Gregory T. Stelzer

Role of intracellular calcium in priming of human peripheral blood monocytes by bacterial lipopolysaccharide

To determine the role of intracellular calcium ([Ca2+]i) in the priming of monocytes (MΦ) by bacterial lipopolysaccharide (LPS), the membrane expression of two functional proteins and phagocytosis and respiratory burst were examined by microfluorimetry. LPS induced a significant increase in HLA-DR and C3bi receptor (CR3) expression within 2 h of its addition to whole blood. The enhanced expression of both antigens by LPS was dose-dependent, with concentrations as low as 0.1 ng/ ml producing a response. The involvement of [Ca2+]i was demonstrated by loading isolated MΦ with the intracellular calcium chelator quin-2 or the inhibitor of intracellular calcium redistribution TMB-8 prior to addition of LPS. Both compounds inhibited the LPS-induced increase in HLA-DR and CR3 expression. No role for extracellular calcium, for calcium slow channel flux, or for the calcium-calmodulin complex in LPS priming was demonstrated when LPS was added in the presence of EGTA, trifluperazine (TFP), or verapamil. The addition of the calcium ionophores A23187 or ionomycin failed to increase expression of either antigen. Prior exposure to LPS primed MΦ for enhanced phagocytosis and respiratory burst activity. These functions were inhibited by TMB-8, but not by TFP or verapamil. Addition of LPS to isolated MΦ increased [Ca2+]i by 23% at 30 sec and 42% at 5 min, as measured by the calcium-sensitive, intracellular probe indo-1. These results suggest that intracellular Ca2+ mobilization is necessary, but not sufficient, for LPS-induced priming of human peripheral blood monocytes.

Treatment of murine immune complex glomerulonephritis with prostaglandin E2: Dose-response of immune complex deposition, antibody synthesis, and glomerular damage

A model of immune complex glomerulonephritis (ICGN) produced in mice by the daily injection of apoferritin was employed to study the effect of treatment with various doses of prostaglandin E2 (PGE2) on glomerular damage, immune complex deposition, proteinuria, and serum anti-apoferritin antibody. Administration of PGE2, 200 micrograms twice daily, resulted in a significant decrease in glomerular damage and immune complex deposition, prevented the development of proteinuria, and significantly reduced serum levels of anti-apoferritin antibody. PGE2, 100 micrograms twice daily, resulted in a decrease in immune complex deposition as assessed by immunofluorescence microscopy, but this dosage did not significantly alter glomerular damage, proteinuria, or antibody levels. PGE2 dosages of 50 and 25 micrograms twice daily had no effect on any of these parameters. The protective effect of PGE2 on the development of ICGN occurred only at dosages that were associated with decreased anti-apoferritin antibody.

Regulation of oxygen radical release from murine peritoneal macrophages by pharmacologic doses of PGE2

The ability of pharmacologic doses of PGE2 to alter the release of superoxide (O2-) and hydrogen peroxide (H2O2) from elicited peritoneal macrophages (M theta) was studied. Twice-daily administration of 200 or 100 micrograms of PGE2 to mice during accumulation of peritoneal M theta resulted in a significant reduction in M theta recovery and in the triggered release of H2O2, but not O2-. Cultivation of elicited M theta from normal mice with concentrations of PGE2 in excess of 10(-7) M for 24-48 h resulted in a significant reduction in the triggered release of H2O2, but not O2-. Cultivation for shorter periods of time or with lower concentrations of PGE2 failed to alter H2O2 release. This effect of PGE2 was reproduced by the phosphodiesterase inhibitor theophylline. The ability of PGE2 to inhibit H2O2 release in the presence of normal production of O2- was not prevented by the addition of superoxide dismutase. Cultivation of peritoneal M theta with 10(-5) M PGE2 for 48 h failed to increase intracellular catalase, although increased H2O2 scavenger activity was demonstrated. The inhibition of extracellular release of H2O2, but not O2-, by pharmacologic doses of PGE2 may be one mechanism for the anti-inflammatory action of this compound.

Alterations in T lymphocyte subpopulations associated with renal allograft rejection

The peripheral blood OKT3 (total T), OKT4 (T helper/inducer), and OKT8 (T suppressor/cytotoxic) cells were determined by flow cytometry on twenty consecutive recipients of HLA-nonidentical cadaveric renal allografts. The absolute number of cells in all three populations decreased significantly posttransplantation, but no differences were found between patients experiencing rejection and those in quiescence. An OKT4/OKT8 ratio of greater than or equal to 1.7, either pretransplant or posttransplant, uniformly identified patients who subsequently experienced rejection. However, an OKT4/OKT8 ratio of less than 1.7 did not identify patients with a low risk of rejection. Pretransplant splenectomy was performed in 6 of 7 patients who rejected despite a low ratio. Serial monitoring of the OKT4/OKT8 ratio posttransplantation determined that an increase in the ratio of greater than or equal to 0.5 was a sensitive (81%) and specific (91%) indicator of a rejection episode. Graft survival was improved in patients with a high posttransplant OKT4/OKT8 ratio. These results indicate that the balance of helper and suppressor cell function may be of critical importance to the fate of an allograft, and that the alterations in this balance can be used to assist in the clinical management of allograft recipients.

Human histocompatibility antigen associations in patients with chronic cutaneous lupus erythematosus

Increased frequency of certain human leukocyte antigens (HLA) has been reported in various subsets of patients with lupus erythematosus (LE). Specifically, HLA-B8 and HLA-DR3 have been associated with subacute cutaneous LE; HLA-DR3 or HLA-DR2 is found in mothers of infants with neonatal LE; and HLA-A1, HLA-B8, HLA-B15, and HLA-DR2 occur in patients with systemic LE. We typed twenty-two white and nineteen black patients with chronic cutaneous (discoid) LE (DLE) for the A, B, and DR loci of the HLA antigens. HLA-DRw6 was found in an increased proportion of our patients of both races compared with controls. HLA-B8 was found more frequently in white DLE patients than in white control subjects. Thus a genetic predisposition may be a factor that explains the variation in disease expression.

Mechanism by which methylprednisolone inhibits acute immune complex-induced changes in vascular permeability

Intravital microscopy was used to quantitate protein leakage which resulted from the deposition of immune complexes in the vasculature of the rat cremaster muscle. Immune complex deposition was initiated by the addition of 80 μg/ ml of ovalbumin to the bath surrounding the muscle, followed by the intravenous administration of antiovalbumin. Administration of 25 mg/kg of antiovalbumin produced significant leakage of protein from the third-order venules, while 7.5 and 2.5 mg/kg had no effect. Administration of methylprednisolone (MP), 30 mg/kg, 1 h prior to the deposition of immune complexes significantly inhibited protein leakage. In separate experiments, MP inhibited intradermal edema formation and protein exudation induced in rats by histamine, platelet activating factor, or C5a. However. MP had no effect on protein exudation or edema produced by xanthine oxidase or glucose oxidase. Intravenous administration of MP inhibited the ability of polymorphonuclear leukocytes (PMNs) to phagocytize bacteria, but failed to alter hydrogen peroxide production. These results suggest that MP prevents acute changes in vascular permeability following immune complex deposition by inhibiting the effects of soluble mediators of edema on vascular endothelium and by inhibiting PMN phagocytosis.

Mechanism of prostaglandin E2 inhibition of acute changes in vascular permeability

In order to determine the mechanism of antiinflammatory activity, prostaglandin E2 (PGE2) or diluent was administered to rats 2 h prior to intradermal injections of various mediators of inflammatory vascular permeability changes. Vascular permeability was measured as the accumulation of [125I]rat serum albumin at the site of mediator injunction. PGE2 at 500μg significantly inhibited protein leakage produced by histamine, platelet activating factor, zymosan, and zymosan-activated plasma. Pretreatment with PGE2 had no effect on protein leakage induced by injection of lysosomal enzymes, glucose oxidase, or xanthine oxidase. The accumulation of polymorphonuclear leukocytes (PMNs) at the site of injection of zymosan or zymosan-activated plasma was not altered by PGE2 administration. In separate experiments, the ability of PGE2 to alter phagocytosis and oxygen radical production by PMN was examined. PGE2 significantly inhibited phagocytosis at 2 h, but this returned to normal by 6 h. Production of hydrogen peroxide by PMN was not affected by PGE2. These results suggest that PGE2 prevents acute changes in vascular protein leakage by preventing endothelial cell contraction and by inhibiting specific PMN functions.

Alterations in serum antibody and peripheral T-lymphocyte subsets resulting from treatment of murine immune complex glomerulonephritis with PGE2

The effects of treatment with 16,16-dimethyl prostaglandin E2 (DMPGE2) on histologic damage, glomerular immune complex deposition, serum total IgG subclass levels, anti-apoferritin IgG levels, and peripheral blood T-lymphocyte subsets were determined in apoferritin-induced immune complex glomerulonephritis of mice. The results demonstrate that doses of DMPGE2 ranging from 2.5 to 10 micrograms twice daily significantly reduced the degree of glomerular damage in a dose-dependent manner. Similarly, these doses of DMPGE2 reduced the amount of immunoglobulin deposition along peripheral capillary loops. Total IgM, IgG1, IgG2a, and IgG2b were unaffected by DMPGE2 administration. Serum anti-apoferritin IgG levels were significantly reduced in mice receiving DMPGE2 at doses of 5 and 10 micrograms twice daily. Nephrotic mice had significantly reduced peripheral blood total T lymphocytes (Lyt-1+) and a reduction of T-suppressor (Lyt-2+) cells. Administration of DMPGE2 at doses of 5 and 10 micrograms twice daily prevented these T-lymphocyte alterations. These studies indicate that treatment of mice receiving apoferritin with DMPGE2 may prevent glomerulonephritis by altering both cellular and humoral immune responses.

Characterization of Suppressor Cells in Mice with a Transplantable Malignant Melanoma

C57BL/6 mice with progressive B-16 melanoma develop a generalized immunosuppression, as measured by their lack of response to SRBC in vivo and in vitro. The severity of immunosuppression increases with the progress of the tumor, and is due to the generation of an antigen-nonspecific suppressor cell. Suppressor cells were first demonstrable 15 days after the appearance of the tumor, and their appearance correlated with the induction of immunosuppression in vivo. The suppressor cell generated by the growth of B-16 melanoma was a T lymphocyte since it was nonadherent to plastic and nylon wool, unaffected by passage through Sephadex G-10, and sensitive to treatment with rabbit antimouse brain serum and complement.

Cultivation and characterization of macrophages from murine embryonic skin. Are they Langerhans cells

Abstract We have observed a population of trypsin-resistant adherent cells in long-term primary cultures of murine embryonic skin. These cells were subsequently demonstrated to share a variety of characteristics with cells of the monocyte/macrophage lineage. The trypsin-resistant adherent cells stained positively for nonspecific esterase, exhibited surface receptors for Fc-IgG, and complement components as well as strong phagocytic activity. Additionally, these cells exhibited membrane ATPase enzyme activity and a large proportion of the cells expressed la antigens as detected by cytotoxicity and membrane fluorescence. The possible relationship between these trypsin-resistant adherent cells and Langerhans cells of the skin is discussed.