Muquarrab A. Qureshi

Genetic variation in the recruitment and activation of chicken peritoneal macrophages.

Abstract Genetic variation in the ability to recruit and activate peritoneal macrophages was examined in seven partially developed 15I5-B congenic White Leghorn chicken lines. While the ability to generate peritoneal exudate cells (PECs) was similar in all lines, major differences were observed in the numbers, composition, and functional activity of harvestable peritoneal adherent cell populations. In response to a general stimulant, Sephadex, lines .7-2 and .6-2 produced the greatest numbers of adherent peritoneal cells while lines .C-12 and .15I-5 were among the poorest responders. Macrophage percentage of adherent PECs varied between lines. 15I5 chickens produced a consistently high percentage of adherent macrophages while .6-2 birds exhibited the lowest macrophage percentage at all ages examined. Phagocytosis was used as one measure of the level of macrophage activation and similar results were obtained using both opsonized and unopsonized sheep erythrocytes; adherent peritoneal cells from lines .6-2, .7-2, and .P-13 exhibited the highest activity and .C-12, .15I-5, and background 15I5 (B 15) lines produced cells with the lowest phagocytic activity. In a second functional assay, the killing of Salmonella typhimurium, macrophage-rich cells from line .P-13 exhibited the lowest activity which was significantly lower than that obtained with cells from lines .6-2 and .15I-5. Antigen-specific stimulation of peritoneal adherent cells by ferritin also showed that .C-12 was a low responder in contrast with other lines. The results indicate that these genetic lines differ in peritoneal macrophage function and suggest that the chicken major histocompatibility complex may influence certain properties of chicken macrophage function.

Avian peritoneal exudate cells: A comparison of stimulation protocols

The influence of different stimulation protocols on the induction of peritoneal exudate cells (PECs) and adherent cells, and on the percentage of different adherent cell types was examined in the chicken and Japanese quail. The results suggest that different protocols may be selected to maximize isolation of specific PEC populations for immunological studies. In the chicken, starch, peptone, glycogen, and Sephadex G-40 were all equally effective and superior to saline in generating PECs. While a single injection of Sephadex produced the highest yield of adherent cells with a maximum percentage of macrophages, repeated injections of Sephadex led to dramatic increases in non-adherent PECs (lymphocytes). In contrast, a single injection of starch was optimum for generating non-macrophage adherent cells (primarily heterophils). Since responses of the Japanese quail to stimulation with starch and saline were similar to those observed for the chicken, it is suggested that these protocols may be generally applicable for use with avian species.

Chemotactic activity of chicken blood mononuclear leukocytes from 15I5-B-congenic lines to bacterially-derived chemoattractants

The chemotactic activity of chicken blood mononuclear leukocytes was examined in partially-developed 15I5-B-congenic chicken lines using Enterobacter cloacae culture supernatant and formyl-methionyl-leucyl-phenylanine (f-met-leu-phe) (10(-5)M). Cells from seven different coded B-congenic lines were used to study each chemoattractant in vitro. Mononuclear cells from lines .15I-B5,.C-B12 and the background line 15I5 (B15) exhibited a significantly greater directed migration to bacterial supernatant than did cells from four lines carrying the B2, B2, B13, and B19 haplotypes, respectively. Similarly response to f-met-leu-phe was greatest in lines .15I-B5,.C-B12 and .N-B21, with the same four lines exhibiting a significantly lower response. Since f-met-leu-phe was originally isolated from bacteria, the results indicate that these lines possess differential chemotactic responses to certain bacterially-derived chemoattractants. Major histocompatibility complex (MHC) differences between the lines may serve as a genetic basis for the differential responses. Extrapolation of these results to other chemotactic-receptor systems would require further examination.

Hematological toxicology following embryonic exposure to aflatoxin-B1.

Abstract The influence of embryonic exposure to aflatoxin-B1 (AF-B1) upon the erythroid system of the maturing chicken was examined using a variety of assays. Since the chick embryo is known to possess mixed-function oxidase activity, this animal serves as an excellent model system for studies of human fetal toxicology. AF-B1 (0.1 μg) was administered to either 6- or 12-day embryos by the air sac method. This level of AF-B1 was highly mutagenic and was found to induce an average of 10.6 sister chromatid exchanges (SCEs) per cell compared with 1.8 SCEs per cell for the acetone control solvent. Despite selection against treated embryos through acute and chronic embryonic toxicity, hatched chicks from AF-B1 treatment groups exhibited erythroid anemia when compared to the acetone controls. Cell count, hematocrit, and hemoglobin concentration were all significantly reduced in the 12-day AF-B1 treatment groups compared with controls. Both sexes were equally affected. While the number of peripheral erythrocytes was reduced following exposure to AF-B1, the differentiation status of erythrocytes was apparently unaltered. Mean cell volume, percentage of circulating reticulocytes, and incidence of an erythroid differentiation marker, chicken fetal antigen, were parameters in which no treatment effects were observed. An apparent maturation effect was noted since adult hematocrits were similar between control and treatment groups. Possible explanations for this age effect are discussed. The ability to detect significant posthatch erythroid toxicity following embryonic exposure to mutagenic levels of AF-B1 suggests the importance of this general approach to perinatal carcinogenic evaluation.

Developmental Expression of Chicken Fetal Antigen on Peritoneal Macrophage: Implications for Hematopoietic Differentiation

Abstract Chicken fetal antigen (CFA) was demonstrated by both complement-mediated microcytotoxicity and immunofluorescence to be present on the cell surface of activated peritoneal macrophage. While CFA-positive macrophage were observed in young birds, the antigen was absent from adult macrophage. A comparison of cell surface CFA on macrophage, lymphocytes, and erythrocytes indicated that unique subsets of CFA determinants are characteristic for each differentiation lineage. As a result, CFA can serve as a cell surface marker for differentiation-specific gene expression. Since CFA determinants are known to be at least partly defined by carbohydrates, it is suggested that hematopoietic heterogeneity for CFA may result from lineage-specific carbohydrate microheterogeneity.

Monoclonal Antibodies Reactive with Chicken Peritoneal Macrophages: Identification of Macrophage Heterogeneity

Abstract Mouse monoclonal antibodies (MCAs) were prepared against chicken inflammatory macrophages for the purpose of analyzing macrophage heterogeneity. Macrophage-rich adherent peritoneal exudate cells harvested from Cornell K-strain chickens 42 hr after stimulation with Sephadex were used as immunogens in the production of the monoclonal antibodies. Eight hybridoma clones producing antibodies reactive with chicken peritoneal macrophages were subjected to characterization. While six of the monoclonal antibodies cross-reacted with various hematopoietic cell types, two MCAs (CMTD-1 and −2) were restricted in reactivity to macrophages. CMTD-1 was found to react with activated peritoneal macrophages generated by several irritants. In contrast, CMTD-2 identified a subpopulation of macrophages elicited by specific carbohydrate-based stimulants. This presumably was based on the cross-reaction of this MCA with specific carbohydrate linkages. Analysis using flow cytometry revealed the time-dependent appearance of CMTD-2-positive peritoneal macrophages between 24 and 52 hr after ip Sephadex injection. This subpopulation of peritoneal macrophages was found to be heterogeneous for the ability to undergo in vitro phagocytosis of sheep erythrocytes. CMTD-2-positive cells were also detected in the thyroids of 2-week-old Obese strain chickens with spontaneous autoimmune thyroiditis and at a low incidence in the spleens of normal chickens.

Effects of Increased Major Histocompatibility Complex Dosage on Chicken Monocyte-Macrophage Function

Abstract The influence of major histocompatibility complex (B complex) dosage on monocyte-macrophage function was examined using 4- to 6-week-old trisomic strain chickens. Di- (B15 B15 ), tri- (B15 B15B15 ), and tetrasomic (B15B15 B15B15 ) progeny were produced from trisomic x trisomic crosses. Although mononuclear leukocytes from tetrasomics exhibited enhanced chemotactic activity in response to both f-met-leu-phe and Enterobacter cloacae culture supernatant as compared with that of cells from other groups, the ability to generate peritoneal exudate cells in response to intraperitoneal Sephadex stimulation was similar in all groups. Among peritoneal exudate cells, tetrasomic birds produced a significantly lower percentage of adherent macrophages with a higher proportion of Fc receptor-positive and CMTD-2-reactive macrophages than either disomic or trisomic chickens. Both tetrasomic and trisomic peritoneal macrophages exhibited a reduced phagocytic activity for unopsonized but not opsonized SRBC than was found with disomic macrophages. Thus, the number of major histocompatibility complex copies present in cells appears to influence monocyte-macrophage function.

Quail Fetal Antigen (QFA): Identification and comparison with chicken fetal antigen

Quail fetal antigen (QFA), an analogous hematopoietic antigen to chicken fetal antigen (CFA), was identified and shown to be a developmental antigen on the blood cells of Japanese quail and other avian species. Rabbit antiserum against 14-day embryonic quail red blood cells (RBCs) was specifically adsorbed to achieve fetal specificity and to eliminate any cross reactivity with the CFA system. Complement-mediated microcytotoxicity and hemagglutination assays were used to detect the presence of QFA on hematopoietic cells. QFA was found on 100% of the peripheral RBCs from 10-day quail embryos but incidence of the antigen gradually declined with increasing development. Complete loss of QFA from RBCs occurred just prior to sexual maturation between 31 and 42 days of age. No qualitative differences in erythroid expression of QFA were observed during development; however, RBCs from both embryonic duck and interspecific quail- chicken hybrids reacted with R-anti QFA. Like CFA, quail fetal antigen was associated with lymphocytes, particularly those from primary lymphoid organs. Similarities in the developmentally controlled tissue distribution of QFA and CFA suggest that developmental hematopoietic antigens are a common feature of avian species and are useful cell surface markers for studies of development and differentiation.