Spatial distribution of growth hormone receptor, insulin-like growth factor-I receptor and apoptotic chondrocytes during growth plate development.

Linear bone growth depends upon proliferation, maturation, and apoptosis of growth plate chondrocytes, processes regulated by growth hormone (GH) and insulin-like growth factor-I (IGF-I). To investigate the contribution of GH, IGF-I and apoptosis to growth plate function, the expression of GH receptor (GHR) and IGF-I receptor (IGF-IR) mRNA were evaluated by in situ hybridization in fractionated costochondral growth plates of growing rats (at 2, 4, and 7 weeks). Apoptosis was determined by TUNEL assay and morphology in histological sections. GHR mRNA was greatest in resting cells with hypertropic cells increasing GHR expression with increasing age. Hypertropic and resting cell IGF-IR mRNA declined over the ages studied. Receptor mRNA expression was altered by exposing cells to GH or IGF-I. GH and IGF significantly decreased GHR mRNA in proliferative cells. GH and IGF also decreased IGF-IR mRNA in resting cells and the 2- and 4-week-old proliferative and hypertropic cells. Treating cells in culture with GH increased the number of apoptotic cells across all ages and zones. Histologically, apoptotic cells were observed at the chondro-osseous junction and within actively proliferating chondrocytes but not in resting cells. Apoptosis was highest at 4 weeks of age with lateral regions displaying the greatest number of cells undergoing apoptosis. These data indicate that apoptosis plays a role in growth plate function, particularly spatial configuration as indicated by the preferential lateral cell apoptosis. The susceptibility of proliferative cells to GHR and IGF-IR down regulation during the period of greatest apoptosis supports a role for the GH-IGF axis in both proliferation and apoptosis during growth plate development.

The kinetics of hemopexin and alpha1-acid glycoprotein levels induced by injection of inflammatory agents in chickens.

The acute phase response to inflammation induces changes in the secretion of hepatic proteins. To examine the time course of an acute phase protein response in broiler chickens, the plasma levels of hemopexin (HX) and alpha1-acid glycoprotein (AGP) and liver HX mRNA were measured at various time points from 3 hr to 336 hr after an intraabdominal injection of either lipopolysaccharide (LPS), complete Freund's adjuvant, incomplete Freund's adjuvant, phytohemagglutin, or mineral oil. Uninjected chicks served as controls. The accumulation of liver HX mRNA began within 3 hr of stimulation and peaked at 12 hr. Relative to control levels, plasma HX and AGP levels increased by 6-12 hr postchallenge and peaked at 24 hr. Complete Freund's adjuvant and LPS treatments induced the greatest increase in plasma HX (threefold; P < 0.05). Plasma levels of HX and AGP returned to control levels at 336 and 168 hr postinjection, respectively. A second experiment demonstrated that turpentine induced a similar AGP response as LPS and that albumin is a negative acute phase protein. The results suggest that plasma levels of HX or AGP could be used as an indicator of the systemic component of a local inflammatory response in chickens.

Soluble type-I interleukin-1 receptor blocks chicken IL-1 activity.

The ligand-binding domain of the chicken type-I interleukin-1 (IL-1) receptor (soluble IL-1R(I); sIL-1R(I)) was cloned into a Pichia pastoris expression system and the resulting sIL-1R(I) binding protein was used to produce antisera in rabbits (anti-IL-1R(I)). Two experiments were conducted to determine the capacity of sIL-1R(I) or anti-IL-1R(I) to block the IL-1 bioactivity (thymocyte co-stimulation) in conditioned media (CM) from HD11 chicken macrophages stimulated with lipopolysaccharide. In the first experiment, pre-incubation of CM with unpurified sIL-1R(I) significantly decreased its thymocyte co-stimulation activity by 57%. Further purification of sIL-1R(I) from other proteins secreted or shed from P. pastoris expression system by size exclusion filtration or ammonium sulfate (60%) precipitation did not influence its capacity to neutralize IL-1 bioactivity. These partially purified sIL-1R(I) preparations significantly decreased thymocyte co-stimulation activity in CM by 70.7 and 77.3%, respectively. In the second experiment, pre-incubation of thymocytes with antisera against the sIL-1R(I) decreased IL-1 activity in CM by 70% relative to control thymocyte cultures that received no antibody and by 59% relative to thymocyte cultures incubated with pre-immune sera. Presumably anti-sIL-1R(I) diminished the IL-1 bioactivity in CM by blocking IL-1 binding to its type-I receptor on thymocytes. Thus, 30% of the IL-1-like activity released by LPS-stimulated HD11 macrophages is probably due to at least one other cytokine. Our data are consistent with the type-I receptor being the primary IL-1 receptor on chicken thymocytes that is capable of providing a signal for proliferation.

Dietary copper level affects copper metabolism during lipopolysaccharide-induced immunological stress in chicks.

Two experiments were conducted to examine the effect of dietary Cu level on Cu metabolism during the acute phase response in broiler chicks with adequate (Experiment 1) or deficient (Experiment 2) Cu. Diets based on cornstarch and isolated soybean protein were used to formulate a basal diet, and basal diet plus either 5, 10, or 15 mg/kg additional Cu as either CuO or CuSO4. Each diet was fed to six pens of five chicks per pen (Experiment 1) or eight pens of five chicks (Experiment 2). Half of the chicks on each diet were injected with Salmonella typhymurium lipopolysaccharide (LPS) on alternate days. In Experiment 1, LPS significantly decreased daily gain, feed intake, and feed efficiency (P < 0.01) and increased the concentration of Cu in blood plasma (P < 0.01). In the uninjected birds, adding 5, 10, or 15 mg/kg Cu as CuO or 15 mg/kg Cu as CuSO4 increased the rate of gain over that of chicks fed the basal diet. In the birds challenged with LPS, 10 mg/kg Cu as CuO increased the rate of gain and efficiency compared to those of chicks fed the basal diet. Addition of CuSO4 to the diet of chicks challenged with LPS did not affect gain, intake, or feed efficiency compared to those of chicks fed the basal diet. Ceruloplasmin levels were higher in chicks challenged with LPS than in control chicks (P = 0.03), and this difference tended to be greater in chickens fed CuO than in chickens fed CuSO4 (P = 0.07). In chicks challenged with LPS, feeding CuO at all levels and feeding CuSO4 to give 10 or 15 mg/kg Cu increased ceruloplasmin levels above that of chicks fed the basal diet. Hepatic Mn superoxide dismutase (SOD) and Cu/Zn SOD were not influenced by dietary Cu level or source or LPS. Results of Experiment 2 were similar to those of Experiment 1 except that supplemental CuSO4 and CuO gave similar increases in gain and CuSO4 was more effective at increasing ceruloplasmin levels. Chicks given supplemental Cu had higher ceruloplasmin levels following challenge with LPS than Cu-deficient chicks fed the basal diet. Apparently, Cu requirements are higher for chicks experiencing an acute phase response than for healthy chicks.

Immunologically mediated growth depression in chicks: influence of feed intake, corticosterone and interleukin-1.

The effects of an immune response on growth and feed efficiency in chicks and the role of interleukin-1 (IL-1) and corticosterone (Cort) as mediators of the response were investigated. Daily injections of either sheep red blood cells or the inflammatory agent Sephadex resulted in significantly (P less than 0.05) lower rates of weight gain, feed intake and efficiency of feed utilization than controls fed ad libitum, indicating an immunologically mediated stress. Feeding control chicks the same amount of diet as that consumed by immunologically challenged chicks did not completely equalize rates of weight gain. Injections of a crude preparation of IL-1, but not Cort, resulted in weight gain, feed intake and efficiency of feed utilization that were similar to those of immunologically challenged chicks. The concentrations of IL-1 and Cort, measured by bioassay and radioimmunoassay, respectively, in serum from immunologically challenged chicks were significantly higher than in nonchallenged chicks. To determine the influence of IL-1 and Cort on protein accretion in skeletal muscles, the extensor digiti communus and ulnaris lateralis were incubated in the presence of these two hormones at concentrations similar to that seen in serum after an immunologic challenge. Cort did not affect the rate of protein degradation but resulted in rates of protein synthesis that were significantly lower than controls. IL-1 did not affect the rate of protein synthesis but resulted in rates of protein degradation that were about 24% greater than controls.

Influence of cell sources, stimulating agents, and incubation conditions on release of interleukin-1 from chicken macrophages.

Experiments were conducted to determine the cell source, stimulating agents, and incubation conditions that maximize interleukin-1 (IL-1) release by chicken macrophages/monocytes. Thymocyte co-mitogen proliferation was used to assay IL-1 activity of conditioned or partially purified supernatants. Monolayers of a transformed chicken macrophage cell line, HD11, released greater amounts of IL-1 than adherent cells isolated from peripheral blood, peritoneal cavity, or spleen. E. coli endotoxin and heat-killed S. aureus induced greater release of IL-1 by HD11 and splenic macrophages than latex or a super induction protocol with mezerien. Blocking macrophage eicosanoid synthesis with indomethacin did not influence IL-1 release from HD11 macrophages. Removing low molecular weight compounds from conditioned supernatants by dialysis did not influence IL-1 activity. IL-1 release was increased by incubating macrophages at 42 C compared to 39 C. Thymocyte co-mitogenic activity of IL-1 was increased by incubating thymocytes at 42 C compared to 39 C. Species cross reactivity between chicken and mammalian IL-1 was also investigated. Chicken IL-1 had slight co-stimulation activity on murine thymocytes, but murine and human IL-1 were without activity on chicken thymocytes.