed by dual-stain immunohistochemistry. The fetal central nervous system cells did not express fas ligand. Our findings indicate that fas expression on central nervous system cells may reflect their state of maturity; expression may not, however, always be coupled to susceptibility to cell death via this pathway.

Lymphokine synthesis in vivo in an acute murine graft-versus-host reaction: mRNA and protein measurements in vivo and in vitro reveal marked differences between actual and potential lymphokine production levels.

The in vivo activation of lymphokine-producing cells was analyzed in the first 7 days of an acute graft-versus-host reaction (GVHR) induced by injection of C57BL/6 spleen cells into irradiated DBA/2 mice. Although the GVHR was accompanied by a 1000-fold increase in serum IL-6 titers, circulating levels of other lymphokines were low (IL-3, IFN-gamma, and GM-CSF) or undetectable (IL-2 and IL-4). Spleen and lymph node cells from these mice nevertheless produced elevated levels of IL-3, IFN-gamma, GM-CSF, and IL-6 when cultured for 24 h without stimulation; culture with anti-CD3 antibody further increased IL-2, IL-3, IL-4, IFN-gamma, and GM-CSF production by at least 20-fold. Both constitutive and anti-CD3-induced synthesis of all the lymphokines was mediated by CD3+ cells. Messenger RNA analyses revealed the presence of IL-6, IFN-gamma, and GM-CSF transcripts in freshly harvested GVHR spleen cells and increased expression of IL-2, IL-3, IL-4, IFN-gamma, and GM-CSF mRNAs following anti-CD3 stimulation in vitro. In vivo, however, IL-3 mRNA was barely detectable even following cDNA amplification by polymerase chain reaction. In vivo restimulation of day 5 GVHR mice by injection of concanavalin A enhanced expression of IL-3, IL-6, IFN-gamma, and GM-CSF mRNAs and markedly increased serum titers of the corresponding lymphokines, which peaked 6-12 h after injection at levels at least 10- to 100-fold higher than in concanavalin A-treated normal mice.2+ for high-level synthesis of all these lymphokines.

Survival of the myeloid progenitor cell line FDC-P1 is prolonged by interferon-gamma or interleukin-4.

Continuous proliferation of the immortalized myeloid progenitor cell line FDC-P1 depends on stimulation with either interleukin-3 (IL-3) or granulocyte-macrophage colony stimulating factor (GM-CSF). Two other cytokines, interferon-gamma (IFN-gamma) and IL-4, were found to prolong FDC-P1 survival for several days. Surviving cells incorporated [3H]thymidine and a minority completed up to 3 cell divisions before dying. This transient proliferative response was a direct effect of IFN-gamma and IL-4 since these cytokines did not induce production of detectable IL-3 or GM-CSF and the response was unaffected by cell concentration. IL-6, a constitutive product of FDC-P1 cells whose secretion was increased by IL-3, GM-CSF and IL-4 but not by IFN-gamma, was not responsible for the proliferative response. FDC-P1 lines that constitutively expressed the cell cycle-associated oncogene myc or the survival-associated oncogene bcl-2 also responded only transiently to IFN-gamma or IL-4, indicating that expression of these genes did not complement the signals delivered by IFN-gamma or IL-4. By contrast, the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) prolonged survival of FDC-P1 cells on its own and potentiated the response to IFN-gamma or IL-4, although the combination of stimuli did not support long-term growth. It is concluded that IFN-gamma and IL-4 trigger only some of the signalling events that lead to mitogenesis; these events are complemented by stimulation with PMA but additional signals are required for sustained proliferation.

Tissue distribution of murine hemopoietic growth factor mRNA production.

We have studied the tissue distribution of interleukin (IL) and hemopoietic colony-stimulating factor (CSF) transcripts in mice by S1-nuclease protection analysis. Accumulation of several of these mRNAs in response to intravenous injection of lipopolysaccharide (LPS) appears to occur in a tissue-specific fashion. IL-1 alpha transcripts accumulate in spleen and lung; IL-6 transcripts accumulate in kidney, heart, and spleen; granulocyte-macrophage-CSF transcripts accumulate in lung and heart; and granulocyte-CSF transcripts accumulate in heart. Three distinct patterns of in vivo mRNA accumulation were detected: 1) silent--interleukins 2-5 showed no transcripts in either LPS-treated or untreated animals; 2) induced--IL-1 alpha, IL-6, granulocyte-macrophage (GM)-CSF, and G-CSF transcripts were increased in abundance in LPS-injected mice; and 3) constitutive--M-CSF transcripts were found in similar amounts in both untreated and treated mice and were present in all tissues examined.

Enumeration of lymphokine mRNA-containing cells in vivo in a murine graft-versus-host reaction using the PCR.

A method of enumerating lymphokine mRNA-containing cells in vivo was developed by combining limiting dilution analysis with PCR amplification of cDNA. Single-hit kinetics revealed that the PCR-limiting dilution analysis could detect a single positive cell among greater than 40,000 negative cells. With this method, spleens and lymph nodes of mice undergoing an acute allogeneic graft-versus-host reaction were found to contain lymphokine mRNA-expressing cells at frequencies of 3% for interferon gamma, 0.05% for granulocyte/macrophage colony-stimulating factor, 0.002% for interleukin 3, and 0.03% for interleukin 4; these frequencies were 20- to 175-fold higher than in lymphoid tissues of normal mice. In contrast to their low frequencies of lymphokine mRNA-containing cells in vivo, graft-versus-host reaction populations restimulated in vitro for 24 hr with anti-CD3 antibody yielded frequencies ranging from 3% for interleukin 4 to nearly 70% for interferon gamma. Furthermore, lymphokine transcripts were also detected in single micromanipulated cells from these populations. Because frequencies of anti-CD3-inducible lymphokine mRNA-containing cells in normal mice were only 0.03-1%, it was concluded that lymphoid tissues of graft-versus-host reaction mice contained high frequencies of cells that had been primed for lymphokine synthesis. Only a small fraction of these cells, however, expressed lymphokine mRNAs at a given time point in vivo.

Evidence for the stochastic acquisition of cytokine profile by CD4+ T cells activated in a T helper type 2-like response in vivo.

The diversity of cytokine production patterns displayed by T cells activated in vivo was investigated by analyzing short-term antigen-specific CD4+ T cell clones and single CD4+ T cells derived from draining lymph nodes of mice undergoing a T helper 2 (Th2)-like response to keyhole limpet hemocyanin (KLH). On average, 2.7% of CD4+ lymph node cells gave rise to clones in the presence of the immunizing antigen and, of these, about 90% secreted interleukin-4 (IL-4) and 20% secreted interferon-gamma (IFN-gamma) when restimulated after 2 weeks in vitro. Almost all IFN-gamma-producing clones co-produced IL-4. The definition of clones as positive or negative for cytokine synthesis depended on assay sensitivity, however, since their titers were distributed continuously from the threshold of detection over at least a 1000-fold range. Reverse-transcription polymerase chain reaction analysis of 59 clones revealed multiple patterns of co-expression of IL-2, IL-3, IL-4, IL-6, IFN-gamma and granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA. Although most clones contained detectable IL-4 and IL-6 mRNA and a minority contained IFN-gamma mRNA, only 1 clone expressed the canonical Th2 cytokine profile. The observed frequencies of mRNA co-expression for most of the six cytokines (including IL-4 with IFN-gamma), and the frequency of co-secretion of IL-4 and IFN-gamma, were not significantly different from those predicted for random association. Independent regulation of IL-4 and IFN-gamma mRNA expression was confirmed at the single-cell level in a polyclonal population of KLH-primed CD4+ cells, among which co-expression of these cytokines again occurred at the frequency predicted for a random event. The data suggest that the polarization of this immune response towards a Th2 cytokine profile is achieved by altering the probabilities of expression of the IL-4, IFN-gamma and other cytokine genes at the population level, rather than by selective expansion of a distinct T cell subset.

Changes in the precursor frequencies of IL-4 and IFN-gamma secreting CD4+ cells correlate with resolution of lesions in murine cutaneous leishmaniasis.

Limiting dilution analysis was used to estimate the frequency of clonogenic Ag-specific CD4+ T lymphocytes in draining lymph nodes of mice over the course of infection with Leishmania major, and to measure the production of IL-2, IL-3, IL-4, IFN-gamma, and TNF by the resultant clones. Infection of both genetically susceptible BALB/c ("non-healer") and resistant C57BL/6 ("healer") mice resulted in at least a fourfold increase in the frequency (to about 0.3%) and at least a 10-fold increase in the total number of lymph node CD4+ cells that formed clones when cultured with L. major Ag in vitro. At 1 wk after infection, the majority of clones from BALB/c mice secreted IL-4 (precursor frequency 0.15%) and fewer secreted IFN-gamma (0.05%); this pattern remained constant for at least 8 wk after infection. In C57BL/6 mice, however, a high precursor frequency of IL-4-secreting clones was measured in the first 1 to 2 wk when the mice had lesions, but resolution of infection was associated with a decrease in the frequency of IL-4-secreting clones (from 0.13% at 2 wk to 0.03% at 4 wk) and an increase in the frequency of IFN-gamma-secreting clones (from 0.08% to 0.22%). At all stages of infection, most clones from either mouse strain secreted IL-3 and very few secreted TNF. Analysis of PCR-amplified cDNA from draining lymph nodes of infected mice also revealed that IL-4 and IFN-gamma mRNA were expressed in both mouse strains early in infection. IL-4 mRNA was the major species at 2 and 6 wk after infection in BALB/c mice, but declined relative to IFN-gamma mRNA over this time in C57BL/6 lymph nodes. Precursor frequency estimates of lymphokine-secreting CD4+ cells in draining lymph nodes therefore correlated with lymphokine expression patterns in vivo. Analysis of a panel of individual short term clones derived from mice 1 wk after infection revealed marked heterogeneity in lymphokine production patterns. In BALB/c mice, 49% secreted IL-4 without IFN-gamma, 18% secreted IFN-gamma without IL-4, and 14% secreted both IL-4 and IFN-gamma. Similarly in C57BL/6 mice, 39% secreted IL-4, 20% secreted IFN-gamma, and 17% secreted both lymphokines. Many of the clones also produced IL-3 and/or IL-2. Together the data suggest that both IL-4 and IFN-gamma are synthesized early in infection of susceptible and resistant mice as assessed by mRNA and precursor frequency analyses.(ABSTRACT TRUNCATED AT 400 WORDS)

Co-engagement of CD3 with LFA-1 or ICAM-1 adhesion molecules enhances the frequency of activation of single murine CD4+ and CD8+ T cells and induces synthesis of IL-3 and IFN-gamma but not IL-4 or IL-6.

Interaction between the cell adhesion molecules lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) augments T cell activation by increasing the avidity of T cell/antigen-presenting cell (APC) binding. To examine whether LFA-1 and ICAM-1 can also contribute to T cell activation in the absence of APCs, single murine CD4+ and CD8+ T cells were cultured with IL-2 and immobilized antibodies to CD3, CD4 or CD8, and LFA-1 or ICAM-1. The combination of anti-CD3, anti-CD4/CD8, and IL-2 stimulated approximately 20% of CD4+ cells and 30% of CD8+ cells to proliferate. Inclusion of anti-ICAM-1 antibody increased these frequencies to 30 and 40% respectively. Maximum activation frequencies were obtained with the combination of anti-CD3, anti-CD4/CD8, and anti-LFA-1 which stimulated cell division by approximately 40% of single CD4+ cells and at least 60% of single CD8+ cells. Under these conditions, 30-40% of the resultant CD4+ clones and greater than 90% of CD8+ clones secreted IL-3 and IFN-gamma. In addition to responding at higher frequencies that CD4+ cells, CD8+ cells formed larger clones which produced 4-fold higher levels of both cytokines. Although the expression of IL-2, IL-3, IFN-gamma, granulocyte-macrophage colony stimulating factor and tumor necrosis factor alpha could be detected in CD4+ and CD8+ clones at the mRNA level following reverse transcription and polymerase chain reaction amplification, neither the secreted nor mRNA expression of IL-4 or IL-6 was detected in any of the tested clones. It is concluded that co-stimulation of T cells via LFA-1 or ICAM-1 can enhance T cell receptor-dependent activation in the absence of accessory cells and that this mode of stimulation leads to the expression of a restricted range of cytokine genes.

Contact sensitization to oxazolone: involvement of both interferon-gamma and interleukin-4 in oxazolone-specific Ig and T-cell responses.

The synthesis and role of several lymphokines were examined during contact sensitization to oxazolone (OX). Application of OX to the skin of mice increased the delayed-type hypersensitivity (DTH) response to challenge, serum titres of OX-specific IgG1 and IgG2a, and draining lymph node cell (LNC) numbers. At day 3, LN contained detectable interleukin-4 (IL-4), interferon-gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) but not IL-2 or IL-3 mRNAs; IL-3 and higher levels of IL-4, IFN-gamma and GM-CSF mRNAs were measured after 24 hr culture with anti-CD3 antibody in OX-primed but not unprimed LNC. As a result of sensitization, LNC secreted IL-3 constitutively and produced elevated levels of IL-2, IL-3, IL-4 and IFN-gamma in response to anti-CD3 antibody; a similar but weaker lymphokine response was recalled by OX-protein conjugate. CD4+ cells were the major source of the anti-CD3-induced lymphokines except IFN-gamma, which was derived mainly from CD8+ cells. Since both IL-4 and IFN-gamma were synthesized by OX-primed LNC in vivo and in vitro, their role was investigated by administering anti-lymphokine antibodies at the time of sensitization. Anti-IL-4 treatment reduced OX-specific serum IgG1 titres without affecting IgG2a titres, whereas anti-IFN-gamma treatment reduced IgG2a but not IgG1 titres. Although neither antibody altered DTH responsiveness, anti-IFN-gamma treatment markedly increased IL-4 production by CD4+ LNC and reduced IFN-gamma production in vitro, particularly by CD4+ cells. We conclude that endogenous IL-4 and IFN-gamma reciprocally influence the isotype of the Ig response to OX and that IFN-gamma also affects the relative levels of IL-4 and IFN-gamma synthesis by CD4+ LNC.

Soluble receptors neutralizing TNF-alpha and IL-1 block stress-triggered murine abortion.

PROBLEM: In several models of abortion in rodents, the success or failure of the implanted embryos is determined by a balance between pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin-2, interleukin-1 (IL-1), and gamma-interferon, and cytokines that counteract the former, such as interleukin-10 and transforming growth factor-beta 2 (TGF-beta 2)-related suppressor factor. Stress can trigger abortions in susceptible strains of mice and is thought to reflect the pathogenesis of some types of miscarriage in human pregnancy. In mice, stress increases levels of the abortogenic cytokine TNF-alpha and decreases the suppressive activity of TGF-beta 2-related factor via a neurotransmitter substance P (SP)-dependent pathway. Evidence for a role of pro-inflammatory cytokines in SP-mediated abortions in vivo is indirect. METHODS: Direct evidence for a role of IL-1 and TNF-alpha in stress-triggered abortions was sought by injecting pregnant female mice with soluble receptors neutralizing TNF-alpha (rhuTNFR:Fc) or IL-1 (rmIL-IR) beginning 1 day after implantation and prior to stress. RESULTS: The stress-triggered abortion rate was reduced by 68% when either TNF-alpha or IL-1 antagonists were injected. The stress-triggered decreased TGF-beta 2-like suppressive activity in the maternal uterine decidua was not restored by injection of either antagonist; indeed the soluble IL-1 receptor significantly reduced suppressive activity in unstressed control mice, and soluble TNF-alpha receptor had a lesser effect. CONCLUSIONS: Both IL-1 and TNF-alpha play a role in the pathogenesis of stress-triggered abortions, and may induce a compensatory physiological increase in suppressive activity in normal pregnancy counteracting pro-inflammatory cytokines.

Limiting dilution analysis reveals the precursors of interleukin-4-producing CD4+ cells induced by protein immunization.

Although cytokine-producing T cells play a key role in the response to vaccination, they are not always revealed by antigen stimulation of primed lymphoid cells in vitro. In this study, mice were immunized subcutaneously with alum-precipitated keyhole limpet haemocyanin (KLH) in adjuvant to activate interleukin-4 (IL-4)-producing CD4+ T cells. IL-4 mRNA was the dominant cytokine mRNA species found in draining lymph nodes (LN) 7 days after immunization and its levels were increased after in vitro stimulation with KLH for 24 hr. IL-4 protein, on the other hand, was not detected in the supernatants of such antigen-stimulated cultures. The presence of T cells primed for IL-4 production was nevertheless suggested by the findings that primed LN cells produced low IL-4 titres in response to anti-CD3 antibody, whereas normal LN cells did not, and primed CD4+ LN cells produced readily detectable IL-4 levels in response to antigen after one or more cycles of in vitro restimulation. Culture at limiting dilution showed that 1-2% of 7 day KLH-primed CD4+ LN cells were clonogenic and specific for KLH without prior expansion in vitro, and that this frequency was markedly increased by repeated stimulation in bulk culture. Most clonogenic cells in primed LN gave rise to IL-4-secreting clones and a smaller number gave rise to interferon-gamma (IFN-gamma)-producing clones. The precursor frequency of IL-4-producing CD4+ cells in primed LN and the average IL-4 titre per cloned cell support the conclusion that these two parameters account for the low levels of IL-4 produced in bulk culture by LN cells from immunized mice.

Rapid establishment of a stable IL-4/IFN-gamma production profile in the antigen-specific CD4+ T cell response to protein immunization.

Development of the antigen-specific murine T cell response to immunization with keyhole limpet hemocyanin (KLH) in adjuvant has been monitored with direct limiting dilution analysis of CD4+ cells in draining lymph nodes (LN) and measurement of the cytokines produced by their clonal progeny. In vivo, the response to immunization suggested a major role for IL-4 and a minor role for IFN-gamma since IL-4 mRNA levels increased and IFN-gamma mRNA levels declined in LN over the first 3 days, and KLH-specific serum antibodies were mainly of IgG1 class with lower levels of IgE and IgG2a. Antigen-specific clonogenic cells were first detected in LN at day 4, at which time they comprised approximately 8% of the total CD4+ LN cell pool, declining to 1-2% from day 7 until at least 6 weeks after immunization. These clonogenic cells expressed high levels of surface CD44 (Pgp-1) both early and late in the in vivo response. Over the whole of the time span from day 4 to 6 weeks after immunization, most antigen-specific cells gave rise to clones that secreted IL-4 and a smaller proportion gave rise to IFN-gamma-secreting clones. By contrast, polyclonally activated CD4+ cells from untreated mice preferentially gave rise to clones with the converse cytokine profile. We conclude that a stable ratio of antigen-specific CD4+ cells committed to IL-4 or IFN-gamma synthesis is established within the first 4 days after KLH immunization and, contrary to prediction, does not evolve towards a more restricted cytokine profile during the primary response.