Bjoern M. Thobe

The role of MAPK in Kupffer cell toll-like receptor (TLR) 2-, TLR4-, and TLR9-mediated signaling following trauma-hemorrhage

Severe injury deranges immune function and increases the risk of sepsis and multiple organ failure. Kupffer cells play a major role in mediating posttraumatic immune responses, in part via different Toll‐like receptors (TLR). Although mitogen‐activated protein kinases (MAPK) are key elements in the TLR signaling pathway, it remains unclear whether the activation of different MAPK are TLR specific. Male C3H/HeN mice underwent midline laparotomy (i.e., soft tissue injury), hemorrhagic shock (MAP ∼35 mm Hg for 90 min), and resuscitation. Kupffer cells were isolated 2 h thereafter, lysed and immunoblotted with antibodies to p38, ERK1/2, or JNK proteins. In addition, cells were preincubated with specific inhibitors of p38, ERK1/2, or JNK MAPK followed by stimulation with the TLR2 agonist, zymosan; the TLR4 agonist, LPS; or the TLR9 agonist, CpG DNA. Cytokine (TNF‐α, interleukin‐6 (IL‐6), monocyte chemoattractant protein‐1 (MCP‐1), and KC) production was determined by cytometric bead array after 24 h in culture. MAPK activity as well as TNF‐α, MCP‐1, and KC production by Kupffer cells were significantly increased following trauma‐hemorrhage. TLR4 activation by LPS stimulation increased the levels of all measured cytokines. CpG‐stimulated TLR9 signaling increased TNF‐α and IL‐6 levels; however, it had no effect on chemokine production. Selective MAPK inhibition demonstrated that chemokine production was mediated via p38 and JNK MAPK activation in TLR2, ‐4, and ‐9 signaling. In contrast, TNF‐α and IL‐6 production was differentially regulated by MAPK depending on the TLR pathway stimulated. Thus, Kupffer cell TLR signaling employs different MAPK pathways in eliciting cytokine and chemokine responses following trauma‐hemorrhage. J. Cell. Physiol. 210: 667–675, 2007.

Impact of Thermal Injury on Wound Infiltration and the Dermal Inflammatory Response

Healing of the burn wound is a critical component of the burn patients successful recovery. While inflammation is a critical component of the healing process, it is unknown whether the inflammatory response differs between non-burn and burn wounds. To study this, mice were subjected to major burn injury or sham procedure. Wound cells were collected by implantation of polyvinyl alcohol sponges beneath the burn site in injured mice or beneath uninjured skin in sham mice (i.e., non-burn wound). Three days thereafter, skin, wound fluid, and infiltrating cells were collected for analysis. Significant levels of tumor necrosis factor (TNF)-alpha, interleukin (IL-6), monocyte chemoattractant protein (MCP)-1, and keratinocyte-derived chemokine (KC) were observed in burn wound tissue and the wound fluid from both non-burn and burn wounds. Burn injury induced 3-fold higher levels of KC and 50-fold higher levels of IL-6 in the wound fluid compared with non-burn injury. Significant numbers of the cells from both burn and non-burn wounds were CD11b(+), GR1(+), and F4/80(+), suggestive of a myeloid suppressor cell phenotype, whereas CD3(+) T-cells were negligible under both conditions. LPS induced TNF-alpha, IL-6, IL-10, MCP-1, KC, and nitric oxide production in both cell populations, however, IL-6, IL-10, MCP-1, and KC levels were suppressed in burn wound cell cultures. These findings indicate that significant differences in the wound inflammatory response exist between burn and non-burn cutaneous wounds and that the unique characteristics of the inflammatory response at the burn site may be an important contributing factor to post-burn wound healing complications.

Regulation of the postburn wound inflammatory response by γδ T-cells

Healing of the burn injury site is a critical component of the patients successful recovery from this form of trauma. Previous studies from our laboratory have demonstrated that &ggr;&dgr; T-cells via the production of growth factors are important in burn wound healing. Nonetheless, the role of these cells in burn wound inflammation remains unknown. To study this, wild-type (WT) and &ggr;&dgr; T-cell receptor-deficient (&dgr; TCR−/−) C57BL/6 male mice were subjected to burn injury or sham procedure. Wound cells were collected by implantation of polyvinyl alcohol sponges beneath the burn site in injured mice or beneath uninjured skin in sham mice. At 3 days after injury, infiltrating cells, wound fluid, and skin were collected for analysis. Burn injury markedly increased skin tumor necrosis factor-&agr; (TNF-&agr;) and monocyte chemoattractant protein 1 levels. In WT mice, the numbers of infiltrating cells were similar between nonburn wounds and burn wounds. In contrast, &dgr;TCR−/−mice displayed a 6-fold reduction in the cellular infiltrate. Burn injury in WT mice caused a marked increase in burn wound TNF-&agr;, monocyte chemoattractant protein 1, and interleukin 6 content as compared with nonburn wounds, whereas in &dgr; TCR−/−mice, the burn-induced increase of TNF-&agr; and interleukin 6 was not observed. The wound cell infiltrate at 3 days postinjury was devoid of &ggr;&dgr; T-cells in WT mice. It was predominately of myeloid origin expressing high levels of CD11b and F4/80. In conclusion, these findings suggest that resident &ggr;&dgr; T-cells are important in the recruitment of inflammatory cells and regulation of the inflammatory response at the wound site after thermal injury.

Are the protective effects of 17β‐estradiol on splenic macrophages and splenocytes after trauma‐hemorrhage mediated via estrogen‐receptor (ER)‐α or ER‐β?

The depression in cell‐mediated immune function following trauma‐hemorrhage is shown to be restored by 17β‐estradiol (E2) administration. However, it remains unknown which of the two estrogen‐receptors, (ER)‐α or ER‐β, plays the predominant role in mediating the beneficial effects of E2. Female B57BL/J6 ER‐β−/− transgenic mice [knockout (KO)] and corresponding ovariectomized wild‐type (WT) mice were subjected to laparotomy and hemorrhagic shock (35.0±5.0 mmHg for 90 min) and treated with E2 (50 μg/25 g) or ER‐α agonist propyl pyrazole triol (PPT; 50 μg/25 g) following trauma‐hemorrhage. Four hours after resuscitation, systemic cytokine concentrations and cytokine release by splenocytes and splenic macrophages were determined by cytometric bead array. Trauma‐hemorrhage resulted in a significant increase in plasma tumor necrosis factor α (TNF‐α), interleukin (IL)‐6, and IL‐10. In contrast, the release of these cytokines by splenic macrophages was decreased significantly in WT and KO animals. Administration of E2 or PPT following trauma‐hemorrhage produced a significant reduction in systemic TNF‐α and IL‐6 concentrations in WT and KO mice. Although the suppression in the productive capacity of these cytokines following trauma‐hemorrhage by macrophages and splenocyte was also prevented in E2‐ and PPT‐treated WT mice, the release of cytokines by macrophages and splenocytes in E2‐ and PPT‐treated KO mice was not restored to the levels observed in sham animals. These findings collectively suggest that both receptors appear to play a significant role in mediating the immunoprotective effects of E2 in different tissue compartments following trauma‐hemorrhage.

Downregulation of TLR4-dependent ATP production is critical for estrogen-mediated immunoprotection in Kupffer cells following trauma-hemorrhage.

Toll‐like receptor 4 (TLR4) mediates mitochondrial DNA (mtDNA) damage and biogenic responses. Mitochondrial transcription factor A (Tfam) is an essential regulator for mtDNA transcription and ATP production. Increased ATP levels were associated with normalization of immune function following trauma‐hemorrhage. Moreover, administration of 17β‐estradiol following trauma‐hemorrhage upregulates cardiac Tfam and ATP levels. We therefore hypothesized that the salutary effect of 17β‐estradiol on Kupffer cell function following trauma‐hemorrhage is mediated via negative regulation of TLR4, which downregulates iNOS, upregulates Tfam and mtDNA‐encoded gene cytochrome c oxidase I (mtCOI), and consequently increases cellular ATP levels. Male C3H/HeN, C3H/HeOuJ (intact TLR4), and C3H/HeJ (TLR4 mutant) mice were subjected to trauma‐hemorrhage (mean BP 35 ± 5 mmHg ∼90 min, then resuscitation) or sham operation. At the beginning of resuscitation, mice received 17β‐estradiol (25 µg/25 g) or vehicle intravenously and were sacrificed 2 h thereafter. Kupffer cell TLR4, iNOS, IL‐6 and TNF‐α production capacities were increased, and ATP, Tfam, and mtCOI levels were decreased following trauma‐hemorrhage. Administration of 17β‐estradiol following trauma‐hemorrhage prevented the increase in Kupffer cell TLR4, iNOS, and cytokine production. This was accompanied by normalized ATP, Tfam, and mtCOI levels. Furthermore, the decreased Kupffer cell ATP and mtCOI levels were not observed in TLR4 mutant mice following trauma‐hemorrhage. Taken together, these findings suggest that downregulation of TLR4‐dependent ATP production is critical to 17β‐estradiol‐mediated immunoprotection in Kupffer cells following trauma‐hemorrhage. J. Cell. Physiol. 211: 364–370, 2007.

Effects of 17β‐estradiol and flutamide on inflammatory response and distant organ damage following trauma‐hemorrhage in metestrus females

We hypothesized that administration of androgen receptors antagonist flutamide following trauma‐hemorrhage (T‐H) in metestrus females will maintain immune function and reduce remote organ damage under those conditions. Female B57BL/J6 mice (metestrus state, 8–12 weeks old) underwent laparotomy and hemorrhagic shock (35.0±5.0 mmHg for 90 min) and then received 17β‐estradiol (E2; 50 μg/25 g), flutamide (625 μg/25 g), or E2 + flutamide. Four hours after resuscitation, plasma cytokine and chemokine (TNF‐α, IL‐6, IL‐10, IFN‐γ, and MCP‐1) concentrations and their release in vitro by hepatic and pulmonary tissue macrophages (MΦ) were determined by flow cytometry. Organ damage was assessed by edema formation (wet‐to‐dry weight ratio) and neutrophil infiltration [myeloperoxidase (MPO) activity]. Administration of E2, flutamide, or E2 + flutamide following T‐H resulted in a significant decrease in systemic TNF‐α, IL‐6, and MCP‐1 concentrations under those conditions. This was accompanied by significantly decreased in vitro TNF‐α release by Kupffer cells after administration of E2, flutamide, or E2 + flutamide. The in vitro release of proinflammatory cytokines by alveolar MΦ, however, was reduced significantly only by the addition of E2 or E2 + flutamide but not by the addition of flutamide. A significant decrease in pulmonary and hepatic edema formation as well as neutrophil infiltration in the lung was observed after E2, flutamide and E2 + flutamide administration. In contrast, hepatic neutrophil infiltration was only significantly reduced following E2 and E2 + flutamide administration. Thus, although flutamide does not produce synergistic, salutary effects with E2, its administration in females following T‐H also produces salutary effects on the immune and organ function, similar to E2 administration under those conditions.