Steroid sex hormones and macrophage function: regulation of chemiluminescence and phagocytosis.

PROBLEM: Female sex hormones modulate a variety of humoral and cell-mediated immunologic functions. In this study, the effects of estrogen, progesterone, and testosterone on the chemiluminescence (CL) response and phagocytic ability of male rat peritoneal macrophages (M luminal diameter) were examined. METHOD: In M luminal diameter pretreated with 10(-2) ng/ml of 17 beta-estradiol (E2) for 20 hours, the CL generated in response to phorbol myristate acetate (PMA), 1,2-dioctanoyl-rac-glycerol (C8:0), or opsonized zymosan (OZ) was significantly increased by 135%, 140%, and 136% of control values, respectively. In addition, M luminal diameter treated with 10(-5) ng/ml or 10 ng/ml of E2 exhibited a significantly greater PMA- or OZ-stimulated CL response than did untreated controls. RESULTS: At 10(-2) ng/ml, progesterone enhanced and testosterone reduced the CL response, but these changes were not statistically significant. In time course studies, the PMA-stimulated CL response of M luminal diameter treated with 10(-2) ng/ml of E2 or progesterone for 5 h was significantly less than that of the untreated group. In the presence of endotoxin (12 pg/ml), the CL response in M luminal diameter treated with E2 or testosterone was significantly depressed as compared to untreated controls. Phagocytosis of opsonized sheep erythrocytes also was significantly enhanced (140% to 190% of control) when M luminal diameter were pretreated with 10(-12) M to 10(-8) M of either E2 or progesterone. CONCLUSIONS: These findings suggest that, at physiological concentrations, E2 is capable of modulating both CL generation and phagocytic uptake by M luminal diameter in a manner not shared by other steroid hormones.

Steroid sex hormones regulate the release of tumor necrosis factor by macrophages.

Generally, females have been found to have a heightened immune response and a concomitantly higher incidence of autoimmune diseases compared to males. We have used male rat peritoneal macrophages (M phi) to study the effect of female sex hormones on tumor necrosis factor (TNF) release. The amount of TNF released by macrophages (M phi) exposed to 10(-2) and 10(-3) ng/ml of 17 beta-estradiol (E2) (35.1 +/- 7.3 and 23.2 +/- 2.5 units/ml, respectively) was significantly (P < 0.05; n = 9) greater than that released by untreated M phi. Progesterone (P) also significantly (P < 0.05; n = 8) stimulated a maximal TNF release (24.4 +/- 2.8 units/ml TNF) at 10(-2) ng/ml. On the other hand, the amount of TNF released by M phi exposed to E2 or P at concentrations greater than 10(-1) or less than 10(-4) ng/ml was significantly (P < 0.05) reduced compared to untreated controls. In contrast, testosterone did not significantly affect TNF release at any concentration. Within the physiological range of E2 and P concentrations, TNF release from M phi is finely regulated and dramatically affected by relatively small changes in hormone concentrations.

Steroid sex hormones and macrophage function: modulation of reactive oxygen intermediates and nitrite release.

PROBLEM: In general, females have a more active immune response than do males. The effects of female sex hormones on lymphocytes have been studied extensively but their effects on macrophages are poorly understood. METHOD: In this study, peritoneal macrophages (M phi) obtained from male rats were treated in vitro with estradiol (E2), progesterone (P), testosterone (TS), or hydrocortisone (HC) and their effects on superoxide, hydrogen peroxide, and nitrite release determined. RESULTS: At concentrations between 10(-10) and 10(-9) M, female and male sex hormones had no significant effect on superoxide release but, at concentrations above or below that range, these hormones stimulated the release of these reactive oxygen intermediates (ROI). In contrast, M phi treated with HC generally exhibited either unaltered or reduced ROI release. CONCLUSIONS: These findings suggest that female sex hormones regulate ROI release by M phi in a manner not entirely shared by other steroid hormones. At most concentrations used, E2, P, TS, and HC significantly inhibited nitrite release by M phi. However, with 10(-10) M of E2 or 10(-9)M of P, nitrite release by M phi was not affected. In the presence of anti-TNF antibody, the amounts of superoxide and hydrogen peroxide release were moderately reduced but nitrite release was dramatically inhibited. The sensitivity of M phi to variations in the concentrations of female sex hormones may contribute to gender-related differences in the immune response.

Modulation of the cytoskeleton and intracellular calcium in leukocytes exhibiting a cancer-associated chemotaxis defect.

Monocyte chemotaxis is severely depressed in patients with advanced tumors, but the cellular basis for this chemotactic defect is not known. Because the actomyosin cytoskeleton is thought to play a primary role in chemotaxis, we have employed flow cytometry to examine several aspects of the contractile machinery including myosin II, myosin light chain kinase (MLCK), actin, and cytoplasmic calcium in unstimulated and in formylpeptide-stimulated neutrophils and monocytes. Serum-pretreated polymorphonuclear leukocytes (PMNs) and monocytes from healthy blood donors or PMNs and monocytes isolated from tumor patients were studied. Leukocytes pretreated with serum from cancer patients exhibited decreased baseline myosin staining and a vastly different response to formylpeptide stimulation compared with leukocytes pretreated with normal human serum. In contrast, similar amounts of MLCK were observed in neutrophils and monocytes preincubated with normal or cancer serum with or without stimulation with formylpeptide. The fluorescent calcium indicator fluo-3 showed that resting and fMLP-stimulated levels of intracellular calcium were not significantly different in control and cancer serum-pretreated human leukocytes or in leukocytes isolated from tumor patients. Similarly, resting and fMLP-stimulated levels of F-actin in cancer patients' leukocytes as assessed by NBD-phallacidin staining did not differ significantly from those of normal leukocytes. Because the actomyosin cytoskeleton is intricately involved in leukocyte chemotaxis, alterations in the cytoskeleton may dramatically affect cell motility. The cytoskeletal alterations and changes in the response of leukocytes pretreated with cancer patients' serum to formylpeptide stimulation as described here may result in decreased chemotaxis by these cells.

Autoradiographic analysis of formylpeptide chemoattractant binding, uptake and intracellular processing by neutrophils.

Formylpeptide chemoattractant binds specifically to leukocyte cell surface receptors and during chemotactic activation, is internalized and processed by these cells. Using electron microscope autoradiographic techniques, we have examined the binding, uptake and disposition of fNle-Leu-Phe-Nle-[125I]Tyr-Lys by rabbit peritoneal neutrophils. Cells were incubated with ligand for 15 min at 4 degrees C, rinsed and then further incubated in buffer at 4 degrees C, 15 degrees C, 24 degrees C, or 37 degrees C for 0-40 min. For all cells incubated at 4 degrees C, grains were predominantly localized on the plasma membrane. Initially, this formylpeptide was seen in small clusters or microaggregates that were not associated with coated pits. Upon further incubation at 37 degrees C for 1 min, formylpeptide clustering on the plasma membrane increased and extensive internalization of formylpeptide was observed. Endocytosis of formylpeptide-receptor complexes clearly involved uncoated membrane pits and vesicles but did not appear to involve coated pits or coated vesicles. In the following 3 min, peptide proceeded in waves through compartments composed of small uncoated endocytic vesicles, then large vesicles, and then into dense granules. After 4 min at 37 degrees C, the most active phase of intracellular processing subsided. The percentage of grains in the cytoplasm, granule and small vesicle compartments very gradually increased during the remainder of the 40 min incubation. Formylpeptide neither bound at 4 degrees C nor accumulated at 37 degrees C on the cell surface in proximity to the underlying Golgi/centrosome region of the cell. At 24 degrees C, processing was slowed but formylpeptide-receptor complexes proceeded through a similar series of compartments. The t1/2 for formylpeptide uptake at 37 degrees C was 15-20 s, whereas at 24 degrees C the t 1/2 was approximately 10 min. No uptake was observed at 15 degrees C. The distinctive characteristics of formylpeptide binding and receptor-complex uptake seen here may be essential in initiating and maintaining continued chemotactic responsiveness.

Definition of fibronectin-mediated uptake of gelatinized latex by liver slices and macrophages.

These studies show that both liver slices and macrophages carried out fibronectin concentration-dependent uptake of 125I-labeled gelatin-coated latex (test latex). Lack of phagocytosis of test latex by liver slices was shown directly by electron microscopy and indirectly by trypsin treatment, which caused the release of all test latex taken up in response to fibronectin. Inhibitors of phagocytosis did not alter this uptake. On the other hand, trypsin released only a portion of test latex from macrophages. Inhibitors of phagocytosis did not effect the released radioactive particles from macrophages but greatly reduced the trypsin-resistant radioactivity, taken as representing phagocytized particles. Opsonization of test latex with fibronectin did not require heparin but its association with liver slices occurred only in the presence of heparin. Macrophages, however, readily bound and internalized the opsonized test latex and heparin only potentiated these reactions. Gelatin competed with test latex for fibronectin for opsonization, but did not inhibit binding and phagocytosis of fibronectin-test latex complexes. Finally, soluble fibronectin-gelatin complexes did not compete for binding and phagocytosis of fibronectin-test latex complexes. Thus, fibronectin concentrated on the surface of latex is preferred for interaction with the fibronectin receptor of macrophages. Gelatin, however, was not essential for this reaction, because fibronectin directly coupled to latex was also readily taken up.

Lymphocyte requirement for the functional development of follicle-associated epithelium.

Follicular development in the bursa of Fabricius was disrupted by testosterone treatment and chorioallantoic membrane grafting. Analysis by a quantitative histological technique demonstrated that testosterone treatment causes a dose dependent delay in development by inhibiting lymphoid proliferation. Inhibition of lymphoid development prevented the development of carbon transport ability by follicle associated epithelium. Reconstitution of follicular development by grafting treated bursae to the chorioallantoic membrane of untreated hosts results in the development of functional follicle associated epithelium. These results establish the lymphoid requirement for the development of transport ability by the follicle-associated epithelium.

The development of transport ability by embryonic follicle-associated epithelium.

The development of transport ability by follicle-associated epithelium was studied in embryonic chick bursae. Developing lymphoid follicles were divided into three morphological types to determine the relationship of epithelial function to lymphoid development. Transport ability was first observed on day 13, two days earlier than obvious morphological evidence of follicle-associated epithelial development. Functional differentiation of the epithelium was, however, closely associated with lymphoid development rather than a day-specific developmental event. It is probable that the lymphoid cells have an inductive influence on the development of the overlying epithelium. Evidence of early embryonic transport by relatively undifferentiated epithelium also suggests that lymphoid development in the bursa may be influenced by epithelial transport.

The influence of embryonic testosterone treatment on bursal epithelial pinocytotic activity.

Developing White Leghorn chicks were treated with testosterone propionate in ovo to effect a partial chemical bursectomy. After hatching, birds were administered carbon particles or horseradish peroxidase via the cloaca, and the transport of these tracers by the bursal epithelium was examined. Uptake of both tracers was inhibited in the treated when compared to untreated birds. The degree of development of interaction between bursal epithelium and underlying lymphoid tissue appeared to play a major role in determining ability of the epithelium to transport these tracers. Epithelium associated with follicles possessing medullary and cortical divisions was able to transport, while intraepithelial follicles or regions of the epithelium overlying diffuse lymphoid tissue did not transport either carbon or horseradish peroxidase. Bursal function in regional defense of the gut may depend on a local interaction of bursal epithelium with lymphoid, and perhaps other cell types.

Antibody production in organ culture by bursae of mature chickens and the development of immunological compentence.

Antibody responsiveness of bursal lymphocytes was studied in vitro. Organ culture of bursal tissue in the presence of antigen, either sheep erythrocytes or bovine serum albumin, results in significant numbers of plaque-forming cells (PFC) compared to controls. The response in organ culture is age-dependent in that only bursae from chickens at least three weeks of age contained significantly increased numbers of secreting cells. Prolonged culture of normally unresponsive bursae from newly hatched birds results in a PFC response to antigen, suggesting that in vitro maturation occurs.