Biomedical subjects

J A Spitzer

Publications and source records attributed to J A Spitzer.

At least 37 records · Page 2Linked to original sources

PubMed1994

Prohormone convertases PC2 and PC3 in rat neutrophils and macrophages. Parallel changes with proenkephalin-derived peptides induced by LPS in vivo.

Prohormone- or proneuropeptide-converting enzymes PC2 and PC3 have been observed exclusively in nervous and endocrine tissues. In this work the presence of these enzymes in cells of the immune system was demonstrated. PC2 was detected in peripheral and liver-infiltrating polymorphonuclear leukocytes (PMN) but not in alveolar macrophages (AM) or spleen mononuclear cells (SMC). PC2 proteins corresponded to 75, 71 and 56 kDa forms. PC3 appeared in AM and SMC but not in PMN, and a 66 kDa protein was the only PC3 form detected. Proenkephalin-derived peptides (PENKp) were observed in PMN and AM, showing peptides of 35, 28, 21, 18 and 14 kDa in the former cells and a doublet of 35 and 32 kDa in the latter. PC2 proteins and PENKp decreased in liver PMN and peripheral PMN 90 min after intravenous (i.v.) infusion of LPS, suggesting an increased release. However, in vitro assays showed that the chemotactic peptide FMLP but not LPS increased the basal secretion of PC2 proteins and PENKp in PMN. These results indicate that PC2 proteins are released from PMN, together with PENKp, and suggest that LPS in vivo may act through an indirect mechanism. Low levels of PC3 and PENK were detected in the AM of rats treated for 90 min with SAL or LPS. However, a significant increase of PC3 and PENKp appeared 30 h after LPS infusion. These results show for the first time that PC2 and PC3 are differentially expressed in PMN and AM, respectively, which were paralleled by the presence of different post-translational products of PENK. In addition, the in vivo effect of LPS on PC2, PC3 and PENKp levels in PMN and AM resembles the effect of LPS on prohormone levels in endocrine tissues, suggesting that similar mechanisms may control the turnover of PENK in endocrine and in these immune cells.

Animals↗

PubMed1994

Increased surface expression of CD11b/c and CD18 appears to be dissociated from anti-CD11b/c monoclonal antibody stimulated O2- anion generation in in vivo Escherichia coli lipopolysaccharide and tumor necrosis factor-alpha-treated rat neutrophils.

The purpose of this investigation was to determine the effect of anti-rat CD11b/c monoclonal antibody (MAb) on in vitro superoxide anion (O2-) generation in in vivo Escherichia coli lipopolysaccharide (LPS) and tumor necrosis-alpha (TNF)-treated rat polymorphonuclear leukocytes (PMN). After a continuous infusion of a nonlethal dose of E. coli LPS (.5 mg/kg) or TNF (6.0 x 10(5) units) into rats, PMN were recovered by centrifugal elutriation and discontinuous density gradient centrifugation from the liver (LPS-treated) and whole blood (LPS- and TNF-treated) and compared for CD11b/c, CD11a, and CD18 upregulation and their capacity for basal and agonist-stimulated O2- production. Immunofluorescence flow cytometry studies of rat whole blood PMN demonstrated that, upon LPS infusion, there was a time-dependent upregulation of CD11b/c and CD18, but not of CD11a. Similarly, TNF infusion upregulated CD11b/c although to a lesser degree. Stimulation of LPS- and TNF-treated PMN with phorbol 12-myristate 13-acetate (PMA), opsonized zymosan (OPZ), and anti-rat CD11b/c MAb triggered O2- generation. Although total O2- generated by OPZ and anti-rat CD11b/c MAb was less than that generated by PMA stimulation, the in vivo LPS- and TNF-induced beta 2 integrin upregulation did not result in a statistically significant enhancement of O2- generation with respect to normal saline-treated PMN. Our results do not appear to support the hypothesis that enhanced expression of CD11b/c or CD18 might be associated with enhanced in vitro anti-CD11b/c MAb-triggered O2- generation in LPS- and TNF-treated PMN in vivo.

Animals↗

PubMed1994

Generation of a CD11b/c upregulating and chemotactic factor by hepatocytes of endotoxic rats--nonidentity with interleukin-8.

To further clarify the mechanism of polymorphonuclear leukocyte (PMN) recruitment into the liver associated with short term endotoxin infusion (1), we investigated the effect of a noval factor generated by hepatocytes of such endotoxic rats on the expression of PMN adhesion molecules CD11b/c and chemotactic activity. Conditioned medium of hepatocytes from endotoxin-infused rats shows a fast induction and dose-dependent activity for upregulating CD11b/c expression in and chemotactic activity for blood PMN of naive rats. Supernatants of naive control rats cultured in the presence of endotoxin and Kupffer cells and liver PMNs of endotoxic rats also produce activation, but to a much lesser extent. The upregulating activity can be reduced significantly by heat inactivation at 100 degrees C for 10 min and by pronase hydrolysis at 37 degrees C for 60 min. Generation of the activity does not depend on cyclooxygenase products or phospholipase A2 activity, and it does not seem to be associated with the complement pathway. The activity is associated with molecular masses of 9-12 and 27-32 kDa and cannot be reduced by antiserum to rat interleukin-8 in serial dilutions ranging from 1:50 to 1:25,600. The results show that hepatocytes from acutely endotoxin infused rats generate a small molecular weight protein factor (or factors) that is capable of upregulating PMN 11b/c expression and chemotactic activity and is seemingly different from rat interleukin-8. Thus, hepatocytes in endotoxemia may play an important role in modulating neutrophil function and contributing to the mechanism of neutrophil sequestration into the liver.

Animals↗

PubMed1994

Hepatic neutrophil sequestration in early sepsis: enhanced expression of adhesion molecules and phagocytic activity.

Abdominal sepsis was produced by cecal ligation and puncture (CLP) in rats to observe neutrophil (PMN) migration into the liver and assess the functional alteration in circulating PMNs, liver sequestered PMNs, and Kupffer cells. 7 h following CLP, rats demonstrated severe leukopenia and major amount of PMNs sequestered into the liver (17.0 +/- 5.5 x 10(6) vs. 3.1 +/- 1.6 x 10(6) in sham-operated rats, p < .01). Light microscopic evidence demonstrated the presence of such PMNs in the sinusoids and in the liver parenchyma. By 20 h following CLP, the number of PMNs in the liver was not different from sham controls. CD11b/c expression on circulating PMNs was significantly upregulated from 1.6 +/- .3 to 7.8 +/- .9 mean channel fluorescence (MCF) in 7 h CLP rats. Liver-sequestered PMNs showed further enhancement of CD11b/c expression to 10.4 +/- 1.9 MCF than the circulating PMNs. However, in the late septic rats, CD11b/c expression on circulating PMNs 2.9 +/- .5 MCF returned to the control level of 1.9 +/- .7 MCF. Liver-sequestered PMNs and Kupffer cells in septic rats exhibited remarkably enhanced phagocytic activities 53.0 +/- 10.8 and 56.9 +/- 7.7% phagocytosis, respectively, regardless of the suppression of phagocytosis in circulating PMNs (16.0 +/- 5% phagocytosis). In 7 h CLP rats, liver-sequestered PMNs exhibited a significantly higher level of superoxide anion generation 21.8 +/- 12.2 nmol/30 min/10(6) cells than did Kupffer cells (4.2 +/- 3.0 nmol/30 min/10(6) cells). These results demonstrate that the liver is a target organ for neutrophil sequestration during the septic response.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Cytokine stimulation of nitric oxide formation and differential regulation in hepatocytes and nonparenchymal cells of endotoxemic rats.

Some disease processes in which increased endotoxin and cytokine levels exist (e.g., sepsis and infantile diarrhea) are also associated with increased levels of blood nitrates, the stable end products of nitric oxide. Available evidence suggests that the effects of an endotoxic environment, with its attendant complex cytokine networks, on liver function are mediated in part by modulation of hepatic nitric oxide synthesis. This hypothesis was tested by means of studying nitric oxide formation and its regulation in liver parenchymal and nonparenchymal cells of rats that had been continuously infused with endotoxin for 30 hr. Hepatocytes of such rats responded to in vitro stimulation for 20 hr by single cytokines, tumor necrosis factor, interleukin-1 beta and interferon-gamma with enhanced nitric oxide formation. In combination, interferon-gamma and endotoxin had greater synergistic effect on hepatocytes than did tumor necrosis factor and endotoxin. Kupffer cells of these endotoxic rats responded to 20 hr of interferon-gamma stimulation with the same enhanced nitric oxide formation we documented previously for endotoxin. Potentiation of the effect, through combination of endotoxin and interferon-gamma, was not as marked as it was with hepatocytes. Challenge of Kupffer cells with tumor necrosis factor or interleukin-1 beta evoked no response. Hepatocytes and Kupffer cells of time-matched, saline solution-treated rats were unresponsive to endotoxin or cytokine stimulation. Small quantities of nitric oxide were produced by endothelial cells spontaneously; this production was somewhat enhanced in cells of the endotoxin-infused rats by a 20-hr in vitro endotoxin challenge. Studies with inhibitors suggest that enhanced nitric oxide formation by endotoxic hepatocytes and Kupffer cells in response to in vitro endotoxin stimulation is differentially regulated. Our findings indicate modulation of nitric oxide generation by cytokines in vitro in various liver cell types of endotoxic rats. A similar scenario may exist in vivo because of the prevailing inflammatory response to endotoxin administration.

Animals↗

PubMed1994

Modulation of superoxide generation in in vivo lipopolysaccharide-primed Kupffer cells by staurosporine, okadaic acid, manoalide, arachidonic acid, genistein and sodium orthovanadate.

Continuous infusion of a nonlethal dose of Escherichia coli lipopolysaccharide (LPS) into rats induced extravasation of mononuclear phagocytes into the liver and the priming of Kupffer cells for in vitro phorbol myristate acetate (PMA)-stimulated superoxide anion (O2-) release. The purpose of this investigation was to determine the role of protein kinase C (PKC), protein serine-threonine phosphatase(s) 1 and 2a, protein tyrosine kinase(s) and phosphatase(s), phospholipase A2 (PLA2), arachidonic acid (AA) and its cyclooxygenase (CO) and 5-lipoxygenase (5-LO) metabolites in the modulation of PMA-stimulated O2-generation in in vivo LPS-primed rat Kupffer cells. The following inhibitors blocked PMA-stimulated O2- generation in the absence (-AA) or presence of AA (+AA) (50 microM): 1) staurosporine, a putative PKC inhibitor (150 nM, 95% inhibition without AA, 88% inhibition with AA); 2) okadaic acid, a protein serine-threonine phosphatase inhibitor (2 microM, 65% inhibition with or without AA); 3) the marine PLA2 inhibitor manoalide (1 microM, 97.5% inhibition without AA, 75% with AA). In addition, it was observed that exogenously added AA enhanced PMA-stimulated O2- generation in a time- and dose-dependent manner (5-50 microM) and partially reversed the inhibitory effect of manoalide. The following inhibitors did not block PMA-stimulated O2- generation in the absence or presence of AA: 1) indomethacin, a CO inhibitor (1-100 microM) and WY-50,295M tromethamine, a novel 5-LO inhibitor (1-100 microM); 2) genistein, a protein tyrosine kinase inhibitor (1-100 microM); and 3) sodium orthovanadate (1-300 microM), a protein tyrosine phosphatase inhibitor. It was concluded that, in in vivo LPS-primed Kupffer cells, PMA-stimulated O2- generation is modulated by PKC, protein serine-threonine phosphatase(s), PLA2 and AA but not by protein tyrosine kinase(s) and phosphatase(s) and CO and 5-LO products. These findings could have implications on the design of novel therapeutic approaches for the modulation of enhanced O2- release by Kupffer cells in endotoxemia.

Alkaloids↗

PubMed1993

Hepatic neutrophil influx: eicosanoid and superoxide formation in endotoxemia.

We examined early changes, at 0.5, 1.5, and 3 hr of infusion of a nonlethal dose of Escherichia coli endotoxin (ET) in neutrophil (PMN) sequestration in the liver and accompanying alterations in [1-14C]arachidonic acid metabolism and superoxide anion release by Kupffer and endothelial cells and PMNs. One hundred fifty micrograms ET (268 micrograms/kg) was infused over 3 hr. Shorter infusions delivered proportionally less ET. By 30 min of ET infusion the number of PMNs in the 45 ml/min fraction was 2.41 x 10(7), eightfold higher than that in the NaCl-infused rats, representing 35.90 +/- 3.49% (n = 7) of the total cell yield vs 8.20 +/- 0.20% (n = 4) in NaCl controls. By 90 and 180 min of ET infusion the number of PMNs and their share of the total cell yield increased significantly further. Cells were separated by elutriation, followed by application of a Ficoll-Hypaque gradient, when appropriate. Kupffer cells and PMNs were recovered in the 45 ml/min fraction, endothelial cells in the 23 ml/min fraction. At 30 min of ET infusion the profile of arachidonic acid metabolites released from [1-14C]arachidonic acid-prelabeled nonparenchymal cells upon A23187 stimulation was not different from that of cells of NaCl-infused rats. Infusion of ET for 90 min accentuated PMN infiltration, and resulted in significant modulation of the eicosanoid profile, consisting of a major shift in PGD2/PGE2 to PGE2, and LTB4 and 12-HETE accounting for 34% of the total eicosanoids, compared to 12.9% in time-matched NaCL controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Chronic endotoxemia reversibly alters respiratory burst activity of circulating neutrophils.

Endotoxin-induced sequestration of neutrophils and ectopic activation of the cytocidal respiratory burst are thought to contribute to the pathophysiology of "endotoxin shock." The effects of endotoxin on the circulating neutrophil population have not been described. A rat model of continuous sublethal endotoxin infusion by minipump was used to study spontaneous and stimulus-specific activation and priming of superoxide anion and chemiluminescence production of circulating neutrophils. At 3 hr of endotoxin infusion there are marked neutropenia, minimal spontaneous activation compared to an active response in the control, and no priming effect. By 30 hr of endotoxin infusion, a neutrophilic leukocytosis, a marked spontaneous activation of superoxide anion and chemiluminescence production are observed, with no priming response and a loss of zymosan-stimulated chemiluminescence. The biologic significance of endotoxin-induced spontaneous activation is unknown. By 78 hr of infusion the host tolerance to endotoxemia is also evident for neutrophils, since neutrophil responses are nearly identical in endotoxin- and saline-infused groups. The loss of zymosan-stimulated chemiluminescence in both groups at 78 hr suggests a foreign-body effect from the minipump and may reflect altered arachidonic acid metabolism.

Animals↗

PubMed1993

Endotoxin infusion primes elicited neutrophils and Kupffer cells for platelet-activating factor-induced and tripeptide formyl-methionine-leucine-phenylalanine-induced basal free intracellular calcium concentration responses.

OBJECTIVE: To determine whether in vivo infusions of bacterial endotoxin prime rat Kupffer cells and elicited neutrophils by altering the basal free intracellular calcium concentration or the response of free intracellular calcium to platelet-activating factor or the tripeptide formyl-methionine-leucine-phenylalanine (f-met-leu-phe). DESIGN: Prospective, randomized, controlled study. SETTING: Laboratory of a university medical school. SUBJECTS: Adult male Sprague-Dawley rats. INTERVENTIONS: Rats were infused with sterile saline or with Escherichia coli endotoxin for 3 or 30 hrs via a venous catheter or an osmotic minipump. Rats were anaesthetized, and the livers were perfused in situ with a collagenase-containing buffer. Kupffer cells and elicited neutrophils were isolated by centrifugal elutriation, and were partially purified over Ficoll gradients. MEASUREMENTS AND MAIN RESULTS: Basal free intracellular calcium concentrations were determined with the calcium indicator, fluo-3. Various concentrations of platelet-activating factor or f-met-leu-phe were then added, and any alterations in free intracellular calcium values were quantified. Neither acute (3-hr) nor chronic (30-hr) infusions of endotoxin altered basal free intracellular calcium values. F-met-leu-phe-induced increases in free intracellular calcium concentrations were positively correlated with the percentage of neutrophils in Kupffer cell fractions. Three-hour infusions of endotoxin, which caused a significant inclusion of neutrophils in the Kupffer cell fraction, resulted in an enhanced response to f-met-leu-phe. This response was considered to be primarily due to the presence of neutrophils, because preparations that were > 95% Kupffer cells did not respond to f-met-leu-phe. Platelet-activating factor-induced increases in free intracellular calcium concentrations were also positively correlated with the percentage of neutrophils. However, Kupffer cells isolated from rats infused for 30 hrs with endotoxin contained < 12% neutrophils, and exhibited a ten-fold greater response to platelet-activating factor than did Kupffer cells isolated from saline-infused rats. CONCLUSIONS: In vivo infusions of endotoxin result in an enhanced response to platelet-activating factor by rat Kupffer cells. F-met-leu-phe does not appear to induce an increase in free intracellular calcium values in rat Kupffer cells, but it appears to do so in endotoxin-elicited neutrophils. These observations indicate that changes in calcium homeostasis may be one mechanism by which endotoxin primes Kupffer cells and elicited neutrophils for enhanced production of superoxide anion and/or nitric oxide.

Animals↗

PubMed1993

LPS inhibits PI-phospholipase C but not PC-phospholipase D or phosphorylase activation by vasopressin and norepinephrine.

Rats were infused with endotoxin (50 micrograms/100 g body wt) for 3 h, and the parenchymal cells of the liver were maintained in primary culture for 1-3 h. The effects of vasopressin, norepinephrine, and glucagon on the activation of phosphatidylinositol (PI)-phospholipase C, phosphatidylcholine (PC)-phospholipase D, and glycogen phosphorylase a were investigated. Activation of PI-phospholipase C was markedly reduced, particularly with norepinephrine. This confirms that one of the early metabolic impairments seen in acute endotoxin treatment is inhibition of PI-phospholipase C activity. However, the ability of vasopressin, norepinephrine, and glucagon to stimulate glycogen phosphorylase a and PC-phospholipase D was not affected by this endotoxin treatment. We conclude that activation of phosphorylase a by vasopressin and norepinephrine is not entirely dependent on the activation of PI-phospholipase C and inositol trisphosphate formation.

Animals↗

PubMed1993

Shift from alpha- to beta-type adrenergic receptor-mediated responses in chronically endotoxemic rats.

Hepatocytes from chronically endotoxemic rats, or appropriate saline controls, were maintained in primary culture for 3 or 20 h. The ability of a variety of hormones to stimulate glycogen phosphorylase a was examined. At 3 h in culture, hepatocytes from endotoxemic rats had lower basal activities and exhibited impaired response to vasopressin, angiotensin II, and, to a lesser extent, norepinephrine and glucagon. The norepinephrine response was predominantly of the alpha-type in the saline rats but mixed alpha- and beta-type in the endotoxic cells. After 20 h in culture, vasopressin and angiotensin II responses were still impaired, while norepinephrine and glucagon responses were similar to those seen in the saline cells. The response to norepinephrine was predominantly of the beta-type in the endotoxic cells but still of the alpha-type in the saline cells. The results show that multiple mechanisms are involved in endotoxin-mediated inhibition of glycogen phosphorylase a activity and that alterations in intracellular calcium homeostasis play more of a significant role than adenosine 3',5'-cyclic monophosphate-mediated processes in diminished responsiveness of the liver seen in endotoxemia.

Angiotensin II↗

PubMed1993

Effect of in vivo TNF administration on superoxide production and PKC activity of rat alveolar macrophages.

After the intravenous injection of recombinant human tumor necrosis factor (TNF)-alpha (6.0 x 10(5) U) into rats, phorbol 12-myristate 13-acetate (PMA)-stimulated superoxide anion (O2-) secretion was enhanced in suspensions of alveolar macrophages (AM phi) compared with saline-treated controls. No enhancement of spontaneous, A23187-stimulated, or opsonized zymosan (OPZ)-stimulated O2- release was observed. Intratracheal injection of TNF-alpha (6.0 x 10(5) U) did not result in enhancement of spontaneous or A23187-, OPZ-, or PMA-stimulated O2- release. Although no TNF-alpha was detected in the bronchoalveolar lavage fluid, small quantities of TNF-alpha and/or other mediators secreted by polymorphonuclear leukocytes present in the lung capillaries, veins, and arteries may have leaked into the alveolar compartment and primed AM phi for enhanced PMA-stimulated O2- release. The respiratory burst in macrophages and neutrophils appears to be dependent on the translocation of protein kinase C. We have demonstrated protein kinase C translocation in both TNF-alpha- and saline-treated AM phi on PMA stimulation, although no differences were observed due to TNF-alpha treatment.

Animals↗

PubMed1993

Steroid hormones inhibit induction of spontaneous nitric oxide production in cultured hepatocytes without changes in arginase activity or urea production.

The spontaneous formation of nitrites was examined in the medium of cultured rat hepatocytes and taken as a measure of nitric oxide generation. The rate of nitrite formation increased after 8-12 hr in culture which was blocked by the addition of dexamethasone, actinomycin D, or cycloheximide. Various glucocorticoids, mineralocorticoids, and sex steroids also inhibited nitrite formation by varying degrees, without affecting arginase activity or urea production. The inhibition of nitric oxide formation appears, therefore, not to be due to changes in the availability of arginine. The results suggest that nitric-oxide synthase is induced in hepatocytes in culture and show that anti-inflammatory glucocorticoids are not the only steroids that inhibit nitric oxide formation.

Animals↗

PubMed1993

Modulation of superoxide anion generation by manoalide, arachidonic acid and staurosporine in liver infiltrated neutrophils in a rat model of endotoxemia.

Early accumulation of polymorphonuclear leukocytes (PMN) in the liver after in vivo exposure to Escherichia coli lipopolysaccharide (LPS) and concomitant in vitro phorbol myristate acetate (PMA)-stimulated superoxide anion (O2-) generation has recently been described in a rat model of endotoxemia. The purpose of this investigation was to study the role of phospholipase A2 (PLA2), arachidonic acid (AA), its metabolites, and protein kinase C (PKC) in the mechanism of PMA-stimulated O2- generation of liver infiltrated PMN as compared to circulating blood PMN. Rat PMN were isolated after a 1.5-h infusion of saline or LPS from the blood (SAL-PMN) or the liver (LPS-PMN), respectively. The following results were observed in both SAL-PMN and LPS-PMN: 1) Inhibitors of cyclooxygenase (indomethacin) and 5-lipoxygenase [eicosatetraynoic acid, WY 50,295 tromethamine and VZ 65, 4-(11-hydroxy-1,9-undecadiin)-brenzcatechin] pathways did not inhibit O2- generation; 2) the potent marine PLA2 inhibitor Manoalide inhibited O2- generation in a dose-dependent manner (IC50 = 0.5 microM); 3) exogenously added AA enhanced PMA-stimulated O2- generation in a time- and dose-dependent manner and partially reversed the effect of Manoalide in LPS-PMN; 4) staurosporine, a putative PKC inhibitor, blocked PMA-stimulated O2- generation completely in the absence of AA and 79% in the presence of AA. It was concluded that LPS-induced liver sequestration of PMN does not alter the role PLA2, AA and PKC play in PMA-stimulated O2- generation. These findings should have implications on the design of novel therapeutic approaches for the modulation of O2- release in the pathogenesis of LPS hepatotoxicity.

Alkaloids↗

PubMed1992

Endotoxin and TNF alpha directly stimulate nitric oxide formation in cultured rat hepatocytes from chronically endotoxemic rats.

We examined the effects of endotoxin on nitric oxide formation in isolated rat hepatocytes in primary culture. Endotoxin was administered either in vivo, by continuous infusion for 30 or 3 h, or in vitro, on cultured cells. The spontaneous production of nitrites in hepatocytes from in vivo ET-infused rats was lower than equivalent saline controls in the absence of added stimuli. However in vitro addition of endotoxin in culture to hepatocytes from 30 h ET-infused rats greatly enhanced production relative to saline controls. This effect was mimicked by TNF alpha, and activators of protein kinase C (PMA and Ca2+ ionophore A23187). The effects of ET were blocked by NMMA, dexamethasone and protein synthesis inhibitors Actinomycin D and cycloheximide. No in vitro effect of ET was observed in the 3 h infusion model. The results show that chronic exposure to sub-lethal levels of ET primes liver parenchymal cells for the production of nitric oxide, when exposed in vitro to ET or TNF alpha.

Animals↗

PubMed1992

Splenocyte blastogenesis suppressed in rats implanted with an osmotic pump.

Rats implanted subcutaneously with an empty osmotic pump connected by a polyethylene catheter to a jugular vein for 5 to 10 days evinced a decreased splenocyte responsiveness to blastogenic stimulation in vitro to bacterial lipopolysaccharide, a known B cell stimulator, as well as to the plant mitogens pokeweed mitogen (PWM), a known stimulator of T and B cells, and Concanavilan A, a known T cell stimulator. The surface of the implanted pumps became infiltrated with lymphoid cells, especially macrophages. Suppression of blastogenic responsiveness after implantation for 10 days with an empty pump or even a pump dispensing pyrogen free saline only in a continuous manner was nearly as marked as that which occurred at 2-5 days after continuous infusion with endotoxin. These depressed blastogenic responses, although less, were also evident when rats were implanted with a catheter into a jugular vein connected by means of a swivel to either an empty pump or one dispensing pyrogen free saline. Suppression of blastogenic responsiveness was not related to alteration in serum complement or corticosteroid levels. Since administration of immunomodulatory substances systemically to individuals often involves implantation of an osmotic pump, investigation into the mechanisms of lymphoid cell suppression associated with an implanted pump itself has potential significance.

Animals↗

PubMed1992

The effect of endotoxemia on concanavalin A induced alterations in cytoplasmic free calcium in rat spleen cells as determined with Fluo-3.

The effects of acute (3 h) and chronic (30 h) in vivo infusions of Escherichia coli endotoxin on the Ca2+ homeostasis of rat spleen cells was investigated. Conditions were established for obtaining reliable estimates of [Ca2+]i in these cells using the newly-developed Ca2+ indicator Fluo-3. The resting [Ca2+]i of splenocytes and T lymphocyte-enriched preparations were 119 +/- 35 and 102 +/- 31 nM, respectively. Treatment of the cells with concanavalin A (Con A) resulted in a rapid increase in [Ca2+]i. The magnitude of the increase was positively correlated with the concentration of Con A, whereas the time required to reach the maximum [Ca2+]i was inversely related to the amount of Con A. The peak [Ca2+]i was attained more rapidly in splenocytes (i.e. less than or equal to 30 s) than in the T cell-enriched fraction (i.e. 1.5-2.0 min). Both the resting [Ca2+]i and the Con A-induced increase in [Ca2+]i were similar to values previously reported for other lymphocyte cell types using different Ca2+ indicators, thereby supporting the values obtained with Fluo-3. Infusions of saline or endotoxin prior to the isolation of the cells did not result in significant alterations of either resting [Ca2+]i or the cells' response to Con A. Since chronic infusions of endotoxin have previously been shown to cause a reduction in blastogenic responsiveness of splenocytes to Con A, these data suggest that the endotoxin-induced lesion occurs distal to the mobilization of intracellular Ca2+.

Aniline Compounds↗

PubMed1992

Continuous infusion of endotoxin depresses splenic blastogenesis.

Continuous infusion of a sublethal dose of bacterial endotoxin into rats via an implanted osmotic pump markedly affected the blastogenic responsiveness of spleen cells to specific endotoxin as well as to the nonspecific mitogens Con A, PHA or PWM. There was also a marked alteration in lymphoid cell type and number in the spleen of the rats after continuous infusion of endotoxin, with a marked increase in plasma cell infiltration and germinal center formation. There was no significant alteration in glucocorticoid steroid levels. Control rats given saline only for a period of seven days via an implanted pump showed no or minimal effect for the first 5 days and then a delayed depression of blastogenic responses to LPS and to Con A, but this time lag contrasted markedly to the much earlier unresponsiveness of splenocytes from rats infused with endotoxin. Only a slight to moderate cellular infiltration occurred in the spleen of control rats implanted with a pump infusing saline only or an empty pump. Thus endotoxin infusion in a continuous manner via an implanted pump accounted for the early and marked suppression of responsiveness, as well as alteration in spleen size and cellularity.

Animals↗