Molecular weight and charge heterogeneity of Thy-1 glycoprotein in different populations of T-cells.

Molecular weight and charge microheterogeneity of Thy-1 glycoprotein expressed on different T-cell populations were compared by two-dimensional gel electrophoresis and endoglycosidase treatment. Thy-1 was immunoprecipitated from detergent lysates of radioiodinated T-cells of spleens, lymph nodes, Peyer's patches, peripheral blood, IL-2-cultured thymocytes and peanut agglutinin (PNA) separated thymocytes. In general, thymocytes and IL-2-cultured thymocytes expressed the highest levels of Thy-1 with a large Mr and charge variation. The Thy-1 of these cells was basic and contained low levels of sialic acid. On the other hand, peripheral T-lymphocytes exhibited a restricted Mr and more limited charge heterogeneity with higher amounts of sialic acid. The Thy-1 glycoprotein of mature, low Thy-1 PNA- thymocytes had Mr and charge heterogeneity identical to peripheral T-lymphocytes. The Mr and charge variation of immature PNA+ thymocytes was essentially identical to that of whole thymocytes. The molecular source of Mr heterogeneity in thymocyte Thy-1 and restricted Mr in lymph node Thy-1 was studied by analysis with endoglycosidase-H (Endo-H) and Endo-D. The results of Endo-H digestion showed that most of the Thy-1 polypeptides from both thymocyte and lymph node contained one high-mannose type oligosaccharide which was relatively small and not heterogeneous with respect to Mr. The complex-type oligosaccharides (Endo-D-sensitive) were larger and were responsible for the broad Mr-heterogeneity found in thymocyte Thy-1. Both thymocyte and lymph node Thy-1 generally appear to contain three N-linked oligosaccharides (one high-mannose and two complex) and sequential hydrolysis with Endo-H and Endo-D resulted in an unglycosylated polypeptide with an Mr of approx. 12,500.

Poly-N-acetyllactosamine alterations of Thy-1 glycoprotein in lymphocyte differentiation.

Developmental changes in murine lymphocyte Thy-1 include both quantitative and qualitative alterations involving N-linked oligosaccharides. Comparison of immature with mature T-cells has shown that the oligosaccharides of Thy-1 are characterized by an increase in the number of sialic acid residues responsible for the acidic pI of peripheral T-cell Thy-1, and a decrease in those oligosaccharides responsible for Mr heterogeneity of thymocyte Thy-1. The research reported here suggests that the basis of the large Mr heterogeneity in Thy-1 of immature T-cells is the presence of repeating N-acetyllactosamine (R'Gal beta 1,4GlcNAc beta 1,3R") units in the oligosaccharide portion of the molecule. Lymphocytes were surface iodinated and 125I-thy-1 was purified by immunoprecipitation and preparative nonequilibrium pH gradient electrophoresis. The minimal Mr of unglycosylated Thy-1 after endoglycosidase F digestion was 15,000-16,000. Digestion of Thy-1 with endo-beta-galactosidase suggested that the complex type N-linked glycans in thymocytes, but not in lymph node T-cells, contained increased levels of polylactosamine. The presence of polylactosamine was confirmed by binding to a Datura stramonium lectin column which retarded and bound approx. 50% of thymocyte Thy-1 and only about 18% of lymph node T-cell Thy-1. Affinity chromatography using anti-i antibody immobilized on agarose beads indicated that the polylactosamine is probably present in a predominantly linear form. Since alterations of polylactosamine structures have been implicated in development and transformation in several systems, the present results suggest an important role for these glycans in immune-cell differentiation.

Glucuronic acid conjugation by hepatic microsomal fractions isolated from streptozotocin-induced diabetic rats.

The hepatic glucuronidation of 1-naphthol and 4-nitrophenol (3-methylcholanthrene inducible substrates of glucuronyltransferase, GT 1) was found to be deficient in freshly prepared untreated "native" microsomes from streptozotocin-induced diabetic male rats. The defect was not observed in female rats. Moreover, the glucuronidation of 1-naphthol and 4-nitrophenol was higher in "native" microsomes from male control rats than in those from female controls. This sex difference in the glucuronidation of the GT 1 substrates was abolished by detergent activation of the transferase enzyme in vitro. Streptozotocin treatment did not alter the glucuronidation of paracetamol or phenolphthalein (phenobarbitone inducible substrates for GT 2). This diabetes-induced defect in the glucuronidation of GT 1 substrates was abolished by insulin treatment of the animals and was diminished or completely abolished by detergent activation of the transferase enzyme in vitro. Increased membrane constraint is proposed as the mechanism responsible for the transferase defect. 3-Methylcholanthrene induction abolished the streptozotocin-induced defect in 4-nitrophenol glucuronidation, whereas phenobarbitone did not. This is attributed to the differential effect of these inducers on the microsomal membrane.

Differential alterations in cyclic nucleotide levels in Kupffer cells and hepatocytes following trauma-hemorrhage and resuscitation.

Although cyclic nucleotides play an important role in regulating the control of metabolism, it is not known whether there are any differential alterations in cyclic nucleotides in Kupffer cells and hepatocytes after trauma-hemorrhage and resuscitation. To study this, rats underwent laparotomy (i.e., trauma-induced) and were rapidly bled to and maintained at a mean arterial pressure of 40 mmHg until 40% of maximum bleedout volume was returned in the form of Ringer's lactate. The animals were then resuscitated with Ringer's lactate, equivalent to four times the volume of shed blood. At the time of maximum bleedout or at 1.5 h postresuscitation, a portion of the liver was removed, and the levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were determined by radioimmunoassay. Moreover, Kupffer cells and hepatocytes were isolated in additional groups of animals and cAMP and cGMP levels were measured. The results indicate that hepatic cAMP decreased, whereas hepatic cGMP increased significantly at the time of maximum bleedout. Although resuscitation normalized hepatic cyclic nucleotide levels, the levels of cAMP and cGMP in Kupffer cells increased significantly at 1.5 h after resuscitation. In contrast, cAMP and cGMP levels in hepatocytes were not significantly different from shams under such conditions. Thus, differential alterations in cyclic nucleotide levels in different liver cell populations occur following trauma-hemorrhage and resuscitation.

Aggressive fluid resuscitation following intestinal ischemia-reperfusion in immature rats prevents metabolic derangements and down regulates interleukin-6 release.

We have recently shown improved survival following intestinal ischemia-reperfusion in a model that utilized aggressive crystalloid resuscitation sufficient to eliminate reperfusion-induced cardiovascular instability. The aims of this study were to determine whether the salutary effects associated with this regimen were due to: 1) prevention of systemic metabolic derangements; 2) attenuation of secondary organ injury; or 3) modulation of the systemic immune response. Under anesthesia, 4-week-old (65-85 g) male Sprague-Dawley rats (N = 63) received crystalloids at either 15 or 65 ml.kg-1.h-1 intravenously and were subjected to 90 min of superior mesenteric artery occlusion followed by 90 min of reperfusion (IR15, IR65) or time-matched sham (SH) operation (SH15, SH65). Results indicate that inadequate fluid resuscitation following intestinal IR was associated with significant serum hyperkalemia and hyperphosphatemia, acute renal insufficiency, and enhanced serum interleukin-6 levels. Maintenance of cardiovascular stability with aggressive fluid resuscitation was associated with an attenuation of these alterations. Therefore, the prevention of circulatory shock and the attenuation of distant organ injury and inflammatory response are associated with improved survival when an aggressive crystalloid resuscitation regimen is applied after intestinal ischemiareperfusion in immature rats.

Role of Kupffer cells in interleukin-6 release following trauma-hemorrhage and resuscitation.

Although interleukin-6 (IL-6) plays an important role in the pathophysiology of trauma-hemorrhage and resuscitation, the cellular origin of this inflammatory cytokine remains unknown. This study was undertaken to determine whether Kupffer cells (KC) are a major source of IL-6 release following trauma-hemorrhage and resuscitation. KC numbers were significantly (p < .05) reduced in vivo with gadolinium chloride (GdCl3; 10 mg/kg IV). KC-reduced (KC(-)) and KC-normal (saline-treated; KC(+)) rats underwent laparotomy (i.e., trauma-induced), followed by either sham operation or hemorrhage. Hemorrhaged rats were bled to and maintained at a mean arterial pressure of 40 mmHg until 40% of the shed blood volume was returned as Ringer's lactate, and then resuscitated with Ringer's lactate (four times shed blood volume over 1 h). Results indicate that KC reduction per se had no effect on any measured parameter at any time. At 0.5 and 2.0 h postresuscitation, mean arterial pressure, heart rate, cardiac output, stroke volume, and hematocrit were reduced to a similar extent in both the KC(+) and KC(-) hemorrhage groups. KC reduction did, however, significantly reduce plasma IL-6 concentration (means +/- S.E.; U/ml) at both 0.5 h (KC(+) = 709 +/- 391 vs. KC(-) = 159 +/- 5) and at 2.0 h (KC(+) = 527 +/- 394 vs. KC(-) = 83 +/- 20) postresuscitation. In conclusion, this study demonstrates that KC are a major source of in vivo IL-6 release following trauma-hemorrhage and resuscitation.

Insights into the mechanisms of defective antigen presentation after hemorrhage.

Although hemorrhage depresses macrophage antigen presentation (AP), a critical component in eliciting an antigen-specific immune response, it is not known which particular step in macrophage AP (i.e., uptake, ingestion, catabolism, or presentation of degraded antigens to T cells) is defective. To study this, C3H/HeN mice were bled to an arterial mean blood pressure of 35 mm Hg, maintained for 60 minutes, and then adequately resuscitated. Peritoneal and splenic macrophage cultures were prepared 2 and 24 hours after hemorrhage. Macrophage AP capacity was measured by coculturing macrophages with the T-helper cell clone D10.G4.1. To gain information about macrophage ability to digest the specific antigen conalbumin, lysosomal activity was bypassed by use of chemically denatured conalbumin peptides. To study macrophage ability to present conalbumin peptides, macrophages were fixed and D10.G4.1 proliferation in response to fragmented conalbumin was determined. AP of native conalbumin by peritoneal macrophages and splenic macrophages was depressed (p less than 0.05) by 50% (peritoneal macrophages) and 55% (splenic macrophages) 2 hours and 57% (peritoneal macrophages) and 35% (splenic macrophages) 24 hours after hemorrhage. In contrast, presentation of conalbumin peptides was only slightly decreased. In addition, the ability of fixed peritoneal macrophages and splenic macrophages to present conalbumin peptides was similar in hemorrhaged and sham mice. Because bypassing of macrophage lysosomal activity with degraded native antigens prevented the suppression of AP, the results suggest that hemorrhage-induced suppression of AP is not caused by a reduced macrophage capacity to present antigenic peptides but by decreased antigen catabolism by macrophages.

Immunoprotective effect of a calcium channel blocker on macrophage antigen presentation function, major histocompatability class II antigen expression, and interleukin-1 synthesis after hemorrhage.

Hemorrhage induces a severe suppression of the immune system resulting in increased susceptibility to sepsis. Although studies indicate beneficial effects of calcium channel blockers on cell and organ functions after low-flow conditions, it remains unknown whether such agents have any effects on different immune responses after hemorrhage. To study this, C3H/HeN mice were bled to a mean blood pressure of 35 mm Hg and were maintained for 60 minutes, followed by resuscitation with their own shed blood and adequate fluid. The mice received either the water-soluble calcium channel blocker diltiazem (400 or 2400 micrograms/kg body weight) or saline solution (vehicle). Peritoneal macrophages were obtained by lavage 24 hours later. Antigen presentation was measured by coculturing peritoneal macrophages with the D10.G4.1 helper T-lymphocyte clone. Immune associated antigen (Ia) expression was determined by direct immunofluorescence. Interleukin (IL)-1, 6, and tumor necrosis factor-alpha (TNF) levels in peritoneal macrophage supernatants were measured by use of cytokine-specific cellular assays. Hemorrhage caused a significant decrease in peritoneal macrophage antigen presentation function, Ia expression, and IL-1 and IL-6 synthesis in the vehicle-treated group, whereas TNF levels were increased. However, both doses of diltiazem significantly improved peritoneal macrophage antigen presentation, Ia expression, and IL-1 synthesis. IL-6 synthesis was only increased with high doses of diltiazem, whereas both diltiazem doses decreased TNF production. These results indicate that the calcium channel blocker diltiazem can markedly improve macrophage functions after hemorrhage. The use of diltiazem might offer a new therapeutic modality in the treatment of immunosuppression and in decreasing the susceptibility to sepsis after hemorrhagic shock.

Mechanism of the beneficial effects of pentoxifylline on hepatocellular function after trauma hemorrhage and resuscitation.

BACKGROUND: The purpose of this study was to determine whether pentoxifylline administration restores the depressed hepatocellular function after trauma hemorrhage and crystalloid resuscitation and, if so, whether this is the result of the down-regulation of inflammatory cytokines, tumor necrosis factor (TNF) and interleukin-6 (IL-6). METHODS: After laparotomy rats were bled to and maintained at a mean arterial pressure of 40 mm Hg until 40% of maximum shed blood volume was returned in the form of Ringer's lactate. They were then resuscitated with Ringer's lactate to four times the shed blood volume. Pentoxifylline (50 mg/kg body weight) or saline solution was infused intravenously for 95 minutes during and after resuscitation. One and a half hours and 4 hours after resuscitation, hepatocellular function (maximal velocity [Vmax] and the efficiency of the active transport [Km] of indocyanine green clearance) and plasma. TNF and IL-6 levels were determined with in vivo hemoreflectometer and cellular assays, respectively. RESULTS: Circulating TNF and IL-6 levels increased significantly after hemorrhage and resuscitation. Pentoxifylline treatment, however, markedly decreased the levels of these cytokines, and the values were similar to those of sham rats. The decreased Vmax and Km values were also restored by pentoxifylline treatment. Moreover, there was a significant correlation between Vmax and TNF or IL-6 levels. CONCLUSIONS: The down-regulation of inflammatory cytokines by pentoxifylline may be the mechanism by which this agent restores the depressed hepatocellular function after trauma hemorrhage and resuscitation.

Interferon-gamma attenuates hemorrhage-induced suppression of macrophage and splenocyte functions and decreases susceptibility to sepsis.

Although it is known that interferon-gamma synthesis and macrophage functions are depressed after hemorrhage, it remains to be determined whether systemic administration of interferon-gamma has any effect on hemorrhage-induced depression of macrophage and splenocyte functions. To study this, C3H/HEN mice were bled to a mean blood pressure of 35 mm Hg, maintained for 60 minutes, and followed by adequate fluid resuscitation. The mice then received either 1000 units interferon-gamma or saline solution (vehicle). Peritoneal (pM phi) and splenic (sM phi) macrophages and splenocytes were isolated 24 hours later. PM phi antigen presentation was measured by coculturing pM phi with the D10.G4.1 cell clone. Major histocompatibility complex class II (Ia) antigen expression was determined by direct immunofluorescence. Cytokine release by pM phi, sM phi, and splenocytes was assessed with specific bioassays. For survival studies, mice were subjected to sepsis 3 days after hemorrhage. Treatment with interferon-gamma restored (p less than or equal to 0.05) hemorrhage-induced suppression of pM phi antigen presentation capacity and Ia antigen expression and increased (p less than or equal to 0.05) interleukin-1 and tumor necrosis factor release by pM phi and sM phi, as well as splenocyte proliferation (p less than or equal to 0.05). Interferon-gamma also decreased (p less than or equal to 0.007) the susceptibility to sepsis after hemorrhage. Thus interferon-gamma represents a potent agent for treating hemorrhagic shock-induced immunosuppression and for increasing the ability of the host defense system to combat bacterial infections after hemorrhage.

Modulation of macrophage membrane phospholipids by n-3 polyunsaturated fatty acids increases interleukin 1 release and prevents suppression of cellular immunity following hemorrhagic shock.

Studies have suggested that the significant suppression of cellular immunity following hemorrhage may be due to an increased release of prostaglandin E2 (PGE2) by macrophages. Since diets high in n-3 polyunsaturated fatty acids decrease PGE2 synthesis, we assessed whether hemorrhage-induced immunosuppression could be prevented by dietary manipulation. C3H/HeN mice were fed for 3 weeks with fat sources derived from corn oil, safflower oil, or fish oil, then bled to a mean blood pressure of 35 mm Hg maintained for 60 minutes. Following this, the animals were adequately resuscitated with fluids and killed 24 hours later. In the corn oil and safflower oil groups, hemorrhage resulted in a significant increase in PGE2 release by peritoneal macrophages, a marked suppression of peritoneal macrophage antigen presentation capacity, interleukin 1 release, splenocyte proliferation, and interleukin 2 secretion compared with shams. However, feeding mice with fish oil for 3 weeks prior to hemorrhage prevented the rise in PGE2 release and maintained normal macrophage and splenocyte functions following hemorrhage. Thus, the elevated release of PGE2 by peritoneal macrophages plays a pivotal role in hemorrhage-induced immunosuppression. Moreover, diets high in n-3 polyunsaturated fatty acids may offer a new therapeutic approach for preventing posthemorrhage immunosuppression and increased mortality from sepsis.

Chloroquine attenuates hemorrhagic shock-induced immunosuppression and decreases susceptibility to sepsis.

Hemorrhagic shock causes a severe suppression of cellular immunity and an increased susceptibility to sepsis that may be due to increased release of prostaglandin E2 by macrophages. Since chloroquine inhibits the secretion of prostaglandin E2 by macrophages in vitro, the effects of chloroquine administration in vivo following hemorrhagic shock on macrophage prostaglandin E2 secretion and on depressed cellular immunity were examined. Inbred C3H/HeN male mice, aged 6 to 8 weeks, were bled to a mean blood pressure of 35 mm Hg, which was maintained for 60 minutes, and adequately, resuscitated. Mice then received intramuscular injections of either saline (vehicle) or chloroquine (10 mg/kg of body weight). Prostaglandin E2 in macrophage supernatants (radioimmunoassay) concanavalin A-dependent splenocyte proliferation, and interleukin 2 in splenocyte supernatants (CTLL 20 interleukin 2-dependent proliferation) were determined 2 or 24 hours later. Hemorrhage caused a significant decrease of splenocyte proliferation (47%) and interleukin 2 release (49%) at 24 hours, while prostaglandin E2 secretion from macrophages was elevated at 2 hours. Chloroquine treatment attenuated depression of splenocyte functions and reduced prostaglandin E2 release. Furthermore, chloroquine treatment decreased the mortality of septic mice after hemorrhage to levels comparable with those of sham-operated mice. Thus, chloroquine may be a useful adjunct in the clinical setting for the treatment of shock-induced immunodepression and increased susceptibility to sepsis following hemorrhage.

Role of interleukin 6 and transforming growth factor-beta in the induction of depressed splenocyte responses following sepsis.

We examined whether (1) there is an association between elevated circulating levels of transforming growth factor-beta (TGF-beta) and splenocyte dysfunction during sepsis, and (2) administration of monoclonal antibodies to interleukin 6 (an inducer of TGF-beta release) or TGF-beta could ablate these changes. Blood and splenocytes were obtained from C3H/HeN mice at 1, 4, or 24 hours following cecal ligation and puncture or sham operation. Only at 24 hours after cecal ligation and puncture was there an association between elevated blood TGF-beta value and depressed splenocyte interleukin 2 release. Administration of monoclonal antibodies against interleukin 6, but not against TGF-beta (intraperitoneally immediately following cecal ligation and puncture), significantly decreased the blood levels of TGF-beta at 24 hours following cecal ligation and puncture and improved splenocyte interleukin 2 release. Thus, the judicious use of monoclonal antibodies against interleukin 6 may block the subsequent elevation of TGF-beta, thereby attenuating host immunosuppression during sepsis.

Glutathione status in primary cultures of rat hepatocytes and its role in cell attachment to collagen.

In primary cultures of rat hepatocytes the intracellular level of reduced glutathione (GSH) declines to approx. 50% of that observed in freshly isolated cells within 1 h of culture. Pretreatment of freshly isolated hepatocytes with diethylmaleate (DEM) to deplete GSH and inhibition of glutathione synthesis by buthionine sulfoximine (BSO) markedly decrease the proportion of cells attaching to the collagen coated culture dishes. A positive correlation between the intracellular content of GSH and the ability of hepatocytes to attach to collagen is observed. Presence of dithiothreitol (DTT) in the culture medium efficiently prevents hepatocyte attachment. A net increase in hepatocyte disulfides is also observed after the first hours of culture. The formation of disulfides seems to be essential for the attachment of hepatocytes to collagen. The depletion of GSH in the early period of culture is probably due to its regulatory function of thiol/disulfide groups in proteins and/or its involvement in the synthesis of essential cytoskeletal proteins.

Dextran 70 administration after trauma-hemorrhagic shock does not impair cellular immune functions.

PURPOSE: Although the effects of the colloid dextran 70 on induction of anaphylactoid reactions or reticuloendothelial phagocytosis have been examined previously, its effects on specific cell-mediated immunity after trauma-hemorrhage shock remain unknown. METHODS: Nonheparinized C3H/HeN mice underwent a laparotomy, were bled, and then maintained at a blood pressure of 35 mm Hg for 60 minutes. Then they were resuscitated with either 4 x the shed blood volume as lactated Ringer's solution (LRS) or 2 x LRS + 1 x dextran 70. Control mice underwent all operative protocols but were neither hemorrhaged, nor resuscitated. At 2 or 24 hours posthemorrhage, serum, splenocytes (SPL), and peritoneal macrophages (pM phi, splenic Mo (sM phi) were obtained. Bioassays were used to determine interleukin-2 (IL-2), IL-3, IL-6, and SPL proliferation. RESULTS: Trauma-hemorrhage markedly depressed lymphokine release, splenocyte proliferation, and IL-6 release at 2 hours after the insult. The combination of LRS + dextran did not restore lymphocyte functions, but also did not further suppress them. The release of IL-6 by pM phi and sM phi at 2 and 24 hours after dextran infusion and serum IL-6 remained at the same level as in LRS-treated animals. CONCLUSIONS: The combination of LRS and colloid dextran 70 does not adversely affect ex vivo cell-mediated immune functions during the first 24 hours after its administration after trauma-hemorrhage. Thus, from the immunological standpoint, dextran is a safe resuscitation adjunct.

The kinetics of 4-nitrophenol conjugation by perfused livers and hepatic microsomes from streptozocin-induced diabetic rats.

The formation of both glucuronide and sulphate conjugates of 4-nitrophenol is deficient in perfused livers from male diabetic rats. Experiments with 'native' hepatic microsomes demonstrated that the defect in glucuronidation is due to a decrease in the maximal velocity of the reaction. There is no alteration in the affinity of the glucuronyltransferase for 4-nitrophenol. Non-linear regression analysis of the 4-nitrophenol liver perfusate concentrations showed that the elimination follows saturable Michaelis-Menten kinetics. Clearance values in 'native' microsomal preparations and in perfused livers were calculated and found to be similar in both systems. This provides evidence that glucuronyltransferase is 'native' in the intact liver.

The development of an outcomes management system for acute medical rehabilitation.

In 1993, Continental Medical Systems, Inc. (CMS), a provider of comprehensive medical rehabilitation, developed the Total Outcomes and Prediction Program (TOPP) to measure and evaluate key medical rehabilitation outcomes, quality indicators, and patient satisfaction at its 37 acute rehabilitation hospitals. The broad purposes of TOPP are to manage patient treatment, improve the cost-effectiveness of care, and provide outcomes reporting for managed care and other interested parties. The challenge was to develop a system which could measure, evaluate, and report medical rehabilitation patient outcomes in a way that could be easily understood by multiple audiences, including payers, accrediting organizations, physicians, patients and families, case managers, and CMS clinical staff. Using data from the Uniform Data System for Medical Rehabilitation database, CMS created descriptive outcomes reports for each hospital and for the corporation overall, including performance statistics, outcomes report cards, and quality report cards. These initial reports, as well as updates, quarterly reports, and special ad hoc requests, provide CMS corporate and hospital staff with statistically valid and reliable information to manage the outcomes of medical rehabilitation treatment. TOPP has assisted CMS with meeting accreditation standards for outcomes management and measurement and has been used in managed care contract negotiations. Future TOPP efforts will integrate resource use data, medical acuity and outcomes from acute, subacute, and outpatient rehabilitation levels into CMS' outcomes reporting system.