Richard J. Cross

Hypothalamic-immune interactions. I. The acute effect of anterior hypothalamic lesions on the immune response

Rats with electrolytic anterior hypothalamic lesions show changes in lymphoid tissue cellularity and a decrease in the response to concanavalin A (Con A). This effect manifests itself maximally 4 days after lesioning, with a return to normal by day 14. The changes are not mediated through the release of corticosteroids. These data indicate the presence of a neuroendocrine pathway that is capable of modulating immune function.

Hypothalamic-immune interactions: Effect of hypophysectomy on neuroimmunomodulation☆

Electrolyte destruction of certain nuclei of the brain cause specific structural and functional changes in the immune system. Lesions in the preoptic-anterior hypothalamic area result in thymic involution and a decrease in the number and blastogenic reactivity of splenocytes. In contrast, lesions in the hippocampus increase thymic and splenic mitogenic responsiveness and cellularity. Hypophysectomy abrogates all changes in splenocyte number and function induced by hypothalamic and limbic lesions. The effects of ablating the hippocampus and amygdaloid complex on thymocyte number and function also are abolished. Hypothalamic lesions in hypophysectomized animals result in an increase in the number of thymocytes but suppressed mitogenic activity. These data indicated that neuroimmunomodulation is mediated predominantly but not exclusively by the pituitary gland.

Combined Hoechst 33342 and merocyanine 540 staining to examine murine B cell cycle stage, viability and apoptosis

A procedure is described for the dual staining of lymphocytes with Hoechst 33342 (Ho342) to examine cell cycle position, and merocyanine 540 (MC540) that allows for the analysis of cells entering the early stages of apoptosis. Ho342 is a DNA specific dye and MC540 detects membrane phospholipid domain changes, some of which are associated with apoptotic cells. Flow analysis of B cells dually stained with Ho342 and MC540 allows for the discrimination of five distinct subpopulations. Two of these subpopulations represent viable, MC540 negative/dull cells with either 2n or 4n DNA. As 2n and 4n DNA B cells become MC540 bright they move into two distinct subpopulations representing cells entering and progressing through the early stages of apoptosis. As the apoptotic, MC540 bright cells move into the latter stages of apoptosis, they localize into a fifth subpopulation displaying reduced staining with Ho342 indicative of late stage apoptotic cells in the process of fragmenting their DNA. This experimental approach enables the characterization of lymphocyte populations for percentages of viable, early apoptotic, and late apoptotic cells. The cells are not fixed during this procedure, and since both dyes are viable dyes there is an additional opportunity to obtain sorted cells from any of the defined subpopulations for reculturing and functional analysis.

Potentiation of antibody responsiveness after the transplantation of a syngeneic pituitary gland

Transplantation of a pituitary graft under the kidney capsule and the resulting elevation of serum prolactin enhances the primary humoral antibody response to sheep red blood cells. Enhancement of the response is not due to marked changes in the percentage of T-cells and their subsets, B-cells, or the number of nucleated spleen cells. Quantitation of serum prolactin levels correlates well with the proportion of enhancement as mice with two grafts and higher levels of prolactin have increased responsiveness compared to mice with one graft. Systemic administration of mouse prolactin at the time of immunization also enhances the humoral immune response; however, if prolactin treatment is delayed and given 24 h after immunization, no potentiation of the response occurs. Thus, prolactin is enhancing the immune response by affecting an early afferent event in the induction of the immune response.

Lymphocyte Subsets in Cord Blood of Preterm Infants: Effect of Antenatal Steroids

The objective of this study was to evaluate prospectively the influence of gestational age (GA) and short-term antenatal steroids on total lymphocyte count and lymphocyte subsets in cord blood from preterm infants. Two-color flow cytometric analyses of lymphocyte subsets were performed on cord blood collected from 67 infants. These infants were grouped according to GA: group I (term, n = 19); group II (GA 33–37 weeks, n = 25); group III (GA <33 weeks, n = 23). The mean absolute lymphocyte counts (ALC) in groups I, II and III were 5.6 ± 2.5 × 103/µl, 4.3 ± 1.5 × 103/µl and 3.5 ± 1.8 × 103/µl respectively. The mean values for CD4+ lymphocytes in groups I, II and III were 2.7 ± 0.8 × 103/µl, 2.0 ± 0.8 × 103/µl and 1.6 ± 0.9 × 103/µl respectively. Mean values for CD8+ lymphocytes were 0.9 ± 0.3 × 103/µl, 0.6 ± 0.3 × 103/µl and 0.5 ± 0.3 × 103/µl respectively. With decreasing GA, there was a statistically significant decrease in ALC (p = 0.0035), CD4+ lymphocytes (p = 0.0013) and CD8+ lymphocytes (p = 0.0064). We then evaluated the effect of antenatal steroids, now routinely administered to women with preterm onset of labor to facilitate fetal lung maturation, and found that after adjusting for GA, infants of women on antenatal steroids had significantly fewer ALC (p = 0.0001), CD4+ lymphocytes (p = 0.02) and CD25+ lymphocytes (p = 0.03). In this population of infants, the decreased number of lymphocytes seen at younger GAs is associated with antenatal steroid use.

THE EFFECT OF THE ENDOPHYTE (ACREMONIUM COENOPHIALUM) AND ASSOCIATED TOXIN(S) OF TALL FESCUE ON SERUM TITER RESPONSE TO IMMUNIZATION AND SPLEEN CELL FLOW CYTOMETRY ANALYSIS AND RESPONSE TO MITOGENS

Experiments were conducted with rats and mice to evaluate the effect of the consumption of endophyte (Acremonium coenophialum) and associated toxin(s) infected tall fescue on humoral and cellular aspects of immune function. Treatment diets were: (1) rodent chow (RC) or (2) rodent chow mixed 1:1 (w/w) with endophyte infected (E+) or (3) non-infected (E-) tall fescue seed. Rats fed the E+ diet in experiment 1 (43 days) exhibited a lower (P less than 0.05) serum titer to sheep red blood cell (SRBC) immunization than those fed the E- diet (38.4 vs 131.3). The E+ rats also had lower (P less than 0.01) white cell counts than either RC or E- groups (5225 vs 8959 and 7491/mm3). Spleen cells from mice fed the E+ diet for 37 days exhibited a reduced (P less than 0.05) response to the mitogens Concanavalin A and lipopolysaccharide. Flow cytometric analysis revealed a significant (P less than 0.01) 42% increase in T suppressor cell numbers in spleens of mice fed the E+ vs RC diets.

Central catecholamine depletion impairs in vivo immunity but not in vitro lymphocyte activation

We have previously shown that depletion of central nervous system (CNS) catecholamines by injecting the neurotoxin 6-hydroxydopamine (6-OHDA) into the cisterna magna of C57B1/6 mice markedly impairs the humoral immune response to sheep red blood cells. This work extends these observations by showing that 6-OHDA treatment also inhibits the humoral antibody response to the T-cell-dependent antigen trinitrophenyl-keyhole limpet hemocyanin, but does not affect the response to the T-independent antigen trinitrophenyl-lipopolysaccharide. This treatment also impairs humoral responsiveness at peripheral lymphoid sites in addition to inhibiting natural killer cell activity. However, 6-OHDA treatment in vivo does not affect in vitro mixed lymphocyte responsiveness, mitogen-induced lymphocyte activation or antigen presentation by macrophages.

Role of T-lymphocytes in the resolution of endotoxin-induced lung injury.

An acute neutrophilic lung injury was compared in Balb/c normal and nu/nu (nude) mice to assess the role of T lymphocytes in the resolution of acute pulmonary neutrophilic inflammation following the administration of endotoxin. Maximal neutrophilic infiltration occurred on day 1 post-endotoxin treatment and declined to near normal levels by day 5. In contrast, the percentage of lymphocytes in the bronchoalveolar lavage (BAL) fluid increased from 1.8% on day 1 post-endotoxin to greater than 11% on days three and five, during which time neutrophil resolution was occurring. On days 1–5 after endotoxin administration, approximately 40% of the CD4 lymphocytes expressed the cell surface activation marker, CD69. Despite being CD69+, CD4 cells did not express the high affinity IL-2 receptor chain, CD25, to any significant extent on any of the days studied. To assess the contribution of T cells to the rate of clearance of neutrophils from the BAL, normal and nude Balb/c mice were compared for the percentage of neutrophils following nasal administration of endotoxin. Endotoxin-treated nude mice did not demonstrate significant differences in either the total white blood cell counts or in the clearance of neutrophils from the BAL, as compared to normal Balb/c mice. These data indicate that the influx of activated T cells during the resolution of neutrophilic pneumonitis does not contribute to the rate of neutrophil clearance during acute lung injury.

Functional characterization of the insulin-like growth factor I receptor on Jurkat T cells

Insulin-like growth factor I (IGF-I) has been shown to be important in the maintenance, development, and proliferation of various types of leukocytes, particularly T cells. Radio-receptor binding assays demonstrate that Jurkat T cells bind 125I-IGF-I with an affinity of 1.77 nM (Kd) and express approximately 230 receptors/cell. Specificity studies show insulin also binds the IGF-I receptor with an affinity 20-fold lower than that of IGF-I. Interaction of IGF-I with its receptor on Jurkat T cells induces the phosphorylation of tyrosine kinase which is detectable by Western blotting. The 95,000 MW protein detected is equivalent to the molecular weight of the beta chain of the IGF-I receptor described in other types of cells. These studies characterize the binding of IGF-I to its receptor on Jurkat T cells, demonstrate that IGF-I binding induces tyrosine phosphorylation, and support the hypothesis that IGF-I is important in the induction of T cell activation.

Transplantation of pituitary grafts fail to restore immune function and to reconstitute the thymus glands of aged mice.

There is evidence to indicate that the neuroendocrine and immune systems can interact. Thus, neuroendocrine hormones can modulate a variety of immune functions and there have been attempts to manipulate the neuroendocrine system of aged animals to enhance immune function. We have previously shown that the transplantation of a syngeneic pituitary gland under the kidney capsule of young adult mice elevates serum prolactin and enhances immune responsiveness. In the present study pituitary glands were transplanted under the kidney capsule of 22-month-old mice to determine if this maneuver can enhance a number of immunologic parameters. The results demonstrate that aged animals bearing transplanted pituitary grafts for 10 days did not exhibit any enhancement in their primary antibody response to sheep red blood cells, splenic T or B-cell mitogen responsiveness or restoration of thymic architecture. When these immunologic assessments are performed on animals bearing pituitary grafts for 28 days, the IgM and IgG primary antibody responses and splenic T-cell responsiveness are enhanced but repopulation of the thymus still does not occur. Importantly, this enhancement does not restore immunocompetence to levels observed in young mice.