Thymic hormone modulation of age-induced changes in the induction of graft-versus-host disease by DBA/2J lymphocytes

Aging induces a number of changes in the immune system, including the involution of the thymus which results in the loss of thymic hormone production and alteration in T cell function. One age-dependent change in immune response is the increasing risk of developing acute or chronic form of graft-versus-host disease (GVHD) following bone marrow transplantation as the age of the recipient increases. A murine model of GVHD that has been extensively studied is one in which injection of C57BL/6 spleen cells into unirradiated B6D2F1 mice results in an acute form of GVHD characterized by cytolytic T lymphocytes (CTL), suppressor cells, runting, and occasionally death. In contrast, injection of DBA/2J spleen cells results in a chronic form of GVHD characterized by a lack of CTL and hyperproduction of immunoglobulin and autoantibodies. This study shows that the GVHD response of DBA/2J spleen cells is dependent on the age of the donor DBA/2J mice. If spleen cells from DBA/2J mice older than 3 months are injected into B6D2F1 recipients, CTL and lack of immunoglobulin production indicative of acute GVHD result. Administration of thymosin fraction 5, a collection of thymic hormones, to DBA/2J mice older than 3 months caused spleen cells from these treated mice to give a GVHD response characteristic of the chronic form of GVHD in B6D2F1 recipients. Thus, thymic hormones were able to modulate the changes in GVHD responses of DBA/2 lymphocytes that occur as the mice age. Preliminary fractionation of TF5 has indicated that there are at least two active thymic peptides present in TF5.     

Identification of a new type of invariant V alpha 14+ T cells and responsiveness to a superantigen, Yersinia pseudotuberculosis-derived mitogen.

We examined the expression of the H4 T cell activation marker in thymic T cell subpopulations and found that TCR-alpha beta+ CD4+ thymic T cells are segregated into three subpopulations based upon H4 levels. Thymic T cells with either no or low H4 expression differentiate via the mainstream differentiation pathway in the thymus. H4int thymic T cells, which express a skewed V beta repertoire of V beta 2, -7, and -8 in their TCRs, show the phenotype of NKT cells: CD44high, Ly6Chigh, NK1.1+, and TCR-alpha beta low. H4high thymic T cells also show a skewed V beta repertoire, V beta 2, -7, and -8, and predominantly express an invariant V alpha 14-J alpha 281+ alpha-chain in their TCRs but constitute a distinct population in that they are CD44int, Ly6C-, NK1.1-, and TCR-alpha beta high. Thus, invariant V alpha 14+ thymic T cells consist of ordinary NKT cells and a new type of T cell population. V beta 7+ and V beta 8.1+ invariant V alpha 14+ thymic T cells are present in DBA/2 mice, which carry mammary tumor virus-7-encoded superantigens, in comparable levels to those in BALB/c mice. Furthermore, V beta 7+ invariant V alpha 14+ thymic T cells in DBA/2 mice are in the immunologically responsive state, and Yersinia pseudotuberculosis-derived mitogen-induced V beta 7+ invariant V alpha 14+ thymic T cell blasts from DBA/2 and BALB/c mice exhibited equally enhanced responses upon restimulation with Y. pseudotuberculosis-derived mitogen. Thus, invariant V alpha 14+ thymic T cells that escape negative selection in DBA/2 mice contain T cells as functionally mature as those in BALB/c mice.     

LPS regulation of the immune response: separate mechanisms for murine B cell activation by lipid A (direct) and polysaccharide (macrophage-dependent) derived from Bacteroides LPS

Lipopolysaccharide (LPS) derived from Bacteroides fragilis has been reported to stimulate mitogenic responses in spleen cell cultures from the classical LPS-hyporesponsive C3H/HeJ mouse strain; however, we have shown that purified splenic B cells from C3H/HeJ mice are hyporesponsive to phenol-water extracted LPS from B. fragilis ATCC 25285 (B-LPS). In the present study, B-LPS and its purified lipid A and polysaccharide components were tested for their ability to induce mitogenic and polyclonal IgM synthesis in spleen cell and purified splenic B cell cultures from classical LPS-responsive and -hyporesponsive mice. Mitogenic responses to B-LPS and E. coli K235 LPS(Ph) of whole spleen cells (2 X 10(5) cells/culture) or purified B cells (5 X 10(5) cells/culture) from classical LPS-responsive mouse strains (C3H/HeN, BALB/c, C57BL/6J, C57BL/10Sn, and DBA/2), F1 mice (derived from crosses between LPS responsive and C3H/HeJ mice), and classical LPS-hyporesponsive mice (C3H/HeJ and C57BL/10ScN) were high, intermediate, and low, respectively. When a higher number of whole spleen cells (5 X 10(5) cells/well) were cultured, B-LPS induced high mitogenic responses in C3H/HeN, intermediate responses in F1, and lower but significant responses in C3H/HeJ cultures. Similar results were obtained when polyclonal IgM synthesis was assessed in cultures containing 1 X 10(6) cells/culture. In contrast, the purified lipid A component of B-LPS failed to induce mitogenic responses in either whole spleen or purified B cell cultures. The addition of purified splenic B cells from C3H/HeJ mice to C3H/HeN or C3H/HeJ splenic adherent cells resulted in mitogenic responses to B-LPS, implying that the hyporesponsiveness to B-LPS seen in whole spleen cell cultures from C3H/HeJ mice at the lower cell concentration was due to limiting numbers of M phi. When splenic B cells and M phi from either C3H/HeN or C3H/HeJ mice were incubated with the lipid A or the polysaccharide moiety of B-LPS, lipid A induced mitogenic responses only in C3H/HeN cultures, whereas the polysaccharide moiety induced similar responses in both C3H/HeN and C3H/HeJ cultures. These results suggest that Bacteroides lipid A does not stimulate B cells from the classical LPS-hyporesponsive C3H/HeJ mouse strain, whereas the polysaccharide moiety of B-LPS is biologically active and mediates B cell stimulation via M phi.     

Restraint stress and anti-tumor immune response in mice

Psychological stress modulates the immune system through the hypothalamic-pituitary-adrenal axis, the sympatho-adrenomedullary axis and the opioid system. According to literature data, restraint stress increases the immune cell apoptosis, decreases the spleen and thymus cell content, the natural killer (NK) activity in the spleen, and it compromises the anti-tumor immune response in mice. We immobilized mice in two consecutive nights, and then determined the cell number, apoptosis, NK cell content, NK activity and the level of cytokine mRNAs (TNF-beta, TNF-alpha, IL-4, IL-5, IL-1alpha, IFN-gamma, IL-2, IL-6, IL-1beta and IL-3) in the thymus and spleen. No consistent changes were detected in any of the immune parameters either in C57Bl/6 or in DBA/2 mice. Stressed or control B6 mice were injected with B16 melanoma cells immediately after the immobilization or one week later. No significant differences were found in the growth of primary tumors and lung metastases in stressed and control animals. Taken together, our mice, kept in a general-purpose non-SPF animal house, seemed to be refractory to the stress-induced immunomodulation. Our interpretation is that stress-induced immunomodulation can occur only in mice isolated from any background stressors, or rather natural stimuli, throughout their life.     

Overexpression of Bcl-2 in transgenic mice decreases apoptosis and improves survival in sepsis

In sepsis there is extensive apoptosis of lymphocytes, which may be beneficial by down-regulating the accompanying inflammation. Alternatively, apoptosis may be detrimental by impairing host defense. We studied whether Bcl-2, a potent antiapoptotic protein, could prevent lymphocyte apoptosis in a clinically relevant model of sepsis. Transgenic mice in which Bcl-2 was overexpressed in T cells had complete protection against sepsis-induced T lymphocyte apoptosis in thymus and spleen. Surprisingly, there was also a decrease in splenic B cell apoptosis in septic Bcl-2 overexpressors compared with septic HeJ and HeOuJ mice. There were marked increases in TNF-alpha, IL-1beta, and IL-10 in thymic tissue in sepsis in the three species of mice, and the increase in TNF-alpha and IL-10 in HeOuJ mice was greater than that in Bcl-2 mice. Mitotracker, a mitochondrial membrane potential indicator, demonstrated a sepsis-induced loss of membrane potential in T cells in HeJ and HeOuJ mice but not in Bcl-2 mice. Importantly, Bcl-2 overexpressors also had improved survival in sepsis. To investigate the potential impact of loss of lymphocytes on survival in sepsis, Rag-1-/- mice, which are totally deficient in mature T and B cells, were also studied. Rag-1-/- mice had decreased survival compared with immunologically normal mice with sepsis. We conclude that overexpression of Bcl-2 provides protection against cell death in sepsis. Lymphocyte death may be detrimental in sepsis by compromising host defense.     

Helper cells active in the generation of cytotoxicity to a syngeneic tumor

We have examined a population of thymic lymphocytes that augment the generation in vitro of specific cytotoxic T cells against P815.X2, a syngeneic tumor in DBA/2 mice. These helper cells are evident in the thymus of DBA/2 mice 5 to 7 days after subcutaneous injection of live P815 cells. They are Thy-1 positive, resistant to gamma radiation, antigen specific, and appear to exert their influence most effectively when added at the beginning of the culture.     

Age- and strain-related differences in murine spleen cell responses to different activation signals.

The effect of age on the response of splenocytes to activation with anti-CD3 mAb and a combination of anti-CD3 mAb and TPA, as evidenced by interleukin-2 (IL-2) and interleukin-4 (IL-4) production and cell proliferation, was examined in the C57BL/6 and DBA/2 murine strains. Depending on the mode of activation, there were age and strain differences in IL-2 and IL-4 production. With all modes of activation, cells from the old C57BL/6 mice produced less IL-2 than their young counterparts. In DBA/2 mice there was no age-related difference in IL-2 production with anti-CD3 mAb activation alone, whereas when the same cell population was activated with anti-CD3 mAb and TPA an age-associated decrease in IL-2 production occurred. In both strains, there was an age-related increase in IL-4 production with anti-CD3 mAb activation. After addition of TPA, however, there was an age-related decrease in IL-4 production. An age-related decline in the proliferation occurred with all modes of activation in both mouse strains. There were also strain-related differences in proliferation after the addition of forskolin, an inhibitor of Th1-cell function. While forskolin inhibited the proliferation of cells from the young C57BL/6 mice only, in the DBA/2 mice proliferation of cells was inhibited in both age groups. There were no strain-related differences in inhibition by anti-transferrin receptor (TrfR) mAb, although cells from the old mice were slightly more sensitive to this inhibition.     

Genetic and nongenetic factors in expression of infectious murine leukemia viruses in mice of the DBA/2 x RF cross

Mice of the RF and DBA/2 strains possess endogenous ecotropic murine leukemia virus (E-MuLV) genomes but express only low to undetectable levels of infectious virus in their lymphoid tissues. F1 mice of this cross showed high levels of infectious E-MuLV if DBA/2 was the maternal parent but very low levels if RF was the maternal parent. E-MuLV expression, if present, was always higher in the spleen than in the thymus. Studies of reciprocal backcross generations with both parental strains indicated that the presence of the virus was governed by a single dominant autosomal locus present in the RF strain, and that RF females, but neither RF males nor DBA/2 females or males, transmitted a non-Mendelian factor which powerfully suppressed virus expression in their progeny. Some but not all (DBA/2♀ × RF♂)F1 females also possessed the capacity to transmit this maternal suppression to their progeny. Xenotropic murine leukemia virus (X-MuLV) showed a different pattern of expression in this cross. In the thymus it was detected in a minority of DBA/2 and in no RF mice; in crosses the presence of X-MuLV in this organ was independent of the presence of E-MuLV. In the spleen, X-MuLV was detected only in a percentage of E-MuLV-positive mice. The maternal factor from RF mothers which suppressed E-MuLV did not suppress thymic expression of X-MuLV. Skin painting with 3-methylcholanthrene induced a high incidence of thymic lymphoma in mice of both parental strains and in F1 hybrids, all of which normally show only low incidences of the diseases; the treatment did not induce markedly increased expression of E-MuLV or X-MuLV in mice of either parental strain, although it did abrogate the diminution of E-MuLV titers seen with age in (DBA/2♀ × RF♂)F1 mice beyond the age of three months.     

Immune complexes, but not streptococcal cell walls or zymosan, cause chronic arthritis in mouse strains susceptible for collagen type II auto-immune arthritis

In this study we investigated mechanisms involved in the chronic character of experimental collagen type II induced arthritis (CIA). We compared the knee joints of mouse strains which are prone to develop this autoimmune disease (DBA/1,B10RIII) with other nonsusceptible mouse strains (C57Bl/6,BALB/c) in their reaction to different stimuli: immune complexes (IC), zymosan and streptococcal cell walls (SCW). Inflammation was evaluated by(99m)Tc uptake measurements and in haematoxylin- and eosin-stained knee-joint sections. Passively induced immune complex mediated arthritis (ICA) in knee joints of C57Bl/6 and BALB/c mice, showed moderate cell influx at day 3, whereas at day 7 only minor amounts of inflammatory cells were observed. In contrast, in arthritic DBA/1 and, to a lesser extent, in B10.RIII joints, a tremendous cell influx was observed at day 3 and even at day 14 there was still significant synovitis. In contrast, if arthritis was elicited by intra-articular injection of zymosan or SCW in C57Bl/6 and DBA/1, the course of inflammation was similar in both strains and no chronic inflammation developed. In line with severe arthritis, chemotactic factor production was dramatically enhanced in ICA in DBA/1 mice, and a prolonged production of IL-1 was evident. When IL-1 was neutralized before or during the ICA using specific anti-IL-1alpha,beta antibodies, inflammation could be blocked completely. Single or multiple injection of IL-1 in the knee joint of C57Bl/6 or DBA/1 showed comparable inflammation, indicating that the chemotactic response per se is comparable in both strains. No prolonged production of IL-1 was found during zymosan or SCW arthritis. Selective removal of macrophages from the synovial intima prior to ICA induction (using clodronate-containing liposomes) prevented the onset of inflammation in C57Bl/6 and DBA/1 mice. It can be concluded that immune complexes, but not zymosan or SCW, cause a more severe and chronic arthritis in mouse strains which are susceptible for collagen type II autoimmune arthritis. This is due to higher and prolonged expression of IL-1 and chemotactic factors, caused by stimulation with immune complexes. The interaction of IC with lining macrophages probably plays a dominant role in development of chronicity.     

Mechanisms of strain-dependent development of mast cells from mouse splenocytes

Mast cell development from spleen cells was not triggered by prostaglandin E1 (PGE1) or dibutyryl cAMP (db-cAMP) during a 12 day culture when the spleen cells were obtained from C57BL/6N and DBA/1 mice, but mast cells did develop when the spleen cells were obtained from C3H/HeN, BALB/c and ICR mice. A lack of endogenous IFN-gamma in the initial 2 days of the culture period was responsible for the failure. This was confirmed by adding neutralizing anti-IFN-gamma antibody and rIFN-gamma to the cultures and by determining IFN-gamma levels in the spleen cell cultures. Th1 cells in the spleens of C57Bl and DBA/1 mice were much more sensitive to PGE1 and db-cAMP than Th1 cells from other inbred mice strains, and consequently, IFN-gamma production was inhibited in spleen cell cultures of C57BL and DBA/1 mice on addition of PGE1 or db-cAMP. Furthermore, the different sensitivities of Th1 cells to PGE and db-cAMP were dependent on the different levels of IL-12 p40 monomers or homodimers in the spleen cell cultures. As the endogenous specific inhibitors of IL-12 p70 (heterodimers of p40 and p35), large amounts of IL-12 p40 monomers or homodimers in the spleen cell cultures of C57BL and DBA/1 mice enhanced the ability of PGE1 and db-cAMP to inhibit IFN-gamma production by antagonizing the activity of IL-12 heterodimers. These results indicate that the strain-dependent development of mast cells from mouse splenocytes is related to endogenous IFN-gamma levels, which are regulated by PGE, db-cAMP, IL-12 p70 and IL-12 p40.     

Mitogenic,immunoadjuvancy, and genetic studies on fatty acyl maltose

Summary Three synthetic glycolipids, maltose tetrapalmitate (MTP), maltose hexastearate (MHS), and maltose hexalinoleate (MHL) prepared as nontoxic lipid A analogs, and Escherichia coli lipopolysaccharide (LPS) were assayed for their mitogenic activity using spleen lymphocytes in nine inbred mouse strains and three F1 hybrids. The MTP and LPS were also assayed for their ability to enhance plaque-forming cell (PFC) responses using sheep red blood cells as the antigen in th same inbred mouse strains and F1 hybrids, The mitogenic activity of synthetic glycolipids was several fold lower than that of LPS and MHL was inferior to MTP and MHS. DBA/2J was the most responsive strain for MTP and DBA/1J and C3H/HeJ the least. The mitogenic activity of MTP was generally in agreement with the PFC response stimulation by it. Lowdose cyclophosphamide treatment of mice synergized MTP for PFC response augmentation. Genetic studies on MTP mitogenicity revealed that 90% of responder DBA/2J X nonresponder C3H/HeJ F1 hybrids had intermediate mitogenic activity. Among F2, 73% had intermediate-high activity and 27% were nonmitogenic. Among F1 X C3H/HeJ backcrosses 11% had high, 56% intermediate, and 33% had no mitogenic activity, whereas, for the F1 X DBA/2J backcross, 14% had high, 36% intermediate, and 50% low or negligible activity. The data favored a single gene for MTP activation of immune cells.This work was supported, in part, by a grant from the National Cancer Institute of Canada, and by grant from the Cancer Research Society Inc.     

The immune response against Francisella tularensis live vaccine strain in Lpsn and Lpsd mice

Abstract The impact of Lps gene on the course of immune response against subcutaneous infection of mice with Francisella tularensis live vaccine strain was studied. Production and specificity of antibodies, cytotoxic responses of macrophages and NK-cells, spontaneous production ex vivo of cytokines IL-1α, IL-2, IL-4, IL-6, IL-10, IFN-γ, and TNF-α in spleen cell cultures in C3H/HeJ ( Lps ^d) mice in comparison with C3H/HeN ( Lps ^r) mice were tested. The value of LD₅₀ was significantly different in the two strains of mice (8.0 × 10³ cfu for C3H/HeJ versus 4.61 × 10⁵ cfu for C3H/HeN mice after subcutaneous inoculation). The production of NO₂ is also impaired in C3H/HeJ mice in the early intervals after infection. Thus, the defective Lps gene of C3H/HeJ mice influences both the level of innate resistance of mice to F. tularensis live vaccine strain infection and the process of induction and regulation of immune response against this intracellular bacterial pathogen.     

Strain dependence of muramyl dipeptide-induced LAF(IL 1) release by murine-adherent peritoneal cells

The capacity of MDP to stimulate LAF(IL 1) production by adherent peritoneal cells (APC) from different strains of mice was examined. It was observed that MDP could stimulate thioglycollate-induced APC from DBA/2, C57B1/6, and CBA/CA mice, but not from C3H mice. Resident APC from DBA/2 or C3H/HeJ mice were also responders and nonresponders, respectively, to MDP for LAF(IL 1) production, the positive effect of MDP being more marked on DBA/2 cells in the presence of indomethacin. Because a LPS high-responder C3H substrain (C3HeB/Fe) did not respond to MDP, it was concluded that the strain dependence with respect to the induction of LAF(IL 1) seen with both MDP and LPS was not linked. Moreover, the unresponsiveness of C3H mice to MDP was not linked to their MHC haplotype, because a second H-2k strain (CBA/CA) was a good responder to MDP stimulation.     

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

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

Brain Adrenoceptor Binding Sites in Mice Susceptible (DBA/2J) and Resistant (C57 Bl/6) to Audiogenic Seizures

We have examined if the age-related susceptibility of DBA/2J mice to audiogenic seizures is the result of an abnormality in the number or sensitivity of brain adrenoceptors. The binding of alpha 1-, alpha 2-, and beta-adrenoceptor ligands to membranes prepared from whole brain or regions of brain of DBA/2J mice was measured at various ages, corresponding to the periods before, during, and after the maximal sensitivity to audiogenic seizures. For comparison, we have studied concurrently age-matched C57 Bl/6 mice, a strain resistant to audiogenic seizures at all ages. There was no difference in the binding of alpha 2- or beta-adrenoceptor ligands to whole brain membranes between the two strains of mice at any age. The maximal number of alpha 1-adrenoceptor binding sites was lower in whole brains of DBA/2J mice than of C57 Bl/6 mice at all ages studied except 13-15 days of age. The differences were small (maximally 17%) but were statistically significant at 21-23 days of age, the time of maximal sensitivity of DBA/2J mice to audiogenic seizures. No difference between the two strains was found in the number or affinity of alpha 1- or alpha 2-adrenoceptor binding sites at any age in any of the brain regions studied. The age-related susceptibility of DBA/2J mice to audiogenic seizures is not the result of an abnormality in number or sensitivity of alpha 2- or beta-adrenoceptor binding sites, but a reduced number of alpha 1-adrenoceptor binding sites may be involved.     

Heterogeneity in the immune response to serotype b LPS of Actinobacillus actinomycetemcomitans in inbred strains of mice

We investigated the heterogeneity of the humoral immune responses to whole cells and lipopolysaccharide (LPS) of Actinobacillus actinomycetemcomitans serotype b and production of cytokines in inbred strains of mice. Nine such strains were tested: A/J (H-2(a)), C57BL/6 (H-2(b)), BALB/c (H-2(d)), DBA/2 (H-2(d)), B10.BR (H-2(k)), C3H/He (H-2(k)), C3H/HeJ (H-2(k)), DBA/1 (H-2(q)) and B10.S (H-2(s)). Mice were immunized intraperitoneally with whole cells of A. actinomycetemcomitans ATCC 43718 (serotype b) in phosphate buffered saline (PBS; pH 7.2) emulsified with an equal volume of Freund's incomplete adjuvant. Serum immunoglobulin G (IgG), immunoglobulin A (IgA) and immunoglobulin M (IgM) levels against A. actinomycetemcomitans were measured by an ELISA system. ELISA analysis, using LPS fractions from serotype a, b or c strains of A. actinomycetemcomitans as the coating antigens, revealed that mice strains C3H/He, C3H/HeJ, B10.BR and B10.S had an extremely high-IgM response against serotype b LPS. High-IgM titer sera contain also elevated levels of IgA antibodies to the antigen. To compare the cytokine production among inbred mice, the amounts of interleukin-4 (IL-4), interleukin-5 (IL-5), and interleukin-6 (IL-6) released from mouse splenocytes were measured using ELISA systems specific for these cytokines. A. actinomycetemcomitans serotype b LPS stimulation induced IL-6 release from murine splenocytes of all tested strains. However, IL-4 and IL-5 were detected only in high-IgM/IgA responders to A. actinomycetemcomitans serotype b LPS, not in low-IgM/IgA responders. Thus, we found a relationship between the humoral immune response to LPS of A. actinomycetemcomitans serotype b and production of type 2 cytokines by splenocytes.     

Human thymic epithelial cells produce interleukin 1

Although the thymus plays a critical role in generation of immunocompetent T lymphocytes, the precise role of the epithelial component of the thymus in the induction of T cell proliferation and maturation remains unknown. Since interleukin 1 (IL 1) is required for mature T cell activation, we have determined whether human thymic epithelial (TE) cells produce IL 1. By using a system for longterm culture of human TE cells, we found that human TE cells produced an IL 1-like factor (TE-IL 1) that augmented the proliferation of C3H/HeJ mouse thymocytes to phytohemagglutinin. IL 1 activity (20 to 200 U/ml) was detected in supernatants of TE cultures from all individuals (2 to 13 yr old) tested. IL 1 activity was also detected in supernatants of TE cultures from a 17-wk fetus but not from a 10-wk fetus. Production of TE-IL 1 was dependent on TE cell density and time in culture with optimal TE-IL 1 activity observed at 10(6) TE cells/ml after 48 to 72 hr of culture. With the use of high performance liquid chromatography, TE-IL 1 chromatographed as a molecule of 18,000 to 20,000 relative molecular mass, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, TE-IL 1 migrated at 15,000 to 17,000 Mr. With the use of isoelectrofocusing gels, charge heterogeneity of TE-IL 1 was demonstrated with two major isoelectric points of 5.7 to 5.8 and 6.9 to 7.0. Polyclonal antibody to human monocyte IL 1 markedly inhibited the TE-IL 1 activity. In indirect immunofluorescence assay of frozen human thymic sections, rabbit anti-IL 1 antibody reacted with epithelial cells in human thymic cortex and medulla. Furthermore, high performance liquid chromatography-purified TE-IL 1 augmented human thymocyte proliferation to suboptimal concentrations of phytohemagglutinin. Thus, thymic epithelial cells are capable of providing an intrathymic source of IL 1-like cytokine (TE-IL 1), which affects thymocyte proliferation. We propose that TE-IL 1 may play an important role in intrathymic proliferation and differentiation of human thymocytes.     

Genetic influences on oestrous cyclicity in mice: evidence that cycle length and frequency are differentially regulated.

Studies in C57BL/6J, DBA/2J and C3H/HeJ mice and in two F1 hybrid strains (B6D2F1 and B6C3HF1) 2-5 months old revealed marked genotypic differences among inbred strains. C57 mice had three times as many regular (3-6 days) cycles as DBA and C3H mice, due largely to fewer pseudopregnant-like (7-14 day) cycles. C57 had longer regular cycles than DBA and C3H mice. Although the frequencies of regular cycles of DBA and C3H mice were similar, the cycles of C3H mice were shorter than those of DBA mice. The results indicated that the genetic determinants of the frequency of regular cycles differ from those specifying cycle length. Frequency of regular cycles of F1 hybrids was either intermediate between the parent strains (B6D2F1) or similar to the C57 strain (B6C3HF1), suggesting that regular cycle frequency shows additive genetic variation in the former crosses, but mostly dominant variance in the latter background. Regular cycles were either shorter than in both parent strains (B6D2F1) or similar to one of them (B6C3HF1), indicating heterosis and dominance for genes specifying short cycles. Although the lack of reciprocal crosses meant that maternal effects and possible genomic imprinting effects could not be assessed, these results reveal marked genetic influences on cycle length and frequency and suggest that some of the genes specifying these two traits differ.     

Peripheral CD4+ lymphocytes derived from fetal versus adult thymic precursors differ phenotypically and functionally

There is growing evidence that the differentiation processes in the fetal and adult thymus are not identical. However, there is little information on whether these developmental differences influence the properties of mature cells that exit the thymus and seed peripheral lymphoid organs. We have addressed this issue by comparing the development of Ag-specific Th1/Th2 function by fetal vs adult thymic derived CD4(+) cells in the same adoptive adult hosts. Host mice were irradiated and transplanted with 14- to 15-day fetal thymic lobes from Thy-1 congenic mice. Ag (keyhole limpet hemocyanin)-specific Th1/Th2 responses of fetal-derived (donor) or adult-derived (host) CD4(+) cells were analyzed by ELISA following primary or secondary immunization. Fetal-derived cells produced up to 10-fold more of both Th1 (IFN-gamma) and Th2 (IL-4) cytokines than did adult-derived cells. Comparisons of the IL-4:IFN-gamma ratios showed that the responses of fetal-derived cells were Th2-skewed in an Ag dose-dependent manner. At low doses of Ag, the fetal-derived ratio was approximately 5 times higher than the adult-derived ratio. As the Ag dose was increased, the differences between the ratios of the fetal- and adult-derived responses were minimized. These relative responses were established initially during the primary effector phase but were maintained for weeks, into the memory phase of the immune response. Importantly, fetal-derived CD4(+) cells showed these properties whether the fetal thymic precursors matured within the fetal or adult thymic microenvironment. These results demonstrate that cells arising from fetal thymic precursors are functionally different both qualitatively and quantitatively from adult-derived cells.