CD14-dependent regulation of Grp78 in the liver and lungs of mice after burn injury

Recent reports have suggested that interactions between the lipopolysaccharide receptor CD14 and heat shock proteins (Hsps) play roles in proinflammatory responses. The involvement of glucose-regulated protein 78 (Grp78), a member of Hsp70 family, and CD14 in signaling events activated in the liver and lungs of mice after burn injury was investigated. Differential induction of Grp78 in the liver of CD14 knockout (KO) mice after 18% total body surface area burn was associated with a transient down-regulation of serum glucose level at day 1 after injury. Subsequent studies revealed that the liver of both CD14 KO and wild-type control mice had a significant induction of Grp78 mRNA at day 1 after injury, while the level of induction was greater in CD14 KO mice. In contrast, in the lungs, there was an up-regulation of Grp78 mRNA only in CD14 KO mice at day 1 after injury. Interestingly, both the liver and the lungs had no apparent changes in Grp78 protein expression after injury. These data demonstrate CD14-dependent and tissue-specific regulation of the Grp78 expression after burn injury. They also suggest potential activation of a CD14-independent signaling pathway involving Grp78 in distant organs after injury.     

CD14-dependent clearance of apoptotic cells: relevance to the immune system

Until very recently, the function of CD14 was thought to be limited to innate immune responses to bacterial and other microbial structures. It is now known that macrophage CD14 serves an additional unexpected function, namely as a receptor involved in the recognition and phagocytosis of cells undergoing apoptosis. In stark contrast to its role in eliciting pro-inflammatory responses following binding of microbial ligands, macrophage CD14 mediates clearance of apoptotic cells without inciting inflammation. Increasing interest in the profoundly important final stage of apoptosis - the engulfment process - together with significant advances in knowledge of the immunological consequences of apoptotic-cell clearance and of the means by which signal transduction may be achieved following CD14-ligand binding have begun to produce a clearer picture of the role of CD14 in the immune system.     

Modulation of LPS-Stimulated Pulmonary Inflammation by Borneol in Murine Acute Lung Injury Model

The object of our study is to investigate the protective effects of Borneol on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. To determine the effects of Borneol on the histopathological changes in mice with ALI, inflammatory cell count in bronchoalveolar lavage fluid (BALF) and lung wet/dry weight ratio were measured in LPS-challenged mice, and lung histopathologic changes observed via paraffin section were assessed. Next, cytokine production induced by LPS in BALF and RAW 264.7 cells was measured by enzyme-linked imunosorbent assay (ELISA). To further study the mechanism of Borneol-protective effects on ALI, nuclear factor-kappaB (NF-κB) and mitogen-activated protein kinases (MAPKs) pathways were investigated. In the present study, Borneol obviously alleviated pulmonary inflammation by reducing inflammatory infiltration, histopathological changes, descended cytokine production, and pulmonary edema initiated by LPS. Furthermore, Borneol significantly suppressed phosphorylation of NF-κB/P65, IκBa, p38, JNK, and ERK. Taken together, our results suggest that Borneol suppressed inflammatory responses in LPS-induced acute lung injury through inhibition of the NF-κB and MAPKs signaling pathways. Borneol may be a promising potential preventive agent for acute lung injury treatment.     

Saturable CD14-dependent binding of fluorescein-labeled lipopolysaccharide to human monocytes.

We used rough lipopolysaccharide (ReLPS) to construct a fluorescein-labeled LPS (FITC-LPS) with a very high labeling efficiency that bound to isolated human monocytes in a CD14-dependent fashion and that in this respect behaved indistinctively from native LPS. The CD14-dependent binding could be inhibited either by a 1,000-fold excess of unlabeled LPS or by polymyxin B, bactericidal/permeability-increasing protein, cationic protein 18, or soluble CD14. Although this FITC-LPS preparation no longer possessed the ability to prime neutrophils for the production of reactive oxygen species or to stimulate human monocytes to produce tumor necrosis factor, activation of the Limulus amoebocyte lysate cascade was comparable to activation by native LPS. Binding to monocytes was enhanced by human pooled serum (HPS) or LPS-binding protein (LBP) for LPS concentrations up to 100 ng/ml and was completely CD14 dependent. For LPS concentrations exceeding 100 ng/ml, binding was still partially CD14 dependent, but not HPS or LBP dependent. CD14-dependent association of LPS with monocytes was shown to be totally saturable. In conclusion, we found an HPS- or LBP-dependent binding of FITC-LPS to monocytes that was CD14 dependent at up to 100 ng of LPS per ml, and saturation of binding was shown.     

CD14-dependent airway neutrophil response to inhaled LPS: role of atopy

BACKGROUND: Inhaled endotoxin (LPS) is associated with airway neutrophilic (PMN) inflammation in both asthmatic and control subjects, with asthmatic subjects demonstrating possibly higher sensitivity. CD14 is the principal receptor mediating LPS responses in vivo. It is unknown whether constitutive CD14 can predict the magnitude of the PMN response after LPS inhalation and whether atopy plays a role in this response. OBJECTIVE: We sought to examine associations between constitutive airway CD14 expression and LPS-induced PMNs after 5 microg of LPS inhalation and to examine associations between markers of atopy (eosinophils and eosinophil cationic protein) and CD14 expression and LPS-induced PMNs. METHODS: Ten atopic asthmatic subjects and 8 healthy control subjects inhaled 0.9% saline and LPS (Escherichia coli 026:B6, 5 microg) separated by 3 weeks. Induced sputum was collected at 24 hours before and 6 hours after inhalation. Induced sputum was analyzed for total and differential cell counts and soluble markers (soluble [s]CD14, eosinophil cationic protein, IL8, and total protein). Flow cytometry was used to analyze membrane-bound CD14 expression. RESULTS: Significant associations were found between the LPS-induced PMN response (PMNs per milligram of sputum) and both constitutive sCD14 (R = 0.7, P =.005) and membrane-bound CD14 (R = 0.9, P =.01). Asthmatic subjects demonstrated significantly higher levels of constitutive sCD14 compared with control subjects, and baseline eosinophils were significantly associated with baseline sCD14 (R = 0.7, P =.01) and LPS-induced PMNs (R = 0.6, P =.03). CONCLUSION: Constitutive airway CD14 expression can predict the magnitude of the PMN response after inhaled LPS. Atopy appears to play a role in the level of CD14 expression and may contribute to LPS sensitivity in asthmatic subjects.     

Interleukin-10 enhances the CD14-dependent phagocytosis of bacteria and apoptotic cells by human monocytes

Monocytes are centrally involved in both specific and nonspecific immunity by secretion of regulatory immune mediators, phagocytosis, and presentation of antigens. Recent work has shown that monocytes can phagocytose bacteria independently from Fc gamma, complement, and scavenger receptors via a CD14-mediated process. Furthermore, incorporation of cells undergoing apoptosis is also mediated by CD14. In this study we investigated the regulation of monocytic CD14-dependent phagocytosis by the immunoregulatory cytokines interleukin-10 (IL-10), interferon-gamma (IFN-gamma) and transforming growth factor-beta1 (TGF-beta1). In this study an in vitro human whole-blood assay was used to test regulation of CD14-dependent phagocytosis of fluorescence-labeled E. coli by IL-10, IFN-gamma, and TGF-beta1 in monocytes from healthy donors. Phagocytosis by monocytes from a patient with paroxysmal nocturnal hemoglobinuria (PNH) and its regulation by IL-10 was also investigated. Finally, regulation of monocytic incorporation of apoptotic Jurkat cells by IL-10 was analyzed. For the CD14 blockade, murine anti-CD14 IgG2a antibody RMO52 was used. We observed that IL-10, suggested to be a monocyte-deactivating cytokine, strongly increased the monocytic CD14-dependent phagocytosis of E. coli. In contrast, IFN-gamma and TGF-beta1 depressed monocytic CD14 incorporation of E. coli. Compatible with this, IL-10 upregulated CD14 expression on monocytes, whereas IFN-gamma and TGF-beta1 downregulated its expression. IL-10 also increased the monocytic CD14-dependent and -independent phagocytosis of apoptotic cells. As expected, IL-10 strongly increased the CD14-independent phagocytosis but had no influence on the CD14-dependent phagocytosis of monocytes from a PNH patient. In conclusion, our data support a general role of IL-10 for activating monocytic scavenger functions, which are at least partly mediated by CD14. This is in line with the fact that IL-10 promotes the development of monocytes to macrophages. The contrasting effects of IL-10 and IFN-gamma on monocytic CD14-dependent phagocytosis may reflect a further mechanism counterbalancing antigen-presentation and nonimmunogenic scavenging of bacterial and cellular debris. TGF-beta, however, may be an inhibitor of both systems.     

人内源性逆转录病毒的生物学功能及与疾病的关系

人内源性逆转录病毒(human endogenousretroviruses,HERV)是逆转录病毒在几百万年前感染人类并整合到人类基因组中,以孟德尔方式遗传至今的残余物.其在人体内数量众多,并且每个家族都存在多拷贝.HERV各家族基因结构基本相同,但许多功能都不明确,过去大多数研究人员将内源性逆转录病毒视为垃圾DNA.但随着研究的深入,人们发现HERV与人类的进化关系密切,是哺乳动物生殖所必需的,并且影响哺乳动物胎盘发育,是妊娠所不可或缺的基因.同时和胎盘共同构建了一个防止微生物感染胎儿的屏障.除了以上生理功能外,HERV还参与人体多种自身免疫性疾病和肿瘤的发生和发展过程.     

Lipopolysaccharide-induced production of tumour necrosis factor and interleukin-1 is differentially regulated at the receptor level: the role of CD14-dependent and CD14-independent pathways.

Cytokine production induced via CD14-dependent and CD14-independent pathways was investigated in mouse peritoneal macrophages incubated with lipopolysaccharide (LPS) or lipid A. Different LPS receptors appear to be responsible for production of tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 alpha (IL-1 alpha) and IL-1 beta. TNF-alpha production is essentially CD14 dependent, both in the presence or absence of plasma. In the presence of plasma, endotoxin-induced IL-1 production is mediated by CD14-dependent mechanisms, while in its absence both CD14-dependent and CD14-independent pathways are involved. Lipid A stimulates cytokine synthesis through both CD14-dependent and CD14-independent mechanisms, but its action is weaker than that of LPS, indicating that the polysaccharide moiety may be necessary for proper stimulation of mouse macrophages by endotoxin.     

Granulocyte-macrophage colony-stimulating factor regulates cytokine production in cultured macrophages through CD14-dependent and -independent mechanisms

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has multiple effects on the antigen phenotype and function of macrophages. In this study we investigated the effect of GM-CSF on cytokine production by macrophages. We found that GM-CSF may modify the tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) response to lipopolysaccharide (LPS) through two different mechanisms. Relatively early in culture, GM-CSF increases the amount of cytokines synthesized by responding cells; this effect appears to be unrelated to modulation of CD14 expression and LPS-binding capacity. After prolonged incubation, GM-CSF up-regulates both CD14 expression and LPS-binding capacity, and the frequency of cytokine-producing cells. Release of CD14 in the culture supernatant was decreased in the presence of GM-CSF, suggesting that a reduced shedding was responsible for the effect of GM-CSF on CD14 expression. Enhancement of cytokine production was also observed in GM-CSF-treated macrophages after stimulation by phorbol 12-myristate 13-acetate (PMA), thus indicating that GM-CSF affects both CD14-dependent and -independent cytokine production. Finally, GM-CSF did not modulate the LPS- and PMA-induced production of IL-10 and IL-12. We conclude that GM-CSF may play a role in manipulating the activation-induced expression of pro-inflammatory cytokines by macrophages. Enhanced production of these cytokines could play an important role in the pathogenesis of Gram-negative septic shock syndrome and in defence against infectious agents.     

Differential expression of CD14-dependent and independent pathways for chemokine induction regulates neutrophil trafficking in infection

Previous studies have shown that CD14(-/-) mice are resistant to peritoneal infection with some clinical isolates of Escherichia coli and that this resistance is accompanied by an enhanced ability to clear the bacteria; in contrast, normal mice expressing CD14 fail to clear the bacteria, causing severe sepsis and death. The enhanced clearance in CD14(-/-) mice is dependent on early neutrophil recruitment to the local foci of infection in the PC. The studies described show that neutrophil recruitment in CD14(-/-) mice occurs as a result of the local induction of the CXCL1 and CXCL2 chemokines, KC and MIP-2. Although local induction of these chemokines also occurs in normal mice, their effects on neutrophil recruitment to the PC appear to be counterbalanced by very high levels of these chemokines in the blood of normal, but not CD14(-/-), mice. Neutrophil recruitment to the PC is also inhibited in normal mice in response to LPS, which also induces high chemokine levels in the blood of normal, but not CD14(-/-), mice. However, MPLA, a monophosphorylated derivative of LPS, is able to induce early neutrophil recruitment in normal mice; this is because MPLA, unlike LPS or E. coli, induces MIP-2 and KC in the PC but not in the blood of normal mice. The pretreatment of normal mice with MPLA is able to protect them from a lethal E. coli infection. Thus, stimulation of a local CD14-independent chemokine induction pathway without triggering a systemic CD14-dependent chemokine pathway can protect against severe E. coli infections.     

Immune Complex Injury of the Lung

Abundant evidence currently exists to suggest that immune complexes play an important role in inflammatory diseases of the lung. Clinically, idiopathic pulmonary fibrosis, eosinophilic granuloma of lung, and systemic lupus erythematosus have been shown to be associated with the presence of immune complexes both in lung and in the serum. Experimentally, there is compelling evidence that acute lung injury can be triggered by the deposition of complexes in vascular walls or by the presence of performed immune complexes instilled into the airways. The observed reactions are, as expected, complement- and neutrophil-dependent. The morphologic changes in lung caused by products of complement activation (C5a and related peptides) depend on whether complement activation occurs within the vasculature or within the airways. Airway activation is associated with intraalveolar accumulations of neutrophils, while intravascular activation leads to intracapillary sequestration of neutrophils. The chronic formation of immune complexes within the vasculature (in the model of “chronic serum sickness”) leads to an interstitial fibrotic reaction and a thickening of basement membranes. Recent studies of intravascularly infused preformed immune complexes indicate a proclivity for certain types of complexes to localize within lung. These “lung-seeking” complexes differ from non-lung-seeking complexes only in the ratio of antigen to antibody. Complement does not seem to alter the tendancy for certain complexes to localize within lung. These studies emphasize the potential importance of immune complexes in lung injury and point out the variety of mechanisms involved in both the localization process and the injury process.     

Priming With Endotoxin Increases Acute Lung Injury in Mice by Enhancing the Severity of Lung Endothelial Injury

Endotoxin‐induced acute lung injury (ALI) is a commonly used model. However, the effect of a priming dose of endotoxin on lung fluid balance has not been well studied. We hypothesized that endotoxin‐induced ALI in mice would be enhanced under a priming condition. Mice were intratracheally (IT) instilled with either a priming dose of endotoxin from E. coli (0.5 mg/kg) or equal volume of PBS. Eighteen hours later, a larger challenge dose of endotoxin (5 mg/kg) was given IT. Control mice received PBS only. After 24 hr, the mice were sacrificed and the degree of lung injury and inflammation were measured. Endotoxin priming increased body weight loss and worsened hypothermia. Extravascular lung water and lung endothelial permeability were higher in the primed group. Priming with endotoxin reduced alveolar fluid clearance; however, there was no effect on bronchoalveolar lavage (BAL) levels of receptor for advanced glycation end products (RAGE). The primed group had increased alveolar inflammation as demonstrated by increased numbers of neutrophils in the BAL. There was no significant difference in NF‐κB p65 in the lung nuclear extract among the experimental groups. Taken together, priming with a small dose of endotoxin followed by a larger challenge dose of endotoxin induces more systemic illness and increased pulmonary edema in mice, largely due to increased lung endothelial permeability and lung inflammation. This model should be useful to investigators studying ALI who want to simulate the clinical setting in which more than one insult often leads to greater clinical lung injury. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.