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Cell Biology International (2008) 32, 11081115 (Printed in Great Britain)
Inhibition of lipopolysaccharide-mediated rat alveolar macrophage activation in vitro by antiflammin-1
Jianzhong Han, Chen Li, Huijun Liu, Dandan Fen, Wanxiang Hu, Yong Liu, Chaxiang Guan and Zi‑qiang Luo*
Department of Physiology, Xiang-Ya Medical School, Central South University, 110 Xiangya Road, Changsha, PR China
Antiflammin-1 (AF-1) is a synthetic nonapeptide with a similar sequence to the conserved sequence of CC10 secreted by lung Clara cells. Studies suggest that it is potent inhibitor of inflammation. We investigated the effects and possible mechanisms of AF-1 on LPS-induced alveolar macrophage (AM) activation in vitro. AMs harvested from the BALF of Sprague-Dawley (SD) rat were treated with various concentrations of AF-1 both simultaneously and after LPS stimulation. The concentrations of the cytokines IL-1β, IL-6, and IL-10 in the supernatant were detected by an enzyme-linked immunosorbent assay. The mRNA expression levels of these cytokines in AMs were analyzed using quantitative RT-PCR. To investigate more fully the possible mechanisms by which AF-1 modulates the expression of cytokines, cells were pretreated with anti-IL-10 antibody. Toll-like receptor-4 (TLR-4) expression on the cell surface was also detected using flow cytometry. The results showed that AF-1 suppressed mRNA expression and protein production of IL-1β and IL-6, while it promoted IL-10 expression in LPS-stimulated AMs, in a dose-dependent manner. The inhibitory effects of AF-1 on IL-1β were significantly decreased when endogenous production of IL-10 was blocked. AF-1 also showed an effect on downregulated TLR-4 expression in LPS-stimulated AMs. The data show for the first time that AF-1 modulates the AM response to LPS by regulating TLR-4 expression and upregulating IL-10 secretion, which could be another important mechanism in the AF-1 inhibiting effect on inflammation.
Keywords: Antiflammin-1, Cytokine, Lipopolysaccharides, Macrophages, Alveolar, Toll-like receptor-4.
*Corresponding author. Tel.: +86 731 2355051; fax: +86 731 2355056.
Regulation of the pulmonary host defense mechanism is crucial for protection of the lung without pathological consequences. This is exemplified in the normal lung by the induction of both pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1, IL-6, IL-8 and IL-12, and the anti-inflammatory cytokine IL-10 by bacterial lipopolysaccharide (LPS) (Lloret and Moreno, 1992; Losa et al., 1999). LPS is an endotoxin from the outer membrane of gram-negative organisms and a primary trigger of innate immunity and acute inflammation (Ulevitch and Tobias, 1999). In many cell types, LPS induces inflammation by binding to soluble LPS-binding proteins, which then facilitates binding to membrane-associated accessory proteins, particularly membrane CD14 (mCD14) and MD-2 and at least one signaling-competent coreceptor where the mammalian toll-like receptor-4 (TLR-4) has been implicated (Poltorak et al., 1998). This LPS receptor complex then triggers activation of myeloid differentiation protein (MyD88), MyD88-associated protein Mal, TNF-receptor-associated factor-6 (TRAF-6) and IL-1-receptor-associated kinase (IRAK), and finally activates transcriptional factors NF-κB, MAPK and IRF3 (Carter et al., 1999; Fitzgerald et al., 2001; Bozinovski et al., 2002, 2004). These signal transduction intermediary molecules in turn upregulate inflammatory mediator and chemokine synthesis and also trigger preformed mediator release. There are also molecules negatively regulating these process, such as macrophage migration inhibitory factor (MIF) (Roger et al., 2001) and MyD88 short (MyD88s) (Janssens et al., 2002), suppressor of cytokine signaling-1 (SCOS-1) (Nakagawa et al., 2002).
Alveolar macrophages (AMs) are the most abundant cells in the alveoli, distal airspaces and conducting airways. They are the first line of defense against infectious agents and other immunologic insults, which constitute 85% of the cells obtained by bronchoalveolar lavage (Grattendick et al., 2002). Normal resting-state AMs have an augmented ability to respond nonspecifically to foreign antigens through phagocytosis but a limited ability to initiate specific immune responses. However, monocytes recently recruited into the alveolar space have an enhanced ability to initiate an acquired immune response (Spiteri et al., 1992; Kradin et al., 2000). This indicates that, under the normal condition, the components in the alveolar milieu play important roles in limiting AM activation. Within these components, CC10 is one that is highlighted at present.
Clara cell 10-kDa protein/uteroglobin/Clara cell secreted protein (CC10, UG, CCSP) have been implicated in the regulation of inflammation processes. CC10 is a homodimeric protein produced by Clara cells, which are nonciliated bronchiolar cells in the lung (Singh and Katyal, 2000). CC10 mRNA is also expressed to a lesser extent in the prostate, thyroid, mammary and pituitary glands as well as in the uterus during pregnancy (Peri et al., 1993, 1995). In the lung, CC10 is one of the most abundant soluble proteins in the extracellular lining fluid of airways (Singh et al., 1990). In in vitro studies, CC10 inhibited the secretory phospholipase A
In vivo, CCSP-deficient mice generated by gene targeting showed increased inflammatory response after lung injury and viral infection (Ikegami et al., 1999). After hyperoxic exposure, CCSP (−/−) mice had reduced survival times and increased pro-inflammatory cytokine production in the lungs (Girod et al., 1996). They also had increased sensitivity to lung injury induced by ozone (Plopper et al., 2006). CC10 also plays a role in immune modulation that follows pulmonary infection. After administration of adenovirus, inflammation, neutrophil migration and pro-inflammatory cytokine production were increased in the lungs of CCSP (−/−) mice (Wang et al., 2003; Plopper et al., 2006). Collectively, these studies suggest a role for CC10 as an important constitutive protein that modulates lung host defense and inflammatory responses after lung injury.
AF-1 is a synthetic nonapeptide with similar sequence to the conserved sequence of CC10 (residues 39–47, MQMKKVLDS). This peptide corresponds to the nine C-terminal amino acids of α-helix 3 of CC10 (Chowdhury et al., 2002). It has been implicated in a broad range of cellular functions, including potent PLA2 inhibition, anti-inflammatory activities and regulating the expression of adhesion molecules on human leukocytes (Miele et al., 1990; Miele, 2000; Zouki et al., 2000). Study in our lab has showed that AF-1 intervention can reduce lung injury and inflammation induced by LPS in naphthalene resulted Clara cell-depleted mice; it also elevated IL-10 mRNA expression in the lung homogenates (data not shown). Although studies report that it shares several properties of CC10, the effect of AF-1 on cytokine expression from activated macrophages is still unclear and the mechanisms by which it modulates these cellular responses need further study.
To gain further understanding of the role of AF-1 in its anti-inflammation function, we investigated the effect of AF-1 on the regulation of cytokines expression in LPS-activated AMs, and demonstrated for the first time that AF-1 exhibits a differential effect on pro-inflammatory and anti-inflammatory cytokines expression in LPS-activated AMs. Its anti-inflammation effect is, at least in part, via upregulating IL-10 secretion and down-regulating TLR-4 expression.
2 Materials and methods
AF-1 (MQMKKVLDS) was synthesized by Huamei Biotechnic Co. (China) with a purity of >90% (HPLC). It was stored in sealed glass vials and desiccated at −20
Anti-mouse toll-like receptor-4 (TLR-4) mAb for flow cytometry and anti-mouse IL-10 mAb were purchased from Boster (China). Escherichia coli 055:B5 LPS was obtained from Sigma; IL-1β, IL-6 and IL-10 ELISA kits from R&D Systems; MMLV reverse transcriptase RNase inhibitor from Promega; cDNA Probe Synthesis from Takara, dalian; Biotin-16-dUTP from Roche; and sheared salmon sperm DNA from Invitrogen. All other reagents were of analytical grade.
2.2 AM isolation and culture
Bronchoalveolar lavage (BAL) was performed to obtain AM in culture for in vitro studies. Sprague-Dawley (SD) rats (from the Experimental Animal Center of Central South University) were euthanized by intraperitoneal (ip) injection of Na pentobarbital (50
For culture supernatant collection and isolation of RNA, cells were plated in a 100-mm tissue culture dish with a ratio of 106
2.3 RNA isolation and detection with quantitive real-time PCR
Total cellular RNA was extracted by Trizol (Qiagen Inc.). The yield and purity of the RNA were measured spectrophotometrically by absorption at 260–280
2.4 Enzyme-linked immunosorbent assay (ELISA) for cytokine proteins
To determine the production of inflammatory mediators in activated macrophages, alveolar macrophages isolated and purified from rat were stimulated with LPS as described above; culture supernatants were collected and assayed for IL-1β, IL-6 and IL-10 using ELISA kits according to the manufacturer's instructions (R&D Systems, USA). Briefly, the 96-well plates were coated overnight at 4
2.5 Flow cytometric analysis for toll-like receptor-4
AMs cultured for detecting TLR-4 expression were treated with different concentrations of AF-1 with or without being LPS-stimulated. Cells were then suspended in 0.5% BSA/PBS and 1
2.6 Statistical analysis
Data were expressed as means
3.1 Dose-dependent effects of AF-1 on LPS-induced cytokine release and expression in alveolar macrophages
To determine an appropriate concentration of AF-1 to be used in studies, AF-1 (0–10
Dose–response inhibition of lipopolysaccharide stimulated by LPS (0.5
The effects of AF-1 on cytokine mRNA expression in LPS-stimulated alveolar macrophages were also detected using quantitive real-time PCR. AF-1 inhibited mRNA expression of IL-1β and IL-6, but elevated IL-10 expression in AMs, in a dose-dependent manner (Fig. 2).
Quantitive real-time PCR analysis of IL-1β, IL-6 and IL-10 mRNA expressed in AMs. The alveolar macrophages isolated from rats were treated with LPS (0.5
In another experiment, cells were pre-incubated with LPS (0.5
Production of cytokine IL-1β and IL-6 by alveolar macrophages in response to exposure for 1
3.2 Effects of AF-1 on TLR-4 expression on LPS-stimulated alveolar macrophages
To further investigate whether the inhibitory effects of AF-1 on LPS-stimulated cytokine release were involved in impaired initiation of LPS signal transduction, LPS receptor toll-like receptor-4 on the cells surface was determined. Compared with the control, the mean fluorescence intensities of AMs were increased significantly in both the LPS-stimulated group and the LPS
TLR-4 expression on the cell surface of alveolar macrophages measured by flow cytometric analysis. Fluorescence intensity (x-axis) of the cells is plotted against cell counts (y-axis). Fluorescence signals from 5000 cells for each condition to determine mean fluorescence in density values for the fluorescence histogram. The mean fluorescence intensities for each profile were given in the up pane. AMs were treated with AF-1 (2
3.3 Endogenous IL-10 involved in the inhibitory effects of AF-1 on pro-inflammatory cytokine release from LPS-stimulated alveolar macrophages
It is known that IL-10 produced by human monocytes following activation by LPS has strong downregulatory effects on the secretion of the pro-inflammatory cytokines IL-1β and IL-6. It is suggested that it is a powerful inhibitor of inflammation (Fiorentino et al., 1991; de Waal Malefyt et al., 1991). In order to investigate whether the inhibitory effects of AF-1 on cytokine release were the result of IL-10 induction, we neutralized the bioactivity of the IL-10 protein with neutralizing monoclonal antibodies. Blockade of IL-10 significantly affects the ability of AF-1 to inhibit pro-inflammatory cytokine IL-1β release (Fig. 5). This suggests that the inhibitory effect of AF-1 is partially dependent on the production of IL-10.
Effect of AF-1 on LPS-induced release production of IL-1β and IL-6 after IL-10 was blocked. The alveolar macrophages isolated from rats were purified and cultured in fresh medium or fresh medium plus AF-1 (2.0
Our study has demonstrated that AF-1 intervention results in changes to cytokine production in LPS-stimulated alveolar macrophages. These changes to cytokine expression and release in AMs suggest that AF-1 has the potential to impact the response of AMs to LPS. In addition, we observed that whereas AF-1 inhibited pro-inflammatory cytokine IL-1β and IL-6 expression in AMs, it promoted IL-10 expression, an endogenous anti-inflammatory cytokine. Our data also suggest that the inhibitory effects of AF-1 on pro-inflammatory cytokines expression, mainly of IL-1, are partly dependent on endogenous IL-10 production. Furthermore, the finding that AF-1 can downregulate toll-like receptor-4 expression on the surface of AMs suggests that AF-1 alters the sensitivity of AMs to LPS. As a consequence, AF-1 reduces the intensity of LPS-induced inflammation by modulating AM response to LPS via regulating TLR-4 expression and upregulating IL-10 secretion.
Bacterial endotoxin/LPS activate inflammatory cytokine release, thus causing tissue injury and acute inflammation. The early release of pro-inflammatory cytokines in response to LPS includes TNF-α, IL-1β and IL-6, which drives a cascade of inflammatory reactions, which in turn amplifies and extends the inflammatory reaction (Lomas-Neira et al., 2006). Monocytes/macrophages play a pivotal role in such processes by coordinating the immune and inflammatory response to infection and tissue injury. The findings that AF-1 inhibits IL-1β and IL-6 and promotes IL-10 production from alveolar macrophages suggest that AF-1, a synthetic peptide derived from CC10, may be a possible agent for treatment of inflammatory diseases.
IL-10 is a potent inhibitor of monocytes/macrophages function and its induction by LPS inhibits the production of pro-inflammatory cytokines (Hofstetter et al., 2005). In addition, IL-10 has been reported to repress the expression of a large fraction of LPS-induced pro-inflammatory genes, while its absence led to the overproduction of lethal cytokines after challenge with small amounts of LPS, or in some infections (Fierer, 2006). Our in vitro observation that AF-1 promoted IL-10 release from LPS-activated alveolar macrophages raised a question as to whether the release of IL-10 was responsible for the inhibitory effects of AF-1 on LPS-stimulated pro-inflammatory cytokine release.
To test this possibility, we blocked IL-10 activity with neutralizing antibodies. The results showed that the inhibitory effect of AF-1 on the release of IL-1β was diminished – but not IL-6 – by blocking the biological activity of IL-10 in LPS-activated AMs. Thus, the present study demonstrates that the inhibitory effect of AF-1 on pro-inflammatory cytokine release is at least partly dependent on it promoting endogenous production of IL-10 in LPS-stimulated alveolar macrophages.
TLRs are a family of proteins that recognize specific patterns of microbial components, especially those from pathogens, and regulate innate and adaptive immune responses (Takeda et al., 2003). The TLR family now consists of at least 13 members (TLR1–TLR13) in the mouse genome, whereas 11 TLRs have been found in humans (Takeda et al., 2003). The ligands for most TLRs have been identified. TLR-4 has been identified as a receptor for LPS, and TLR-4-deficient mice are hyporesponsive to LPS, demonstrating that TLR-4 is a critical receptor for LPS signaling (Hoshino et al., 1999). Studies have shown that many factors increase TLR-4 expression in lung tissue stimulated by LPS (Bozinovski et al., 2004). Our data demonstrate that LPS alone does alter the expression of TLR-4 on AMs surface, as showed by Fig. 4. This is supported by Zhang et al. (2003a), who reported that increased proteins of TLR-4 in alveolar macrophages stimulated with single-dose LPS (100
The molecular function of AF-1 is not fully understood. Previous studies indicate that AF-1 possesses biological properties including inhibition of phospholipase A2, anti-inflammatory activities and regulation of the expression of adhesion molecules on human leukocytes. A contradictory effect of AF-1 on phospholipase A2 has also been reported (Rivers et al., 2002). Meanwhile, the relation of sPLA2 activation with cytokine release is still undetermined. Recent studies have proved that sPLA2 induced the production of cytokines and chemokines in human macrophages by a nonenzymatic mechanism (Moreno, 2006). Recently, researchers found that LPS-induced MAPK activation, the production of endogenous IL-10 and STAT-3 activation all play critical roles in SOCS-3 expression, which provides for feedback attenuation of cytokine-induced immune and inflammatory responses in macrophages (Qin et al., 2007). Ray et al. (2006) reported that UG (CC10) can downregulate SOCS-3 gene expression. These studies suggest that the inhibitory effects of AF-1 might be via SOCS-3 expression regulation. Our recent findings, that AF-1 can specifically bind to uteroglobin-binding protein and induce extracellular signal-regulated kinase 1 and 2 (Erk1/2) phosphorylation in NIH3T3 cells (Li et al., 2007), provide another possible signal transduction pathway to explain the mechanism of AF-1 inhibitory effects. Overall, the inhibitory effects of AF-1 on pro-inflammatory cytokines release are complicated and need to be further investigated.
In summary, this study provides new insights into the anti-inflammation mechanism of AF-1, by which AF-1 modulates AMs response to LPS and, as a consequence, inhibits inflammatory response in the lung after infection. It also suggests that AF-1 may be a potential candidate for the treatment of lung inflammation.
This work was supported by the National Natural Science Foundation of China (Proj. nos. 30440027 and 30400190).
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Received 13 December 2007/11 March 2008; accepted 25 April 2008doi:10.1016/j.cellbi.2008.04.023