Dietary arginine intake alters avian leukocyte population distribution during infectious bronchitis challenge.

Although dietary arginine is a factor in immune function and disease resistance, the full range of effects has yet to be described. In this study, the effects of dietary arginine on leukocyte population changes were examined in the peripheral blood and the respiratory tract of chickens inoculated with infectious bronchitis virus (IBV) strain M41. At 2 wk of age, female line P2a White Leghorn-type chickens were randomly assigned to one of three diets with different arginine levels: a marginally deficient diet (0.5%), an adequate diet (1.0%), and a diet containing a high level of arginine (3.0%). All birds were inoculated with IBV at 4 wk of age, and then the peripheral blood and the respiratory lavage were collected at 1 and 7 d postinfection (DPI). The growth rate of birds that received 0.5% arginine was significantly lower than that of birds receiving 1.0 or 3.0% arginine, whereas the growth of the latter groups did not differ. The percentage and absolute number of heterophil (H) and the H/lymphocyte (L) ratio in the peripheral blood at 1 DPI significantly increased as dietary arginine increased. In the respiratory lavage at 1 DPI, the percentage of H also increased with dietary arginine increase. At 7 DPI, the percentage of CD8+ cells from birds fed the deficient diet was lower than those from birds fed the adequate diet and the diet containing a high level of arginine, whereas the cell surface density of CD8 antigen did not vary among groups. These results show that dietary arginine influences the character of the chicken cellular response to IBV and the distribution of responding leukocyte subpopulations in a target tissue for the infection.

Developmental windows of differential lead-induced immunotoxicity in chickens.

The developing immune system of rodents has been shown to exhibit increased sensitivity to lead-induced immunotoxicity compared with that of adults. However, little is known about potential windows of increased vulnerability during discrete periods of embryonic development. To investigate differential embryonic sensitivity to lead-induced immunotoxicity, sublethal doses of lead ranging from 5 to 400 microg/egg were introduced into fertilized Cornell K Strain White Leghorn chicken eggs via the air sac at one of four different stages of embryonic development (5, 7, 9, and 12 days of incubation, designated as E5, E7, E9, and E12, respectively). Lead levels of blood and bone were determined at hatching and lead-induced immunotoxicity was evaluated in 5-6 week old young chickens using a delayed-type hypersensitivity (DTH) reaction against bovine serum albumin (BSA), macrophage production of nitric oxide, and interferon-gamma (IFN-gamma) production by splenic lymphocytes as immune indicators. Splenic lymphocyte production of IFN-gamma was significantly suppressed (measured for E7 and E9 exposures only, P<0.05) among lead treated groups when compared with controls. Macrophage production of nitric oxide (measured as nitrite production) was significantly depressed (P<0.05) following E5, E7, and E9 lead exposures but not following E12 lead exposure. In contrast with this pattern, DTH function was unaltered following the E5, E7, and E9 exposures, but was significantly depressed (P<0.05) after E12 exposure to lead. Since the same lead dose (200 microg/egg) given at E9 and E12 produced the same blood and bone lead levels and resulted in a different outcome regarding DTH function, the capacity of lead to influence DTH function appeared to emerge between days 9 and 12 of in ovo development. Based on these results, it is hypothesized that lead exposure during different windows of embryonic development is likely to result in different immunotoxic outcomes in the juvenile.

Persistent effect of in utero meso-2,3-dimercaptosuccinic acid (DMSA) on immune function and lead-induced immunotoxicity.

Meso-2,3-dimercaptosuccinic acid (DMSA) is a drug currently employed for cheltion therapy in lead poisoning; however, little is known about its potential effects on the immune system. To examine the effect of DMSA and its capacity to reverse immunotoxicity resulting from exposure to lead in utero, female Fischer 344 rats were administered lead acetate in drinking water from 2 weeks prior to mating until parturition; DMSA was given by gavage on days 6-21 of gestation. The immune function of the female offspring was tested at 13 weeks of age. The results showed that lead (250 ppm) suppressed Th1-type responses (delayed-type hypersensitivity (DTH), interferon gamma (IFN gamma) production), enhanced a Th2-type response (interleukin-4 (IL-4) production), and increased tumor necrosis factor alpha (TNF alpha) production from macrophages. DMSA treatment (60 mg/kg per day) during pregnancy significantly lowered the blood lead levels of both the embryos and the lactating dams as well as the milk lead level of lactating dams. The chelation treatment also reversed the lead-induced alterations in pup body weight, relative spleen weight, TNF alpha, and IL-4 production. But in utero exposure to DMSA alone resulted in decreased DTH response in adult offspring. This was likely due to a reduced cell recruitment, since plasma monocyte chemoattractant protein-1 (MCP-1) levels were decreased. The DMSA-exposed offspring also demonstrated increased interleukin-2 (IL-2) production. These results suggest that DMSA reverses some of the lead-induced immunotoxicity; however, this treatment itself during embryonic development produces subsequent adult immunomodulation.

Avian macrophage metabolism.

This review considers the role of avian macrophages as a source of immune effector and immunoregulatory metabolites. Although considerable attention has been given to the importance of leukocytic cytokines, particularly the monokines such as interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and transforming growth factor-beta (TGF-beta), metabolites produced by macrophages appear to be of equal importance in determining the progression of immune responses. The three metabolite categories that have received the greatest attention are the reactive oxygen species (ROS), the reactive nitrogen intermediates (RNI), and the eicosanoids. Additionally, the xenobiotic metabolites produced via cytochrome P450 activity mediate some immune-environmental interactions. Each of these four metabolite categories is subject to different requirements for metabolite production, and each has distinct effector functions. An understanding of macrophage metabolite regulation could allow improvements in avian health management and production via the effective control of metabolite production. The present review considers prior and recent information on the production of the metabolites by avian macrophages. Additionally, the potential ramifications of metabolite production and regulation are discussed.

Developmental exposure to lead causes persistent immunotoxicity in Fischer 344 rats.

Lead has been shown to exert toxic effects during early development. In these in vivo and ex vivo experiments, the effect of lead on the immune system of the developing embryo was assessed. Nine-week-old female Fischer 344 rats were exposed to lead acetate (0, 100, 250, and 500 ppm lead) in their drinking water during breeding and pregnancy (exposure was discontinued at parturition). Offspring received no additional lead treatment after birth. Immune function was assessed in female offspring at 13 weeks of age. Dams in lead-exposed groups were not different from controls with respect to the immune endpoints used in these experiments; however, in the offspring, lead modulated important immune parameters at modest exposure levels. Macrophage cytokine and effector function properties (tumor necrosis factor-alpha and nitric oxide production) were elevated in the 250 ppm group, while cell-mediated immune function was depressed, as shown by a decrease in delayed-type hypersensitivity reactions in the 250 ppm group. Interferon-gamma levels were decreased in the 500 ppm treatment group. Serum levels of IgE were increased in rats exposed to 100 ppm lead. These results indicate that exposure of mothers to moderate levels of lead produces chronic immune modulation in their F344 rat offspring exposed in utero. Since the mothers were not susceptible to chronic immune alterations, a developmental bias to the immunotoxic effects of lead is indicated. The differences observed are consistent with the possibility that lead may bias T helper subset development and/or function, resulting in alterations in the balance among type 1 and type 2 immune responses.

Characterization of leukotriene production in vivo and in vitro in resident and elicited peritoneal macrophages in chickens and mice.

Previously, we reported differences in arachidonic acid metabolism in elicited chicken peritoneal macrophages when compared with murine resident and elicited peritoneal macrophages. We now describe leukotriene (LT) production in the same systems, using resident (murine) and inflammatory macrophages (from both species). Inflammatory (4- or 42-g Sephadex-elicited) peritoneal macrophages from chickens lacked the capacity to produce LT in vivo (following opsonized zymosan [OZ] stimulation) or in vitro, in response to A23187. In addition, chicken macrophages were unable to metabolize exogenously added LTC4 or LTD4 in vitro. In contrast, resident murine peritoneal macrophages produced measurable quantities of LTs (in vivo) within 5 min with an 8-fold increase after 45 min. LTC4 was effectively converted to LTE4 in vivo in a time-dependent manner (65% LTC4/35% LTE4 after 5 min stimulation with OZ and 6% LTC4/94% LTE4 after 60 min stimulation), but no in vitro. The lack of LTC4 metabolism to LTE4 in vitro could not be explained by cell-cell interaction between adherent and nonadherent cells. LTD4 was not detected under any experimental condition. Murine peritoneal cells incubated with LTD4 (with or without agonist) produced LTE4 in a time-dependent fashion. Addition of L-cysteine (a dipeptidase inhibitor) did not explain the lack of detectable levels of LTD4 following intraperitoneal stimulation with OZ. These results suggest that elicited chicken peritoneal macrophages are incapable of producing LTs compared to murine peritoneal macrophages. In addition, these studies fail to explain the different product profiles with in vivo stimulation of murine peritoneal macrophages as compared to in vitro stimulation.

Inflammatory function of macrophages from chickens with B-recombinant haplotypes.

Both in vivo macrophage activation and in vitro monocyte activation were compared using chickens homozygous for each of two biochemically and serologically similar B-complex recombinant (B(F2-G23)) haplotypes. Chickens carrying the parental (non-recombinant) B haplotypes (B2 and B23) were included for relative comparison, although the genetic backgrounds for these strains were different from the background of the recombinants. Elicited peritoneal macrophages from R4/R4 (international designation B(2r3)) chickens expressed levels of sheep erythrocyte phagocytosis which were significantly higher (P< 0.05) than those from R2/R2 (B(2rl)) chickens. Differences between chickens with B genotypes were analogous to the differences demonstrated previously between B2/B2 and B23/B23 chickens. Similarly, lipopolysaccharide (LPS)-activated monocytes from R4/R4 chickens also expressed significantly higher (P< 0.05) levels of phagocytosis when compared with R2/R2 and B23/B23. In both cases, the functional level of macrophages from R2/R2 chickens was similar to that of B23/B23 cells, whereas macrophages from R4/R4 chickens were similar in functional capacity to those from B2/B2 chickens. These results suggest that R2 and R4 recombinants, despite their demonstrated similarities, may differ in DNA regions which include genetic factors controlling macrophage responsiveness.

Environment-immune interactions.

The need for effective immune function for the maintenance of health has been clearly established in both agriculturally significant animal species and humans. Intensive agricultural practices present production species with numerous disease challenges during the rearing period. Environmental factors represent a ubiquitous, yet frequently manageable, category of immunomodulators that can influence immune performance and ultimately disease susceptibility or resistance. However, strategies for assessing overall immune potential have not been widely implemented for agricultural species. This is in contrast to the use of immune evaluation for human health considerations. Immune assessment relative to environmental-immune interactions can produce benefits in two areas. First, the efficiency of the production operation can be enhanced. Second, the welfare of the animals during the production cycle can be optimized. This paper presents an overview of environmental factors known to influence the immune function of poultry and the opportunities to manage environmental factors to benefit the health of the animals. In addition, the paper discusses the status of immunological assessment for humans and laboratory animals and proposes potential immune assessment panels that could serve as a tool to optimize the environmental management of poultry populations.

Long-term intake of a low-casein diet is associated with higher relative NK cell cytotoxic activity in F344 rats.

Earlier studies in our laboratory showed that animals exposed to aflatoxin B1 (AFB1) develop fewer gamma-glutamyl transpeptidase-positive preneoplastic foci and tumors when fed 6% dietary casein than when fed 22% casein during promotion; mechanisms underlying this effect have not been elucidated. We examined natural killer (NK) cell activity, mitogenic responses, and lymphocyte surface antigen profiles in male Fischer 344 rats dosed with AFB1 or dosing vehicle alone and then fed 6% or 22% casein isocaloric diets for one year. Mean body weights and food intake did not differ significantly among the groups during the study. NK cells purified from peripheral blood of rats fed 6% casein mediated higher specific lysis (p < 0.0001) against YAC-1 target cells than cells obtained from animals fed 22% casein. Mitogenic responses of splenic lymphocytes to concanavalin A and lymphocyte subpopulations, identified by flow cytometry, did not differ significantly among dietary groups. Hepatic tumors were detected in 27% of the 22% casein AFB1-treated group and in 6% of animals in the 6% casein AFB1-treated group. The association between long-term intake of a 6% casein diet and higher relative NK cell cytotoxic activity suggests a potentially important mechanism that may help protect against the development of hepatocellular tumors. Further study of this mechanism as a causal factor in limiting tumor development is required.

Dietary arachidonate enhances tissue arachidonate levels and eicosanoid production in Syrian hamsters.

Eicosanoids are oxidative derivatives of arachidonic acid. When produced in excess many of them are proinflammatory agents. This study investigates whether dietary arachidonic acid enhances arachidonic acid phospholipid content of various tissues and whether this enrichment increases eicosanoid production. Male Syrian hamsters were divided into four groups and fed diets supplemented with ethyl esters of oleic acid, linoleic acid, arachidonic acid or eicosapentaenoic acid. Differences in the composition of the phospholipid fatty acids were monitored in liver, lung, heart, spleen, kidney, testes, macrophages and platelets. In all tissues analyzed, the phospholipid content of arachidonic acid was significantly higher in the arachidonic dietary group compared with all other dietary groups (average > 50% higher). In contrast, increasing dietary linoleic acid by 50% had little effect on altering tissue arachidonic acid levels. Following in vitro stimulation, macrophages and platelets from animals maintained on arachidonic acid produced, in general, the highest levels of eicosanoids compared with cells from animals fed the other diets. Significant differences were observed in prostaglandin E2 (macrophages) and thromboxane B2 (platelets) formation when compared with the oleic acid and eicosapentaenoic acid dietary groups. The data demonstrate that including low to moderate levels of arachidonic acid in the diet increases macrophage and platelet arachidonic acid levels and may augment eicosanoid production.

Distribution and inducibility of a P450I activity in cellular components of the avian immune system.

The level of expression of the cytochrome P450 system in an immune tissue could influence the sensitivity of that immune tissue to damage by xenobiotics. The capacity of immune organs and their cellular components for P450I-catalyzed metabolism was assayed in the 4-week-old chicken using the P450I-specific ethoxyresorufin-O-deethylase (EROD) assay and the P450I-inducer, 3,4,3',4'-tetrachlorobiphenyl (TCB). After induction by TCB, EROD was detectable in microsomes from whole thymus, bursa and in peritoneal exudate cells (containing primarily macrophages) at levels of 28.3, 7.2 and 1.3 pmol/mg microsomal protein/min, respectively; the level in control liver was 89.9 pmol/mg microsomal protein/min. No activity was detected in these immune tissues without induction. The P450I specific in vitro inhibitor, alpha-naphthoflavone (NF) inhibited the TCB-induced liver and immune tissue EROD by 50% at concentrations in the range of 0.07-0.1 microM. The cellular distribution of EROD in the bursa and thymus was studied in lymphocytes and supporting tissue cells after their separation by density gradient centrifugation. Much higher TCB-induced EROD was detected in immune tissue supporting cells than in lymphocytes, particularly in the thymus. The P450I in the supporting tissue of the bursa and thymus at 1 week post-hatch was also measured after eradication of the lymphocytes in both immune tissues by in ovo administration of CP. TCB-induced EROD was 12-fold higher in the lymphocyte-depleted thymus than in normal thymus, with a less marked but similar pattern in the bursa.(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of neonatal avian inflammatory macrophage function following embryonic cyclophosphamide exposure.

Inflammatory macrophage recruitment and function were examined in 4-5-week-old chickens which had received two doses of cyclophosphamide (CP) or vehicle (dH2O) during late embryogenesis (18 and 19 days of incubation). Mononuclear leukocyte chemotaxis to N-formyl-methionyl-leucyl-phenylalanine (FMLP) and hematological parameters were unchanged in CP-treated vs control chickens. Peritoneal exudate cells (PECs) elicited in response to intraperitoneal (i.p.). Sephadex injection did not differ in CP-treated vs control chickens with respect to cell number, cell type, superoxide anion production, or cell surface expression of Ia and transferrin receptor (TfR) antigens. The CP-treated chickens did exhibit the expected decrease in bursa weight; male chickens exposed to CP also had inhibited testes growth. Although embryonic exposure to CP at this dose results in irreversible bursal damage and subsequent impaired humoral immunity, it appears that there are no long-lasting effects on avian inflammatory macrophage function.

Avian inflammatory macrophage function: shifts in arachidonic acid metabolism, respiratory burst, and cell-surface phenotype during the response to Sephadex.

The avian inflammatory response to intraperitoneal (i.p.) Sephadex injection produces macrophages which display characteristics of an increasingly activated state over time. We examined elicited chicken peritoneal exudate cells (PECs) with respect to superoxide anion production, arachidonic acid metabolism and cell surface Ia and transferrin receptor (TfR) expression from 4 to 96 h after i.p. stimulation. Avian PECs showed the highest level of superoxide release when harvested just 4 h after injection, and did not produce PGE2 or 6-keto PGF1 alpha. Early (4-h) PECs produced elevated amounts of thromboxane as compared to later (42-h) macrophages. Expression of both Ia and TfR increased between 4 and 24 h after Sephadex stimulation; TfR remained elevated through 96 h, but Ia declined after 42 h. Some aspects of chicken macrophage regulation of superoxide anion, thromboxane release, and surface antigen expression are in contrast with those reported for mouse macrophages.

Induction of hepatic metallothionein by intraperitoneal metal injection: an associated inflammatory response.

The nature of hepatic metallothionein (MT) induction by several metals and its relationship to an inflammatory response was studied in chicks. Intraperitoneal (ip) injection of chromium (Cr), managanese, and iron (Fe) caused a much greater increase in hepatic MT (10.2-, 9.0-, and 6.8-fold) compared with cobalt and nickel (2.5- and 2.9-fold); thus not all transition metals are effective. Cr3+ caused markedly greater hepatic MT accumulation than Cr6+, suggesting that the ionic nature of the metal is an important factor. Small organic complexes of Fe (ferrous gluconate or lactate, 6.2-fold) caused significantly greater accumulation of hepatic MT than ferric dextran (1.4-fold), a large organic aggregate. In vitro data from chick hepatocytes and/or fibroblasts clearly indicated that Fe does not effect the induction of MT directly. The role of inflammation, as measured by recruitment of peritoneal exudate cells (PEC), was examined. Endotoxin (LPS), Sephadex (S), and Fe elicited significant elevations in PEC number at 24 h posttreatment (S), and Fe elicited significant elevations in PEC number at 24 h posttreatment (S = Fe greater than LPS much greater than control). The percentage of heterophils but not macrophages was significantly correlated with the accumulation and induction of hepatic MT. In a similar experiment with Cr, we demonstrated that Cr3+ but not Cr6+ stimulated MT messenger RNA accumulation and concomitant hetereophil infiltration at 3 h after injection. Our results indicate that the induction of hepatic MT by the parenteral administration of a number of metals is dependent on the chemical nature of the metal and is associated with an inflammatory response.

Dynamics of avian inflammatory response to cross-linked dextran. Changes in avian blood leukocyte populations.

A model inflammatory compound, cross-linked dextran (Sephadex), which preferentially recruits macrophages, was used to examine shifts in chicken leukocyte distribution and monocyte functional status during the inflammatory response. Following intraperitoneal Sephadex administration, total leukocyte counts were transiently elevated because of a fivefold increase in circulating heterophils (granulocytes). While both heterophils and monocytes may become demarginated, primarily monocytes enter the peritoneal cavity in response to Sephadex. Since leukocyte chemotaxis is an important feature of the inflammatory response, this parameter was analyzed during the recruitment period. The optimum conditions for in vitro chicken mononuclear leukocyte chemotaxis to N-formyl-methionyl-leucyl-phenyl-alanine (FMLP) were defined and compared with those for the chicken macrophage-like cell line, HD11. Mononuclear leukocytes from birds injected 6 or 12 h previously with Sephadex were significantly decreased in chemotactic ability when compared with controls; at 24 h after injection, the response to FMLP had returned to normal. This system offers an opportunity to examine differential effects of inflammatory stimuli upon leukocyte function and distribution.

Kinetics of inhibition of in vitro interferon-alpha/beta production after benzo-(a)-pyrene exposure.

L-929 cells were treated with benzo-(a)-pyrene (BP), washed, and then interferon(IFN)-alpha/beta was induced with polyriboinosinic-polyribocytidylic acid (poly-I-C). IFN-alpha/beta production by these cells was measured at 6-hour intervals. Up to 24 h after commencement of IFN induction, IFN production was severely inhibited in BP-treated cells as compared to controls. After 24 h, IFN-alpha/beta production in BP-treated cells began to rise and reached levels seen in untreated cells by 48 h. These data indicated that IFN induction was not completely inhibited by BP treatment, but, rather, the peak of IFN production was delayed. Mouse embryo cultures from SENCAR mice showed the same pattern of delayed IFN production when treated with BP. Direct exposure of the cells to BP for at least 15 min prior to removal of the carcinogen was required for IFN induction to be inhibited; however, at least 24 h of further incubation in fresh medium before addition of poly-I-C was required for inhibition of IFN induction by BP.

Reversibility of inhibition of interferon-alpha/beta induction by benzo[a]pyrene.

Mouse L-929 cells were treated with benzo[a]pyrene [(BP) CAS: 50-32-8] for 24 hours. Several cultures of cells were immediately treated with polyriboinosinic-polyribocytidylic acid (poly I:C) to induce interferon-alpha/beta (IFN). Other sets of cultures were washed and allowed to incubate with fresh culture medium for 3-18 hours before addition of poly I:C to induce IFN. The IFN induction was severely inhibited in cells treated with BP and immediately challenged with poly I:C as compared to non-BP-treated cells. Up to a 6-hour delay in addition of poly I:C still resulted in severe inhibition of IFN induction; however, a delay of 9 hours or longer allowed the cells to recover their IFN production capacity. These data indicate that the inhibition of IFN induction by BP was reversible. The number of cells laid down in the cultures was approximately 6 X 10(5). In another experiment, the number of cells laid down in culture was serially decreased in several different cultures. These normal cells were then treated with poly I:C to induce IFN. A 50% decrease in the number of cells was required to reduce IFN production by 50%. In BP-treated L-929 cells, viabilities ranged from 88 to 90%, but IFN induction was inhibited by greater than 50%. These results suggest that toxicity of carcinogens to cells does not play a major role in inhibition of IFN induction by carcinogens.

Genetics of susceptibility of mice to inhibition of interferon induction by benzo-(a)-pyrene.

Embryonic fibroblasts were prepared from four different strains of mice: DBA/2, noninducible at the Ah locus for aryl hydrocarbon hydroxylase for carcinogen activation and, therefore, relatively resistant to tumor induction; C57BL/6, normal at the Ah locus; SENCAR, mice innately highly sensitive to tumor induction; and ICR, normal controls for SENCAR mice. The fibroblasts were exposed to the carcinogen benzo-(a)-pyrene at varying dosages. Alpha/beta IFN was next induced with poly(I)(C). Interferon production by cells from C57BL/6 mice was severely inhibited. Interferon production by DBA/2 cells was equally susceptible to inhibition by BP as IFN production by C57BL/6 cells, perhaps because of alternative pathways of activation of BP. Interferon production by SENCAR mouse cells was, on the other hand, much more susceptible to inhibition by BP than was IFN production by ICR mouse cells. These results suggest parallels between genetically controlled sensitivity to tumor induction by BP and genetically controlled susceptibility to inhibition of IFN induction by BP.

Effect of 7,12-dimethylbenz[a]anthracene on production and action of gamma interferon.

Spleens were removed from Swiss Webster mice, and cultures of spleen cells were prepared. Gamma (ty pe II immune) interferon (IFN) production was induced in these cells by addition of the purified protein form of phytohemagglutinin (PHA-P) to the cultures. When 7,12-dimethylbenz[a]anthracene (DMBA) was added to the cultures either before or after addition of PHA-P, the production of gamma IFN was inhibited. The degree of inhibition was greater when the cells were treated with DMBA before addition of PHA-P. In experiments to determine the effects of DMBA on the antivirus activity of previously prepared gamma IFN preparations, DMBA was either added to target cells before the addition of exogenous gamma IFN or mixed together with exogenous gamma IFN. In both cases. the antivirus activity of this exogenous gamma IFN was not affected. These data suggest that DMBA treatment can inhibit the production but not the antivirus activity of murine gamma IFN.