Lipopolysaccharide-binding protein accelerates and augments Escherichia coli phagocytosis by alveolar macrophages.

BACKGROUND: The first step in bacterial clearance by leukocytes is attachment and phagocytosis. Although lipopolysaccharide-binding protein (LBP) is best known for potentiating LPS-induced cytokine production through a CD14-dependent pathway, recent studies suggest that LBP plays a critical role in clearance of gram-negative bacteria and is essential for survival after bacterial challenge. We therefore sought to examine LBP's effect on Escherichia coli phagocytosis by alveolar macrophages (AMs) and to determine if this effect is mediated through CD14. MATERIALS AND METHODS: Phosphatidylinositol-specific phospholipase C (PIPLC)-treated and untreated rat AMs were incubated in the presence of increasing doses of recombinant LBP or negative control protein (choramphenicol acetyltransferase) prior to E. coli-FITC (Ec-F) BioParticle challenge. Phagocytosed bacteria were assayed by fluorescence measurement. A time course study was also performed. RESULTS: LBP potentiated phagocytosis of Ec-F BioParticles by AMs in a dose-dependent fashion. Kinetic studies showed that LBP augmented Ec-F phagocytosis by 76% at 30 min. Treatment of AMs with PIPLC to remove CD14 resulted in only a partial decrease in LBP-mediated enhancement of phagocytosis. CONCLUSION: These results clearly demonstrate that LBP plays an important role in enhancing Ec-F binding and phagocytosis in a time- and dose-dependent manner. This observed increase may not require the presence of CD14 as significant potentiation of phagocytosis still occurred after PIPLC treatment. We postulate that the LBP-mediated increase in Ec-F phagocytosis can occur in the absence of CD14 through the presence of another receptor.

Skin lipopolysaccharide-binding protein and IL-1beta production after thermal injury.

In response to a burn injury, skin can have an inflammatory response characterized by the production of inflammatory cytokines, recruitment of immune cells, containment of invading organisms, and clearance of noxious substances from the wound. Lipopolysaccharide-binding protein (LBP) is a molecule that is capable of coordinating all 4 functions; we previously found evidence that suggested that LBP is produced within surgical wounds. Because of the central role of LBP in the response to bacterial infection, as well as in the high rate of infection after burn injuries, we sought to determine whether a thermal injury could affect wound LBP production and thereby affect host responses against bacterial infection. Rats were given either a burn or a sham burn and were killed 24, 48, and 72 hours after the injuries. Wound specimens were assayed for bacterial counts and for the presence of LBP, messenger (m)RNA, and interleukin (IL)-1beta mRNA. Wound LBP mRNA was significantly upregulated at 24 hours in the group with burn injuries (P < .05; burn vs sham burn); this was followed by decreases at 48 and 72 hours. Immunohistochemistry showed LBP protein in the epidermis of animals with burns. Bacterial counts increased in the group with burn injuries (P < .05; burn vs sham burn) and continued to rise for 72 hours. IL-1beta mRNA levels were elevated at all time points in the group with burn injuries (P < .05). These results suggest an inverse correlation between burn wound LBP expression and bacterial wound counts. This failure to maintain local LBP production after severe thermal injury despite localized inflammation shown by high IL-1beta levels may predispose local wounds to bacterial invasion.

Kupffer cell activation by lipopolysaccharide in rats: role for lipopolysaccharide binding protein and toll-like receptor 4.

Lipopolysaccharide (LPS) binding protein (LBP) is a key serum factor that mediates LPS activation of mononuclear cells. In the presence of LBP, 1/1,000 the concentration of LPS is sufficient to activate peripheral blood monocytes. Previous studies with Kupffer cells have shown a variable effect of serum on LPS activation of these cells and led to the conclusion that, unlike extrahepatic mononuclear cells, Kupffer cells do not respond to LPS in an LBP-dependent fashion. Because there are multiple components in serum other than LBP that might affect LPS activation, these reports with serum are difficult to interpret. To investigate the specific role of LBP in LPS activation of Kupffer cells, we produced a functional recombinant rat LBP using a baculovirus expression system, which we used to selectively examine the role of LBP's on Kupffer-cell function. Isolated Kupffer cells exposed to increasing concentrations of LPS (0, 1, 10 ng/mL) showed a dose-dependent increase in TNF-alpha production, which was augmented and accelerated by the presence of LBP. The effects of LBP on Kupffer cell activation by LPS are dependent on a functional Toll-like receptor 4 (Tlr 4) because Kupffer cells from C3H/HeJ mice failed to respond to LPS in the presence of LBP. LBP plays an important role in mediating Kupffer cell activation by LPS, and these effects are dependent on the presence of functioning Tlr 4.

Can the clearance of tumor necrosis factor alpha and interleukin 6 be enhanced using an albumin dialysate hemodiafiltration system?

Patients with acute hepatic failure (AHF) have elevated levels of inflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6). Recently, we have shown selective hemodiafiltration with albumin dialysis, as an extracorporeal liver support device (ECLVS), to be effective in the clearance of multiple toxins that are elevated in AHF. Our objective was to evaluate whether ECLVS would be effective in the clearance of TNF-alpha and IL-6. An in vitro continuous hemodiafiltration circuit was used with single pass counter-current dialysis. A known amount of recombinant rat TNF-alpha and IL-6 was added to heparinized bovine blood and filtered across a polyalkyl sulfone hemofilter using matched filtration and dialysate flow rates. During 4 hours, the serial TNF-alpha and IL-6 concentrations were measured in the circulating blood, and the content of each cytokine was calculated using mass balance. For each cytokine, clearance was determined for two dialysate groups at constant temperature and pH (group 1: dialysate = 0.9 normal saline, n = 5; group 2: dialysate = albumin 2 gm/dl, n = 5). Analysis of data was performed using ANOVA and Student's t-test. There was improved clearance of TNF-alpha and IL-6 when albumin was used in the dialysate (81+/-0.09% of the initial TNF-alpha and 77+/-0.04% of the IL-6 quantities) compared with when 0.9 normal saline was used as the dialysate (58+/-0.14% of the initial TNF-alpha and 56+/-0.18% of the IL-6 quantities); p < 0.03. An ECLVS utilizing hemodiafiltration with albumin dialysis is more effective than conventional hemofiltration in the clearance of TNF-alpha and IL-6 and, therefore, may benefit patients with acute hepatic failure.

Pulmonary LPS-binding protein (LBP) upregulation following LPS-mediated injury.

BACKGROUND: The acute respiratory distress syndrome (ARDS) causes significant morbidity and mortality among trauma patients. Although multiple factors have been implicated, pulmonary injury in this population may be due to inflammatory mediators released in response to stimuli such as endotoxin (LPS). LBP plays an integral part in LPS-mediated release of inflammatory cytokines and increased local expression of LBP as the result of a primary injury may prime the lung to secondary LPS-mediated damage. MATERIALS AND METHODS: To determine the magnitude of pulmonary LBP upregulation following LPS injury we challenged rats with either intravenous (IV) or intratracheal (IT) LPS. Animals from each group were euthanized at 1, 2, 4, and 8 h postchallenge. Lung LBP and CD14 mRNA levels were assayed by Northern blot. Serum and bronchoalveolar lavage (BAL) fluid were assayed for inflammatory cytokines (TNF-alpha, MCP-1, IL-1beta, IL-6, and IL-10) by ELISA. RESULTS: LBP and CD14 mRNA levels were found to increase significantly in lung tissue after both IV and IT LPS with the IV LPS animals having a greater increase over 8 h. Serum TNF-alpha was significantly elevated in the IV LPS group whereas very low levels were detected in the BAL. Only BAL TNF-alpha was increased in the IT group at 8 h. CONCLUSION: Local pulmonary LBP and CD14 mRNA are both upregulated after either systemic or local LPS exposure. Such upregulation may render thelung more susceptible to local immune overactivation and injury during subsequent exposures to LPS.

Tissue coexpression of LBP and CD14 mRNA in a mouse model of sepsis.

BACKGROUND: Lipopolysaccharide binding protein (LBP) markedly increases the sensitivity of immune cells to LPS and CD14 expression correlates with cellular responsiveness to LPS. LBP gene expression can be induced in multiple organs following injury and CD14 upregulation on monocytes correlates with the infection and mortality rates in severely injured patients. We sought to determine the time-course induction of LBP and CD14 gene expression following experimental peritonitis. MATERIAL AND METHODS: BALB/c mice were subjected to laparotomy alone or laparotomy with cecal ligation and puncture and treated with Imipenem. At serial time points, animals were sacrificed and tissues harvested for isolation of RNA and protein. LBP, CD14, and cytokine mRNAs were analyzed by Northern blot analysis and TaqMan fluorescent quantitative RT-PCR. RESULTS: LBP and CD14 mRNA levels were significantly increased in all three organs from CLP mice compared to sham-operated mice. IL-1 mRNA levels increased in all three organs following CLP with significantly higher levels found in the lungs compared to the kidney and liver. No significant differences were noted in local TNF mRNA levels. CONCLUSIONS: LBP, CD14, and IL-1 mRNA levels are induced concurrently in the lung, kidney, and liver after cecal ligation and puncture. Given the synergistic affect of LBP and CD14 in potentiating LPS-induced production of inflammatory cytokines and the hypothesized role of such cytokines in the etiology of MSOF following injury and sepsis, our findings suggest a mechanism by which these organs may be rendered more susceptible to a "second hit" from endotoxemia after initial injury.