Gerald Weissmann

Tumor necrosis factor provokes superoxide anion generation from neutrophils

We report that tumor necrosis factor (TNF) provokes superoxide anion generation from human neutrophils. Superoxide anion generation was provoked at TNF concentration of 1 X 10(-11) M and maximal generation was attained at TNF concentration of 1 X 10(-9) M. We also show that movements of intracellular calcium may mediate the TNF-stimulated superoxide anion generation because 8-(diethylamino) octyl 3,4,5-trimethoxybenzoate hydrochloride--but not extracellular EGTA--inhibited the generation of superoxide anion. These results suggest that TNF may mediate some mechanisms of host defense by provoking superoxide anion generation from neutrophils.

Periodontal Disease and the Oral Microbiota in New-Onset Rheumatoid Arthritis

OBJECTIVE To profile the abundance and diversity of subgingival oral microbiota in patients with never-treated, new-onset rheumatoid arthritis (RA). METHODS Periodontal disease (PD) status, clinical activity, and sociodemographic factors were determined in patients with new-onset RA, patients with chronic RA, and healthy subjects. Multiplexed-454 pyrosequencing was used to compare the composition of subgingival microbiota and establish correlations between the presence/abundance of bacteria and disease phenotypes. Anti-Porphyromonas gingivalis antibody testing was performed to assess prior exposure to the bacterial pathogen P gingivalis. RESULTS The more advanced forms of periodontitis were already present at disease onset in patients with new-onset RA. The subgingival microbiota observed in patients with new-onset RA was distinct from that found in healthy controls. In most cases, however, these microbial differences could be attributed to the severity of PD and were not inherent to RA. The presence and abundance of P gingivalis were also directly associated with the severity of PD and were not unique to RA. The presence of P gingivalis was not correlated with anti-citrullinated protein antibody (ACPA) titers. Overall exposure to P gingivalis was similar between patients with new-onset RA and controls, observed in 78% of patients and 83% of controls. The presence and abundance of Anaeroglobus geminatus correlated with the presence of ACPAs/rheumatoid factor. Prevotella and Leptotrichia species were the only characteristic taxa observed in patients with new-onset RA irrespective of PD status. CONCLUSION Patients with new-onset RA exhibited a high prevalence of PD at disease onset, despite their young age and paucity of smoking history. The subgingival microbiota profile in patients with new-onset RA was similar to that in patients with chronic RA and healthy subjects whose PD was of comparable severity. Although colonization with P gingivalis correlated with the severity of PD, overall exposure to P gingivalis was similar among the groups. The role of A geminatus and Prevotella/Leptotrichia species in this process merits further study.

Leukotriene B4 is a complete secretagogue in human neutrophils: A kinetic analysis

The interactions have been studied of leukotriene B4 (LTB4) and 20-COOH-LTB4 with human neutrophils (PMN). Kinetic studies, utilizing continuous recording techniques, showed that LTB4 activates PMN with respect to aggregation, mobilization of membrane-associated Ca2+, −˙ generation, and degranulation within seconds of exposure. Dose-response studies indicate 1) that LTB4 is much more potent than its dicar☐ylic acid derivative (20-COOH-LTB4) or its all trans-isomer, and 2) that PMN responses to these agents are largely dependent upon pretreatment of the cells with cytochalasin B. These properties were similar to those of the microbial ionophores, ionomycin and A23187. Results demonstrate that LTB4 rapidly activates PMN and indicate that LTB4 serves as a complete secretagogue. Moreover, they provide additional evidence that oxidized fatty acids activate human PMN.

ACID PHOSPHATASE-RICH GRANULES IN HUMAN LYMPHOCYTES INDUCED BY PHYTOHEMAGGLUTININ.

Human lymphocytes, cultured in the presence of phytohemagglutinin, undergo morphologic transformation and subsequent mitosis. Before mitosis (48 to 72 hours), a sharp increase in acid phosphatase activity occurs in cells stimulated with phytohemagglutinin. Histochemical examination of these cells demonstrates that innumerable granules containing acid phosphatase develop in the cytoplasm before mitosis. It is possible that enzymes present in granules which stain for acid phosphatase activity (lysosome-like) may play a role in phytohemagglutinin-stimulated cell division.

Studies of lysosomes—VI: The effect of neutral steroids and bile acids on lysosomes in vitro☆

Abstract The effect of steroid hormones and bile acids on the release of hydrolases from lysosomes in granular fractions of rabbit liver and leukocytes has been studied in sucrose suspensions. At concentrations above 2.5 × 10 −4 M, steroids such as etiocholanolone, pregnanedione, pregnanolone, lithocholic acid, and progesterone were found to release β-glucuronidase and acid phosphatase activity from granules of rabbit liver. Metabolic precursors of etiocholanolone, such as testosterone, dehydroepiandrosterone, or 11-desoxycortisol, were active at 37° but not at 20°. Whereas these 5-β-H and Δ4,5 or Δ5,6 steroids released enzymes from the granules into the suspending medium, no 5-α-H isomer, such as androsterone, had comparable activity. Oxygenation or hydroxylation of carbon-11 of pregnanolone or etiocholanolone decreased the activity on the granules of the steroids. Pretreatment of liver granules with cortisol acetate, cortisone acetate, or chloroquine diminished the release of acid hydrolases by etiocholanolone or progesterone. Both etiocholanolone and progesterone reduced the apparent absorbance of suspensions of isolated leukocyte granules, and released β-glucuronidase from the particles, without physically disrupting the majority of granules. The data are compatible with the hypothesis that lysosomes are disrupted by pyrogenic steroids; the relationship of this action in vitro to their fever-provoking capacity in man has yet to be determined.

Serum Complement Values (C3 and C4) to Differentiate between Systemic Lupus Activity and Pre-Eclampsia

It is often difficult to differentiate between an exacerbation of systemic lupus erythematosus (SLE) and intercurrent pre-eclampsia in a patient with SLE since the manifestations of both entities include proteinuria and hypertension. This study was undertaken to determine wether serum C3 and C4 values can help distinguish SLE activity from pre-eclampsia. In 21 nonpregnant women of child-bearing age, the mean C3 level was 124 +/- 5 mg/dl and the mean C4 was 31 +/- 1 mg/dl. In 24 normal women in the third trimester of pregnancy, the C3 and C4 levels were elevated (165 +/- 4 mg/dl, p less than 0.001 versus nonpregnant control women; 37 +/- 2 mg/dl, p less than 0.01 versus nonpregnant control women, respectively). In 17 women in the third trimester of pregnancy with documented pre-eclampsia, the mean C3 level was 162 +/- 4 mg/dl, no different from that in normal pregnant women (p less than 0.001 versus nonpregnant control women; p = NS versus normal pregnant women), and the mean C4 was 29 +/- 3 mg/dl, lower than that found in normal pregnant women (p less than 0.02 versus normal pregnant women). Antinuclear antibody was absent at titers of less than 1:20 in all of these pre-eclamptic patients. In contrast, pregnant women with SLE has significantly lower C3 (103 +/- 13 mg/dl) and C4 (15.3 +/- 3.6 mg/dl) values during the third trimester of pregnancy than either normal pregnant women (p less than 0.001 for C3 and C4) or women with pre-eclampsia (p less than 0.001 for C3 and p less than 0.004 for C4) during the third trimester of pregnancy. Of the eight women with SLE in whom serial complement values were determined, three had falling C3 or C4 levels, and in each, there was a flare of SLE activity either during or immediately after pregnancy. None of the five patients with a rising C3 concentration had a flare of disease activity; however, pre-eclampsia developed in one of these patients, characterized by hypertension and proteinuria. Thus, measurement of serum C3 and C4 can help differentiate between SLE activity and pre-eclampsia, since both C3 and C4 are significantly lower in women with SLE than women with pre-eclampsia, and serum C3 and C4 concentrations rise during uncomplicated or pre-eclamptic pregnancy in women with SLE.

Nonsteroidal antiinflammatory agents inhibit stimulated neutrophil adhesion to endothelium: Adenosine dependent and independent mechanisms

All nonsteroidal antiinflammatory drugs (NSAIDs) inhibit neutrophil aggregation (homotypic cell-cell adhesion) and do so without affecting expression of CD11b/CD18. Since the first step in acute inflammation is a critical interaction between neutrophils and the vascular endothelium (heterotypic cell-cell adhesion), we determined whether NSAIDs diminish the adherence of neutrophils to the endothelium. At antiinflammatory concentrations (0.5–5 mM) sodium salicylate, an NSAID that does not inhibit prostaglandin synthesis, inhibited stimulated but not unstimulated neutrophil adherence to endothelial cells (IC50 < 1 mM,P < 0.00001). Salicylates have previously been shown to inhibit oxidative phosphorylation and, predictably, sodium salicylate inhibited oxidative phosphorylation, as evidenced by depletion of ATP stores (875±75 pmol/106 PMN, [2.92±0.25 mM]) in stimulated (FMLP, 0.1μM) but not resting neutrophils treated with antiinflammatory doses of sodium salicylate (EC50=1 mM,P < 0.00001). Indomethacin and piroxicam (10 and 30μM) only minimally decreased ATP concentrations in stimulated and resting neutrophils. ATP is metabolized to adenosine, and we have previously demonstrated that both endogenously released (180–200 nM) and exogenous adenosine (IC50=250 nM) inhibit stimulated neutrophil adherence to endothelial cells. To determine whether the increased metabolism of ATP and the resultant increase in adenosine release were responsible for inhibition of neutrophil adhesion to endothelium, we determined whether addition of adenosine deaminase (ADA, 0.125 IU/ml), an enzyme that converts extracellular adenosine to its inactive metabolite, inosine, affected inhibition of neutrophil adhesion to endothelium by stimulated neutrophils. ADA significantly reversed inhibition of neutrophil adherence to endothelium by sodium salicylate (0.5–5 mM,P < 0.00001). This suggests that sodium salicylate inhibits neutrophil adherence by increasing adenosine release. Whereas indomethacin and piroxicam (10–50μM) also inhibited stimulated neutrophil adherence to endothelial cells, ADA did not affect their inhibition of adherence. These studies demonstrate a heretofore unexpected antiinflammatory mechanism for salicylates: salicylates increase ATP hydrolysis and thereby enhance release of adenosine. Moreover, these data are consistent with the hypothesis that NSAIDs differ from one another with respect to their mechanisms of action.

Engagement of adenosine receptors inhibits hydrogen peroxide (H2O2−) release by activated human neutrophils☆

Adenosine and its analogs, acting at specific cell surface receptors, inhibit generation of superoxide anion by neutrophils. Since it has been suggested that hydrogen peroxide (H2O2) release may not be contingent upon superoxide anion release, we studied the effects of 2-chloroadenosine, a potent adenosine receptor agonist, on the formation of H2O2 by neutrophils exposed to various stimuli: n-formyl-methionyl-leucyl-phenylalanine (FMLP), concanavalin A, phorbol myristate acetate (PMA), serum-treated zymosan particles (STZ), and immune complexes. 2-Chloroadenosine (0.01-10 microM) inhibited formation of H2O2 by neutrophils exposed to FMLP, concanavalin A, and STZ particles. As we have found with O2- generation, 2-chloroadenosine failed to inhibit H2O2 release by neutrophils stimulated by either phorbol myristate acetate or immune complexes. The data show that whereas adenosine and its analogs inhibit neutrophil release of H2O2 and superoxide anion in response to most ligands, they fail to inhibit activation of neutrophils by immune complexes. Nor do they inhibit neutrophil activation by PMA, an agent which bypasses cell surface receptors by direct activation of protein kinase C. Surprisingly, we found that adenosine deaminase activity was adsorbed onto zymosan particles during opsonization and enhanced release of H2O2 by neutrophils exposed to STZ. These studies with yeast cell walls suggest that if microorganisms adsorb adenosine deaminase from serum, then the intracellular microbicidal activity of neutrophils is enhanced.

EFFECT OF DMSO ON THE STABILIZATION OF LYSOSOMES BY CORTISONE AND CHLOROQUINE IN VITRO

Previous reports from this laboratory have documented tha t cortisol and its analogues have the capacity to stabilize lysosomes in ~ i t r o . ’ ~ This direct effect of glucocorticoids upon the membranes of lysosomes, first suggested by deDuve et ~ l . , ~ is perhaps the explanation for the ability of cortisone to stabilize lysosomes of living cells against a wide variety of injurious agents, i.e., excess vitamin A, UV irradiation, endotoxins, streptolysin 0, CC14, excess oxygen, antigen-antibody reactions, e t ~ . ’ ~ ’ ~ The glucocorticoids share this property with chloroquine, another agent which is useful in inflammatory disease^.^ One major objection, however, to the hypothesis tha t glucocorticoids and chloroquine act as antiinflammatory agents by virtue of their stabilization of lysosomes has been tha t the concentrations of these agents which are required for in vitro action are far above the physiologic, or even pharmacologic, range: 2.5-7.5 x M.6 But steroids and chloroquine may act on the lipid membranes which bound lysosomes; therefore, their sparing solubility in aqueous media might account for the necessity to employ such artificially high concentrations. We studied the effect of DMSO upon the capacity of glucocorticoids and chloroquine to stabilize lysosomes in vitro, in order to determine whether DMSO might render these agents available to lipid membranes at lower concentrations.

Staphylococcal Alpha-Toxin: Effects on Artificial Lipid Spherules

Staphylococcal alpha-toxin induces the release of previously sequestered anions or glucose from artificial phospholipid spherules, an effect abolished by specific antitoxin. Alphatoxin resembles streptolysin S in releasing anions or glucose from spherules prepared without cholesterol, and can be distinguished from the membrane-active polyene amphotericin B, which preferentially disrupts spherules containing cholesterol. It may affect biological structures by a similiar interaction with membrane phospholipids.