Mechanism of splenic immunosuppression during sepsis: key role of Kupffer cell mediators.

Studies indicate that the liver, in particular the Kupffer cells, appear to be key contributors in the systemic inflammatory mediator response associated with shock and sepsis. Although several of these agents have been implicated as mediators of depressed immunoresponsiveness observed during sepsis, it remains unknown whether or not mediators released specifically by Kupffer cells play any significant role in producing the cellular dysfunction in distant organs. The aim of this study, therefore, was to determine whether or not acute Kupffer cell reduction before the onset of sepsis would protect splenic lymphocyte function. Kupffer cell number was reduced by prior (48 hours) treatment of mice with gadolinium chloride (GdCl2, 10 mg/kg of body weight, intravenously) or saline vehicle. Animals were then subjected to either sham-CLP (sham) or polymicrobial sepsis in the form of cecal ligation and puncture (CLP). Plasma and splenocytes were harvested at 2 or 24 hours after CLP. Splenocyte cultures were exposed to 2.5 micrograms concanavalin A/mL to assess their ability to release lymphokines. Cytokine (interleukin (IL)-2, IL-6, interferon-gamma, tumor necrosis factor-alpha) concentration in plasma or cell supernatants was assessed by bioassay. The results indicated that GdCl2 treated mice exhibited a marked reduction in circulating IL-6 levels at both 2 and 24 hours after CLP. Furthermore, the reduction of Kupffer cell number before the onset of sepsis completely prevented the depression of splenocyte IL-2 and interferon-gamma release, capacity. Thus mediators released by Kupffer cells during the systemic inflammatory response to polymicrobial sepsis play a significant role in producing immune dysfunction in resident splenic lymphocytes. In view of this, it appears that modulation of Kupffer cell hyperactivity during sepsis may be a novel approach for maintaining distant organ host defense mechanisms.

Differential induction of apoptosis in lymphoid tissues during sepsis: variation in onset, frequency, and the nature of the mediators.

Apoptosis (Ao), is a process by which cells undergo a form of nonnecrotic cellular suicide. Although for most cells this is a constitutive process, it can be induced in immature and differentiating immune cell populations by stress mediators associated with inflammation. This inducible form of A(o) is referred to as programmed cell death. However, it is not clear whether hematopoietic cell populations such as the thymus and bone marrow are induced to undergo A(o) during polymicrobial sepsis. To assess this, thymocytes, bone marrow cells, or splenocytes (as a source of comparative nonhematopoietic cells) were harvested from C3H/HeN mice at 1, 4, or 24 hours after cecal ligation and puncture (CLP; to induce polymicrobial sepsis) or sham-CLP (Sham). The results showed that mixed bone marrow cells ex vivo, although not to the same extent as thymus, showed a marked increase in the percentage of cells in A(o), increased endonuclease activity, and a significant decrease in cell yield at 24 hours but not at 4 hours after CLP. Similar changes were not evident in splenocytes. Phenotypic, as well as morphologic assessment, indicated that most of the increase in apoptotic cells in the thymus was associated with the immature T cells (CD4+CD8+) and CD8-CD4- cells. In contrast, the increase in bone marrow cell A(o) was associated with only the B220+ cells, with no significant contribution from myeloid cells. Treatment of CLP mice in vivo with either RU-38486 or PEG-(rsTNF-R1)2 was unable to reverse the increased A(o) in the bone marrow of these animals. Taken together, these findings indicate that A(o) as a process induced by polymicrobial sepsis is not limited to the thymus, but can also be detected in the bone marrow. However, unlike thymic A(o), bone marrow is not affected directly/indirectly by glucocorticoids or tumor necrosis factor released during sepsis.

Is sepsis-induced apoptosis associated with macrophage dysfunction?

Apoptosis (A O) is a pathological process by which cells undergo a form of inducible nonnecrotic cellular suicide. In vitro studies suggest that changes in the rate of macrophage (Mo) A O may be associated with elevated proinflammatory cytokine secretory capacity, such as interleukin-1 beta (IL-1 beta) (via IL-1 converting enzyme activation). Furthermore, it has been reported that Mo are activated during early (0-4 hours) experimental septic insult to act as sources of proinflammatory cytokines, such as IL-1. However, with the progression of sepsis, these same cells become refractory to further stimulation (appearing dysfunctional). Nonetheless, it remains unknown if this acquired immunosuppression (dysfunction) is associated with an acceleration in macrophage A O. To determine this, male C3H/HeN mice were subjected to sepsis (cecal ligation and puncture, CLP) or sham-CLP and 4 or 24 hours thereafter Mo were isolated from the peritoneum (PMo) and liver (KMo). Macrophage monolayers were lysed either after stimulation with lipopolysaccharide (LPS) (10 microgram/mL, 24 hours) in vitro or immediately (ex vivo) before LPS stimulation and the cytoplasmic cell fraction was retained. The extent of A O was determined using a cell-death enzyme-linked immunosorbent assay, which detects the presence of cytoplasmic oligonucleosomes and changes in the propidium iodide staining intensity. The results indicate that, early after CLP (4 hours) only PMo stimulated with LPS in vitro showed evidence of increasing A O. At 24 hours (late) after the onset of sepsis, the ex vivo extent of A O in PMo was increased but it was decreased in KMo. However, the addition of LPS in vitro results in a marked increase in both septic PMo and KMo A O. This latter result suggests that the inability of Mo to release cytokines in response to stiumulants, such as LPS during late sesis (24 hours), may be because of induciton of accelerated A O in these Mo populations.

Does endotoxin play a major role in inducing the depression of macrophage function during polymicrobial sepsis?

BACKGROUND: Endotoxin (ETX) is thought to be the primary inducer of proinflammatory mediator release associated with bacterial sepsis. Furthermore, a number of studies indicate that preexposure of animals to high doses of ETX produces macrophages (M luminal diameters) that are refractory to ex vivo stimulation with ETX. However, it is unknown if levels of ETX comparable to those typically encountered in sepsis induce a similar refractory state in M luminal diameters. DESIGN: To assess this, peritoneal M luminal diameters (PM luminal diameters) were harvested from C3H/HeN mice (ETX sensitive) at 1 hour (early) or 24 hours (late) following cecal ligation and puncture (CLP) to induce polymicrobial sepsis, sham CLP, or laparotomy followed by peritoneal implantation of a minipump delivering either saline or ETX (0.025 microgram/g of body weight, every 24 hours). Peritoneal M luminal diameter cultures were incubated with ETX, either 0 or 10 micrograms/mL, for 24 hours, and their ability to release interleukin-1, interleukin-6, and tumor necrosis factor was assessed by bioassay. RESULTS: Chronic low-dose ETX with 0 microgram of ETX media added produced early (at 1 hour) in vivo activation of PM luminal diameter interleukin-1 release, which was comparable to that seen in mice subjected to CLP. However, unlike PM luminal diameter taken from CLP mice, PM luminal diameters from mice implanted with the ETX minipump at 1 or 24 hours showed no marked decline in their ability to respond to ETX (10 micrograms). Comparable changes were seen for interleukin-6 and tumor necrosis factor release. CONCLUSIONS: Bacterial component(s) other than ETX per se induces the sustained dysfunction in PM luminal diameter capacity to produce proinflammatory cytokines during sepsis and/or peritonitis. Thus, agents directed against ETX alone may not be adequate in the treatment of polymicrobial sepsis.

The induction of accelerated thymic programmed cell death during polymicrobial sepsis: control by corticosteroids but not tumor necrosis factor.

Thymic programmed cell death (PCD) or apoptosis (Ao) is elevated during inflammation by a variety of stressors in vitro (i.e., glucocorticoids, tumor necrosis factor (TNF), prostanoids, etc.), however, little or no information is available concerning its presence in polymicrobial sepsis. To establish whether or not PCD is accelerated in the thymus following the onset of sepsis, thymocytes were harvested from C3H/HeN mice at 1, 2, 12, and 24 h following cecal ligation and puncture (CLP; to induce sepsis) or Sham-CLP (Sham), and assessed for changes in thymocyte viable cell yield, increased Ao + cells based on FACS analysis (propidium iodide staining) or by evidence of fragmentation of the genomic DNA. The results indicate that at 1 h post-CLP there were no marked changes in any of these parameters. However, by 4 h post-CLP the percentage of Ao + thymocytes increased and the septic mouse genomic DNA exhibited trace amounts of fragmentation. These changes increased in the septic animals cells through both 12 and 24 h. Alternatively, thymic viable cell yield did not significantly decrease until 12 h. Marked changes in systemic mediators, corticosterone and TNF, were also detected in septic mouse blood at all time points. In an effort to determine the contribution of these two agents to the induction of the accelerated PCD seen here, mice were randomized to receive either RU-38486 (11 beta-[p-(dimethylamino)phenyl]-17 beta-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one (Mifepristone); a steroid receptor blocker), polyethylene glycol (PEG)-(rsTNF-R1)2 (a TNF inhibitor) immediately following CLP.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of enhanced susceptibility to sepsis following hemorrhage. Interleukin-10 suppression of T-cell response is mediated by eicosanoid-induced interleukin-4 release.

OBJECTIVES: To determine (1) whether interleukin-10 (IL-10) contributes to depressed T-cell responses observed following hemorrhage and (2) what effect other immunosuppressive agents known to play a role in hemorrhage have on IL-10 release. DESIGN: Hemorrhage was induced in C3H/HeN mice. The mice were resuscitated and then killed 2 hours after hemorrhage to obtain plasma, splenocytes, splenic macrophages, and splenic T cells. RESULTS AND CONCLUSIONS: Decreased splenocyte/T-cell proliferation was associated with enhanced release of IL-10 by cells from hemorrhaged mice. However, unlike T cells, IL-10 release by macrophages was not comparatively elevated. While no changes were seen in systemic plasma levels of IL-10, the role of IL-10 as a localized immunosuppressant was demonstrated by the ability of IL-10 monoclonal antibody to restore T-cell proliferation following hemorrhage. Furthermore, elevated IL-10 release was prevented by the addition of ibuprofen or monoclonal antibody against transforming growth factor beta or IL-6. Since these agents have direct or indirect influences on prostanoid synthesis, studies were carried out examining the capacity of varying concentrations of prostaglandin E2 (PGE2) to augment IL-10 release by murine cloned Th2 cells (D10.G4.1) and by T cells from sham-operated or hemorrhaged mice. While the addition of PGE2, 10(-9) mol/L, potentiated the release of IL-10, this effect appears to be indirect, since the incorporation of monoclonal antibody to IL-4 prevented the release of IL-10 by PGE2-treated cells from sham-operated or hemorrhaged mice. Such a mechanism of eicosanoid-induced IL-4/IL-10 cell-mediated immunosuppression may directly contribute to the decreased capacity to ward off infectious challenge seen following hemorrhage.

Pentoxifylline attenuates oxygen-induced lung injury.

Patients requiring mechanical ventilation can develop severe pulmonary injury. Although pentoxifylline (PTX) is known to attenuate endotoxin and tumor necrosis factor (TNF)-induced lung injury, as well as decrease interleukin-6 (IL-6) levels following hemorrhage and resuscitation, it remains unknown if this agent has any beneficial effects against O2-induced lung injury. The aim of this study, therefore, was to determine if PTX attenuates pulmonary oxygen toxicity. To investigate this, male Sprague-Dawley rats were injected subcutaneously with 1 ml saline or 1 ml PTX (50 mg/kg) and immediately exposed to either 21% O2 or > or = 95% O2 for 52 hr. The animals were then reweighed and euthanized. Pleural fluid was collected, blood samples were obtained, and lung lavage was performed. Lactate dehydrogenase (LDH) activity, protein content, and IL-6 concentrations were determined in the lavage fluid and serum. The supernatant LDH activity, protein content, pleural fluid accumulation, and IL-6 concentration were significantly decreased (P < 0.05) in those animals pretreated with PTX prior to exposure to hyperoxia compared to those animals exposed to hyperoxia and not treated. Furthermore, the hematocrit and serum IL-6 concentration were also decreased in the treated group and not significantly different from the controls. Thus, PTX appears to attenuate O2-induced lung injury and may play a role in protecting those patients at risk for developing pulmonary oxygen toxicity.

Polymicrobial sepsis but not low-dose endotoxin infusion causes decreased splenocyte IL-2/IFN-gamma release while increasing IL-4/IL-10 production.

Although studies indicate that polymicrobial sepsis produces marked depression in lymphocyte functions, it remains unclear whether this dysfunction is due to the chronic exposure of immune cells to endotoxin (ETX; a product of the gram-negative bacterial cell wall) at levels typically encountered in the septic state. The aim of this study, therefore, was to determine whether the changes in lymphokine release seen during polymicrobial sepsis are comparable to those observed with chronic ETX infusion. To assess this, splenocytes were harvested from C3H/HeN mice (ETX-sensitive) at 1 or 24 hr following cecal ligation and puncture (CLP; to induce polymicrobial sepsis), Sham CLP (Sham), or laparotomy followed by peritoneal implantation of a mini-osmotic pump which delivered either saline vehicle (Sal-pump) or ETX (ETX-pump; 0.025 micrograms lipopolysaccharide/25 g body wt/24 hr). Splenocytes were then stimulated with concanavalin A (2.5 micrograms/ml/48 hr) and their capacity to release interleukin (IL)-2, interferon (IFN)-gamma, IL-4, and IL-10 was determined by bioassay or ELISA. The results indicated that there were no changes in lymphokine release capacity at 1 hr after CLP or ETX-pump implantation. However, prolonged sepsis (i.e., at 24 hr) caused a marked suppression of IL-2 and IFN-gamma release (immune-enhancing lymphokines characteristic of Th1-cells), while enhancing the release of immunosuppressive Th2-cell products IL-4 and IL-10. Chronic exposure to ETX at a level comparable to that seen in CLP caused no depression in lymphokine (IL-2/IFN-gamma) release. This implies that a bacterial component other than ETX mediates the differential alterations observed in lymphokine release during prolonged polymicrobial sepsis.