Beginning at hatching, male Cornell K strain single comb white leghorn chickens were fed a basal diet, with or without vitamin E (100 IU/kg) and/or selenium (Se, 0.2 ppm). After 3 weeks of treatment, animals fed either the Se-deficient or basal diet had significantly reduced plasma Se-dependent glutathione peroxidase activities when compared to those fed a vitamin E and Se-supplemented diet. Similarly, animals fed the vitamin E-deficient or basal diet had significantly reduced plasma α-tocopherol levels. The effect of these treatments on plasma concentrations of thyroid hormones (T3/T4), growth hormone (GH), and thymic hormone (thymulin) was determined using radioimmunoassay and ELISA. A deficiency in Se, but not in vitamin E, resulted in an increase in plasma T4 concentrations while plasma T3 concentrations were decreased. Plasma GH levels showed some fluctuation as a result of the dietary treatments but there was no significant correlation between plasma GH levels and any of the other variables. A significant decrease in plasma thymulin levels was observed in Se-deficient birds compared to those receiving adequate Se in the diet. A vitamin E deficiency had no measurable effect on plasma thymulin levels. From these studies, we conclude that plasma thymulin concentrations directly correlate with plasma T3 concentrations which are negatively affected by a Se deficiency.

Cyclosporin-A (CYP-A) is a widely used immunosuppressive drug. Yet, information on the long-term impact of embryonic exposure is relatively scarce. The effects of CYP-A on reproductive and immunologic parameters in CD strain female offspring exposed in utero at doses of 0, 0.2, 2, 10, or 20 mg/kg/day (from gestational day 6 to 21) were compared against identically dosed CD adult rats. Embryotoxicity was seen at the two highest doses. CYP-A was acutely immunotoxic in adults (tested at 20 mg/kg/day dose) but with minimum long-term effects. In contrast, the offspring experienced relatively persistent alterations. CYP-A exposure increased ano-genital distance in the neonates. In the 5-week-old offspring, the delayed type hypersensitivity (DTH) response and splenic B cell number (determined by flow cytometry) were both decreased at the 2 mg dose level. IL-4 level was reduced and blood monocytes were increased at both exposure doses. All other parameters were unchanged. In the adult offspring (13-week-old), no difference was seen in either the DTH response or B cell ratios, but IL-4 level was increased at 2 mg/kg/day, and anti-KLH IgG titer decreased at both doses. In exposed non-pregnant adults, changes were minimal following a 13-week recovery period. Blood neutrophils were increased at all doses of the drug and flow cytometry data suggested some perturbation in CD4(+)CD8(+) cells, macrophages, and B-cells. All other parameters were unchanged. In conclusion, the adult rodent immune system largely recovers from CYP-A exposure given sufficient time. However, embryonic exposure appears to produce a series of immune perturbations including functional impairment during postnatal maturation.

Preincubation with either thymulin or IFN-γ can enhance NK activity. In addition, overnight in vitro pre-treatment with thymulin and IFN-γ increases NK activity further than either treatment alone. It has been hypothesized that thymulin increases the responsiveness of immune cells to IFN-γ by either increasing the expression of IFN-γR or by increasing the production and/or secretion of IFN-γ. The effects of thymulin on IFN-γ production and secretion were examined in this study. While an overnight incubation with the polyclonal activator Con A increased the number of cells positive for intracellular IFN-γ, a similar incubation with thymulin produced no change in the percentages of cells labeling positive for intracellular IFN-γ when compared to the media control cells. In addition, IFN-γ was not secreted by splenocytes following an overnight incubation with thymulin, but increased secretion was induced by Con A stimulation. Taken together, these results suggest that thymulin does not increase IFN-γ production or induce IFN-γ secretion by avian splenocytes.

The effect of in vivo and in vitro thymulin treatments on macrophage responsiveness to interferon-gamma was evaluated in chickens. Seven-week-old chickens were treated with 0, 1, 10, or 100ng thymulin per 100g body weight. Abdominal exudate cells (AEC), a source of macrophages, were harvested and cultured in the presence of graded levels of recombinant chicken interferon-gamma (ChIFN-gamma). Responsiveness to ChIFN-gamma was determined by measuring the induction of nitric oxide production. One and 2-day thymulin treatment at 10 and 100ng per 100g body weight doses significantly increased responsiveness to ChIFN-gamma while 1ng per 100g body weight had no effect. Other experiments compared the effect of thymulin treatments in Cornell K strain chickens, having normal serum thymulin levels with sex-linked dwarf (SLD) chickens which are deficient in serum thymulin. The dose of thymulin treatment required to significantly increase responsiveness to ChIFN-gamma differed between strains. Finally, the effect of direct in vitro thymulin treatments on macrophage responsiveness to ChIFN-gamma was evaluated. There were no significant increases in responsiveness to ChIFN-gamma between treatment groups within the macrophage cell line, HD-11, when cultured in the presence of 0-200pg thymulin/ml. These data suggest that the effect of thymulin on AEC responsiveness to ChIFN-gamma is indirectly mediated.

Results arising from investigation of the immunotoxic. effects of early (in ovo) Pb exposure on juvenile immune function suggested that Pb might represent a useful chemical probe of avian immune development. This paper reviews the finding of two studies comparing the effects of developmental timing of 1 Pb exposure and gender-based susceptibility to Pb-induced immunotoxicity. Based on the findings, it is suggested that critical immune components responsible for some cell-mediated immune functions (e.g., delayed-type hypersensitivity function) do not arise until later than 9 days of embryonic development. In contrast, Pb-sensitive immune elements linked to certain macrophage-associated functions are present much earlier in avian development. Additionally, altered antibody-producing functions may be manipulated throughout embryonic development, although gender-based differences appear to exist.

The ability of thymulin to directly enhance NK cell-mediated cytotoxicity was examined. Specific cell population depletions were done in K and SLD chicken splenocyte preparations using anti-CD3, CD4, and CD8 monoclonal antibodies and secondary complement-fixing polyclonal antibodies. The remaining cells were incubated overnight with in vitro treatments of thymulin and IFN-gamma, either separately or together, followed by an assay for cytotoxicity. Although the control K-strain had higher overall NK cell-mediated cytotoxicity than the thymulin-deficient SLD-strain, the following trends were seen in both strains. Thymulin continued to enhance NK activity following CD4 or CD3 cell depletion, but not after CD8 or CD8 and CD4 cell depletion. Since avian NK cells express CD8 alpha, but not CD3 or CD4 on their surface, these results suggest that the ability of in vitro thymulin treatments to enhance NK activity is not mediated by T-cells but may be due to direct effects on NK cells.

The effect of thymulin on IL-2 receptor (IL-2R) expression by avian splenocytes was examined in the functionally hypothyroid sex-linked dwarf (SLD) and in normal euthyroid K strain chickens. Daily thymulin injections of 0, 0.05 and 5.0 ng/100 g body weight were given from hatching until 4 weeks of age. ConA-treated and non-stimulated splenocytes from these animals were analyzed by flow cytometry for their expression of IL-2Rα, CD4 and CD8 cell surface molecules. ConA activation increased the number of IL-2R+ cells within K strain more than in the SLD. Thymulin treatment increased the number of IL-2R+ cells in the SLD but had the opposite effect in K strain chickens. Mitogen activation or thymulin treatment had little effect on the IL-2R density within small cell populations. In contrast, mitogen activation increased the density of IL-2R on larger cell populations in both K and SLD. IL-2R densities on non-stimulated larger cells decreased in the SLD after thymulin exposure. Thymulin treatment produced no effect on the mean IL-2R densities for large activated cells. ConA stimulation increased the number of CD4+ cells in both strains. The density of CD4 expression was modulated by both mitogen activation and thymulin treatment. ConA stimulation produced an increase in the number of CD8+ cells. The SLD had fewer CD8+ cells than did the K strain and thymulin treatment had little effect on this population in either strain. Mitogen stimulation increased the density of CD8 on CD8+ cells but again thymulin treatment had little effect. These results suggest that thymulin can modulate IL-2R expression on splenocytes and that this effect may be dependent upon the thyroidal status of the animal. Further, these data suggest that thymulin has a differential effect on the CD4 and CD8 T-cell subpopulations.

Lead has been shown previously to induce immunotoxic effects on macrophage and T-cell-associated functions after full-gestational exposure. To gain a better understanding of a single developmental exposure and the potential role of gender in immunotoxic responses to low levels of lead, 5-d-old avian embryos were injected once with lead acetate (5 or 10 microg). As juveniles (4 wk of age), animals were immunized with a foreign antigen, bovine serum albumin (BSA). At 6 and 8 wk, animals were sensitized with a self antigen, thyroglobulin (Tg). Immune parameters were examined at 6 and 10 wk of age. In males, anti-BSA immunoglobulin G (IgG) levels were significantly increased at the highest lead treatment level compared to sodium acetate controls, while female antibody production was unaltered. Similarly, after early exposure to lead, males (which were noninducible for anti-thyroglobulin antibodies in sodium acetate controls) were induced to produce autoanti-thyroglobulin IgG. Lead exposure did not markedly alter autoantibody levels in females, although, unlike males, control females could be induced to produce autoantibody to thyroglobulin. Males differed significantly in total leukocyte counts between treatment groups, whereas females did not. No marked differences were observed in males or females in the delayed-type hypersensitivity response, lymphocytic infiltration of thyroids, or in spleen, thymus, or bursa weights following exposure to lead. These results suggest that there is a differential immunotoxic effect based on gender after a single in ovo exposure to lead. Therefore, when examining the developmental immunotoxic effects of a metal such as lead, gender is a potential risk factor.

Sex-linked dwarfism (SLD) in chickens is characterized by impaired growth despite normal or supranormal plasma growth hormone (GH) levels. This resistance to GH action is thought to be due to mutations of the GH receptor (GHR) gene that reduce or prevent GH binding to target sites. The genetic lesion causing GH resistance in Cornell SLD chickens is, however, not known. Previous studies have shown that hepatic GH-binding activity is abnormally low in these birds, yet the GHR gene is transcribed into a transcript of appropriate size and abundance. Point mutations or defects in translation could therefore account for the impaired GHR activity in this strain. These possibilities were addressed in the present study.A missense mutation resulting in the substitution of serine for the conserved phenylalanine was identified in the region of the GHR cDNA encoding the extracellular domain. Translation of this mutant transcript was indicated by the presence of GHR/GH-binding protein (GHBP)immunoreactive proteins in liver (55, 70 and 100 kDa) and serum (70 kDa) of normal (K) and SLD birds. Radiolabelled GH did not, however, bind to the hepatic membranes of most SLD chickens. Serum GH-binding activity, in contrast, was readily detectable, although at significantly lower levels than in normal birds. The missense mutation in the SLD GHR gene may thus affect targeting of GHRs to hepatic plasma membranes.

Mutations in the genes for high mobility group protein I-C (HMGI-C) and insulin-like growth factor 1 (IGF1) are known to be responsible for dwarf phenotypes in the mouse. Because the locus for autosomal dwarfism (adw) in the chicken maps to a region which is syntenic to a region in the human and mouse in which the HMGI-C and IGF1 genes are located, HMGI-C and IGF1 are likely candidate genes for adw in the chicken. In this study their possible role in the establishment of this phenotype has been investigated. We have cloned and sequenced the complete coding region of the chicken HMGI-C cDNA. Comparison with its human counterpart revealed a nucleotide sequence conservation of 84%. Only nine amino acids are present principally in the N-terminal segment before the first DNA-binding domain. Northern blot analysis showed no difference in the expression of the HMGI-C gene between adw and wild-type chicken embryos. Also no mutations in either the HMGI-C or the IGF1 RNA nucleotide sequence were detected in adw chicken embryos.

Comell K strain chickens received a diet supplemented with 0, 0.1 and 1.0 ppm T3 from the day of hatching. At 28 days of age, splenocyte suspensions were prepared and analyzed by flow cytometry for interleukin-2 receptor (IL-2R) and CD3 expression. The low T3 dose increased the percentage of resting small cells expressing IL-2R while the mean fluorescence for this marker was enhanced only after mitogenic activation. This treatment did not alter the number of larger cells positive for IL-2R but did increase their mean fluorescence following mitogenic activation. The high T3 dose depressed the numbers of cells positive for IL-2R and their mean fluorescence amongst all splenocyte preparations. Both levels of T3 enhanced the numbers of CD3-positive cells in all cell preparations. These results suggest that the IL-2R expression can be modulated by in vivo T3 supplementation and that these correlate with the previously demonstrated changes in IL-2-like activity. The regulation of IL-2R expression provides one mechanism through which thyroid status may regulate immune function.

In contrast to the mammalian system, avian species lack the so-called ''resident'' or ''harvestable'' macrophage population in the abdominal exudate. However, macrophages can be recruited into the chicken's abdominal cavity (presumably from the blood monocyte pool) if an inflammatory agent such as Sephadex is injected. The kinetics of inflammatory cell recruitment in terms of time, cell type, and state of activation to perform a particular effector function is currently an active area of research. This report will provide information on several chicken macrophage effector functions, including in vivo chemotaxis, phagocytosis, bacterial uptake and killing, biosynthesis of nitric oxide and various enzymes, and monokines such as interleukin-1 and granulocyte colony-stimulating factor.

One-day-old chickens were treated with varying levels of triiodothyronine (T3) added to the diet. At 28 days of age, the IL-2-like activity in the splenocyte culture supernatants were assessed. The lowest dose of T3 (0.1 ppm) enhanced IL-2-like activity while the highest dose (1.0 ppm) was significantly suppressive. The intermediate dose elicited varying effects. Recombinant chicken growth hormone (rcGH) was added to some cultures 24 h prior to IL-2 assay. In vitro rcGH significantly depressed the IL-2-like activity of splenocytes from animals given the low T3 diet. The addition of varying concentrations of T3 in vitro to splenocytes from non-T3-supplemented chickens had no effect on the IL-2-like activity. These results indicate that in vivo supplementation of low dietary T3 but not in vitro T3 is effective in enhancing avian IL-2-like activity. The addition of rcGH in vitro can modify this response.

The role of the neuroendocrine system in influencing both immune development and function has become an area of active research within many model systems, including the chicken. It is now clear that the neuroendocrine system can exert immediate feedback regulation on the immune system as well as control specific aspects of immune differentiation and development. The primary lymphoid organs of avian species (i.e., the thymus and the bursa of Fabricius) are also known to function as endocrine organs. These produce hormonal products that influence the development of lymphoid cells and that may feed back on the neuroendocrine system. In conjunction with the endocrine activities of the primary lymphoid organs, immune and accessory cells are known to produce a variety of secreted products or cytokines that have the potential not only for the regulation of immune function but also for mediating neuroendocrine activities. Finally, it has been demonstrated in a variety of species that leukocytes are capable of producing endocrine mediators previously believed to be produced only under the direct control of the hypothalamic-pituitary axis. Thus, there are numerous possibilities for bidirectional interactions between the immune and neuroendocrine systems. This discussion focuses primarily on these interactions with an emphasis on the means by which the hormonal mediators, growth hormone and thyroid hormone, may affect the thymus and the thymic microenvironment. The role of the adrenocorticoids and gonadal steroids in regulating immune function and their involvement in immune feedback circuits are also discussed.

GH receptor (GHR) mRNA has been identified in peripheral (liver and muscle) and central (brain and hypothalamus) tissues of sex-linked dwarf (SLD) Leghorn chickens. Total RNA was extracted from the tissues of immature (1 week, 4 week), pubertal (16 week) and adult (> 24 weeks) SLD and K (the normally growing strain) Leghorn chickens. In both groups and all tissues, an mRNA moiety of 4.4 kb hybridized with cRNA probes derived from the rabbit hepatic GHR sequence. An additional low-abundance transcript of 2.8 kb was also identified in some tissues. An age-related increase in expression was observed in K and SLD hepatic GHR mRNA, suggesting normal regulation of SLD GHR gene transcription. Amplification of cDNA from K and SLD tissues in the presence of oligonucleotide primers coding for the intracellular or extracellular domains of the chicken GHR generated electrophoretically separable fragments of expected size. Restriction enzyme digestion of the products with EcoRI, BstNI, HaeIII, NcoI or BamHI produced smaller moieties of expected sizes in both strains. These results demonstrate that, in contrast to broiler SLDs, a GHR gene deletion is not responsible for the GHR dysfunction in Leghorn SLDs. Although the actual defect in GHR gene expression in SLD Leghorns remains to be identified, this study demonstrates that sex-linked dwarfism, like Laron dwarfism, is due to a heterogeneity of lesions.

The effects of the in vitro exposure of avian bone marrow (BM) cells and thymocytes to synthetic thymulin were studied. Two T-cell differentiation markers, PNA binding site and CT-1a expression, were used to examine cell maturation. Enhanced PNA binding to both BM cells and thymocytes resulted following an in vitro thymulin exposure but cell proliferation was not affected. Scatchard analysis supported the conclusion that PNA binding affinity was significantly increased and thus responsible for the observed increase in PNA binding. Flow cytometric analysis suggested that the induced PNA+ thymocyte population may be a different population from the one exhibiting enhanced CT-1a expression following thymulin exposure. Taken together, the observations suggest that cells can express their further differentiation states without undergoing proliferation following in vitro thymulin stimulation.