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Originally published online as doi:10.1189/jlb.0204065 on April 23, 2004

Published online before print April 23, 2004
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(Journal of Leukocyte Biology. 2004;76:135-144.)
© 2004 by Society for Leukocyte Biology

Interleukin-17 regulates expression of the CXC chemokine LIX/CXCL5 in osteoblasts: implications for inflammation and neutrophil recruitment

Matthew J. Ruddy*, Fang Shen{dagger}, Jeffrey B. Smith{ddagger}, Ashu Sharma{dagger} and Sarah L. Gaffen*,{dagger},1

* Departments of Microbiology and Immunology, School of Medicine and Biomedical Sciences, and
{dagger} Oral Biology, School of Dental Medicine, University at Buffalo, SUNY, New York; and
{ddagger} Division of Neonatology of the Department of Pediatrics, David Geffen School of Medicine and Mattel Children’s Hospital at UCLA, University of California, Los Angeles

1Correspondence: Department of Oral Biology, University at Buffalo, SUNY, 36 Foster Hall, 3435 Main Street, Buffalo, NY 14214. E-mail: sgaffen{at}buffalo.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interleukin (IL)-17 is the founding member of an emerging family of inflammatory cytokines whose functions remain poorly defined. IL-17 has been linked to the pathogenesis of rheumatoid arthritis, and numerous studies implicate this cytokine in inflammation-induced bone loss. It is clear that a major function of IL-17 is to amplify the immune response by triggering production of chemokines, cytokines, and cell-surface markers, ultimately leading to neutrophil chemotaxis and inflammation. As an IL-17 signaling deficiency in mice causes a dramatic reduction in neutrophil chemotaxis and a consequent increased susceptibility to bacterial infection, it is important to define gene targets involved in IL-17-mediated neutrophil trafficking. Here, we demonstrate that IL-17 and tumor necrosis factor {alpha} (TNF-{alpha}) cooperatively induce the lipopolysaccharide-inducible CXC chemokine (LIX; a.k.a., CXC chemokine ligand 5, Scya5, or murine granulocyte chemotactic protein-2) in the preosteoblast cell line MC3T3. LIX is induced rapidly at the mRNA and protein levels, likely through the activation of new gene transcription. Conditioned media from MC3T3 cells treated with IL-17 and/or TNF-{alpha} stimulates neutrophil mobility potently, and LIX is a significant contributing factor to this process. In addition, IL-17 cooperates with bacterial components involved in periodontal disease to up-regulate LIX expression. This study is the first demonstration of LIX expression in bone cells and has implications for inflammatory bone diseases such as arthritis and periodontal disease.

Key Words: TNF-{alpha} • cytokine • synergy • bone cells • GCP-2


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Defense against invading pathogens requires that the innate and adaptive immune responses act in a highly coordinated manner. Innate immunity functions by recognizing microbial determinants through relatively nonspecific mechanisms such as Toll-like receptors and other pattern recognition receptors. This is achieved in part by the synthesis and release of chemokines such as interleukin (IL)-8 and proinflammatory cytokines such as tumor necrosis factor {alpha} (TNF-{alpha}) and IL-1ß. These factors subsequently cause the recruitment of neutrophils and thus the rapid onset of inflammation. The adaptive immune response, characterized by specificity and memory, is mediated primarily by T and B lymphocytes, which also secrete immunoregulatory cytokines. A great deal of research has focused on individual components of these systems, but far less is known about how they work in concert.

IL-17 is the founding member of an emerging family of inflammatory cytokines whose biological activities remain incompletely defined. IL-17 is a T cell-derived cytokine produced predominantly by the T memory compartment [1 , 2 ] (reviewed in ref. [3 ]). In contrast, its receptor (the IL-17R) is expressed in a ubiquitous manner, making almost any cell type a potential target of this cytokine [4 ]. Although the functions of IL-17 are not fully understood, it is clear that IL-17 amplifies the immune response by inducing the expression of other cytokines (e.g., IL-6, TNF-{alpha}, IL-1ß), chemokines [e.g., regulated on activation, normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), macrophage-inflammatory protein-2 (MIP-2)/IL-8, KC/growth-related oncogene (GRO){alpha}], inflammatory cell-surface markers [e.g., receptor activator of nuclear factor (NF)-{kappa}B ligand (RANKL), intercellular adhesion molecule-1 (ICAM-1)], and inflammatory mediators {e.g., prostaglandin E2, nitric oxide, cyclooxygenase (COX)-2; reviewed in refs. [3 , 5 ]}. Furthermore, IL-17 exhibits a remarkable capacity to modulate the immune response by cooperating with other cytokines to promote inflammation. For example, IL-17 and TNF-{alpha} synergistically induce the production of IL-6, suggesting a bridge between innate and adaptive cytokines [6 7 8 ]. Mechanistically, IL-17 and other cytokines have been shown to enhance the transcription and mRNA transcript stability of IL-6, COX-2, and GRO{alpha} [9 10 11 ], suggesting that costimulation of cytokines works at multiple levels of regulation. In addition to TNF-{alpha}, IL-17 has been shown to synergize with interferon-{gamma} in keratinocytes, corneal fibroblasts, and intestinal epithelial cells [12 13 14 ]. Thus, it is clear that IL-17 collaborates with numerous cytokines to coordinate immune responses.

IL-17 plays a key role in the pathogenesis of a number of chronic inflammatory diseases. For example, bioactive IL-17 has been detected in rheumatoid arthritis (RA) and osteoarthritis synovial fluids [7 , 15 , 16 ]. Indeed, IL-17 appears to play a causative role in arthritis, as blocking TNF-{alpha} and IL-17 in a mouse model of RA is more effective in controlling synovial inflammation and bone resorption than blocking TNF-{alpha} alone [17 ]. In addition, collagen-induced arthritis is suppressed in IL-17-deficient mice [18 ]. Furthermore, IL-17 plays a role in inflammatory pulmonary diseases that are mediated by the recruitment of neutrophils. Specifically, IL-17 levels are increased in the lungs of patients with asthma and in airways inflamed as a result of exposure to organic dust [19 , 20 ]. IL-17 overproduction has also been associated with systemic sclerosis, psoriasis, and tumor growth [21 22 23 24 25 26 27 28 ].

Several lines of evidence implicate IL-17 in host immunity to infection. Elevated levels of the cytokine have been found in the mucosa of Helicobacter pylori-infected patients [29 ]. In addition, IL-17 is overexpressed in corneas of patients suffering from herpetic stromal keratitis. Notably, IL-17 stimulation of human corneal fibroblasts exhibited a strong, synergistic effect with TNF-{alpha} in the induction of IL-6 and IL-8 [13 ]. The most convincing evidence of a role for IL-17 in host defense comes from studies in IL-17R-deficient (IL-17R–/–) mice. These mice are highly sensitive to intranasal Klebsiella pneumoniae infection, showing enhanced mortality in comparison with wild-type controls [30 , 31 ]. One mechanism of this mortality is a significant delay in neutrophil recruitment, as a result, in part, of a reduction of granulocyte-colony stimulating factor (G-CSF) and MIP-2.

In an effort to understand how IL-17 works with other inflammatory cytokines, we recently performed microarray experiments to identify genes regulated cooperatively by IL-17 and TNF-{alpha} [6 ]. As described here, one of the genes found in this search was lipopolysaccharide (LPS)-induced CXC chemokine, or LIX [also known as CXC chemokine ligand 5 (CXCL5), Scyb5, or murine granulocyte chemotactic protein-2 (GCP-2)]. LIX was originally cloned as a glucocorticoid-attenuated response gene induced in Swiss 3T3 cells by LPS [32 ] and was subsequently shown to be expressed in multiple tissues during endotoxemia [33 ]. Like other members of the glutamate-leucine-arginine (ELR)+ subset of CXC chemokines, of which the best studied is IL-8 [34 , 35 ], LIX has potent, chemoattractant activity for neutrophils in vitro and in vivo [36 , 37 ]. LIX is the sole murine homologue of two closely related human chemokines, epithelial cell-derived neutrophil-activating peptide-78 (ENA-78; CXCL5) and GCP-2 (CXCL6), which appear to have originated via an evolutionarily recent gene duplication [38 39 40 ]. It is interesting that GCP-2 is induced by IL-17 in human bronchial epithelial cells [41 ], and ENA-78, like IL-17, appears to play a significant role in RA [42 ]. Because of the importance of IL-17 and LIX in neutrophil recruitment as well as their roles in inflammatory bone disease, we sought to define further the mechanisms by which IL-17, TNF-{alpha}, and bacterial components regulate LIX expression and biological function in a bone cell background.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture reagents and stimulations
Mouse calvaria-derived osteoblastic MC3T3-E1 cells were cultured in {alpha} minimum essential medium ({alpha}MEM; Sigma Chemical Co., St. Louis, MO) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gemini Bioproducts, Woodland, CA), penicillin, streptomycin, and L-glutamine (Gibco/Invitrogen, Carlsbad, CA). Recombinant human IL-17-, TNF-{alpha}-, and IL-6-neutralizing antibodies were obtained from R&D Systems (Minneapolis, MN). Polymyxin B (PMB) was from Sigma Chemical Co. and was used at a concentration of 10 µg/ml. BspA was purified as described previously [43 ]. For stimulations, cells were seeded onto 10 cm dishes and grown to confluence in {alpha}MEM/10% FBS. Following attachment, cells were washed twice in phosphate-buffered saline (PBS), incubated in {alpha}MEM/0.3% FBS overnight, and then stimulated with the indicated cytokines or other reagents for the designated time periods.

Microarray analysis
MC3T3 cells were grown to confluence in T175 flasks (~107 cells per sample). They were incubated for 16 h in {alpha}MEM/0.3% FBS and stimulated with TNF-{alpha} (2 ng/ml) or TNF-{alpha} (2 ng/ml), together with IL-17 (200 ng/ml) for 2 h, and total cellular RNA was prepared using the RNeasy kit (Qiagen, Valencia, CA). Relative mRNA levels were assessed using the Affymetrix murine gene chip U74v2, processed and performed by the Roswell Park Cancer Institute Affymetrix Microarray facility (Buffalo, NY). Data sets were analyzed using the Microarray Suite software (version 5.0).

Northern blotting
Total cellular RNA was prepared using the RNeasy kit (Qiagen). RNA (10 µg per sample) was separated on a 1.4% denaturing formaldehyde agarose gel, transferred to nylon membrane (Zeta Probe, Bio-Rad, Hercules, CA), and probed with 32P-labeled cDNA probes corresponding to LIX or glyceraldehyde-3-phosphate dehydrogenase (GAPD; American Type Culture Collection, Manassas, VA). Probes were labeled using the Megaprime labeling system (Amersham, Piscataway, NJ).

Enzyme-linked immunosorbent assay (ELISA) analysis
For stimulations, cells were seeded at 1 x 106 cells/ml in {alpha}MEM/10% FBS. Following attachment, cells were washed twice in PBS, incubated in {alpha}MEM/0.3% FBS overnight, and then incubated with the indicated cytokines for 24 h. ELISAs were performed in triplicate using commercial kits (R&D Systems) according to the manufacturer’s instructions. Plates were analyzed on an ELISA plate reader at OD450.

Real-time polymerase chain reaction (PCR)
Total RNA was isolated using RNeasy kit (Qiagen). Single-stranded cDNA was synthesized using reverse transcriptase (M-MuLV, Fermentas, Hanover, MD) and random hexamer primers. Real-time PCR was performed using the iCycler iQ real-time PCR detection system and iQ SYBR green supermix (Bio-Rad) according to the manufacturer’s protocol. Each reaction was performed in triplicate. For the negative control, MuMLV was omitted, and a control cDNA dilution series was created for each gene to establish a relative standard curve. PCR reactions consisted of one cycle at 95°C for 360 s, followed by 40 cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 20 s. Each reaction was subjected to melting temperature analysis to confirm the presence of unique, amplified products. For quantification, target genes were normalized to GAPD controls. Primer pair sequences were as follows: Gro{alpha}, 5'-CACCCAAACCGAAGTCATAG-3' and 5'-AAGCCAGCGTTCACCAGA-3'; MIP-2, 5'-CGCCCAGACAGAAGTCATAG-3' and 5'-TCCTCCTTTCCAGGTCAGTTA-3'; LIX, 5'-GGTCCACAGTGCCCTACG-3' and 5'-GCGAGTGCATTCCGCTTA-3'; MCP-1, 5'-GCCTGCTGTTCACAGTTGC-3' and 5'-TGTATGTCTGGACCCATTCCT-3'; RANTES, 5'-CACCACTCCCTGCTGCTT-3' and 5'-ACACTTGGCGGTTCCTTC-3'.

Neutrophil migration assays
MC3T3 cells were seeded at 1 x 106 cells/ml in {alpha}MEM/10% FBS. Following attachment, cells were washed twice in PBS, incubated in {alpha}MEM/0.3% FBS overnight, and stimulated with the indicated cytokines for 24 h, and supernatants were collected. Bone marrow-derived neutrophils were isolated by magnetic bead separation using anti-Gr-1 antibodies (BD/PharMingen, clone RB6-8C5) and a magnetic cell sorter mass spectroscopy column (Miltenyi Biotec, Auburn, CA). Conditioned supernatants were added to the lower well of a transwell chamber (Corning polycarbonate membrane transwell 3421) and ~6 x 104 neutrophils were added to the upper chambers. For blocking experiments, 30 µg neutralizing LIX antibodies and/or 30 µg KC/Gro{alpha} antibodies (MAB433 and MAB4531, R&D Systems) were added to the lower chambers. After a 10- to 20-min incubation at 37°C, neutrophils in the lower chamber were counted under an inverted microscope under 40x magnification in 10 randomly chosen fields.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To identify genes regulated by IL-17, we previously conducted an Affymetrix microarray study in the MC3T3-E1 preosteoblast bone cell line [6 ]. As much previous work has demonstrated that IL-17 induces the strongest signals when working in concert with other cytokines, we analyzed mRNA samples from cells stimulated with TNF-{alpha} alone versus TNF-{alpha} + IL-17 for 2 h. It is important that the combinatorial activity of cytokines better reflects the in vivo inflammatory environment, where cytokines and chemokines influence each other’s activities in a highly complex network. The Affymetrix MG-U74v2 chip was hybridized with separately prepared samples on two different occasions with highly similar results. As we previously reported, a number of genes known to be regulated by IL-17 and/or TNF-{alpha} were up-regulated in cells treated with TNF-{alpha} + IL-17 as compared with cells treated with TNF-{alpha} alone, including IL-6 and the chemokines Gro{alpha} (KC) and RANTES [6 ]. In addition, the CXC chemokine LIX (CXCL5) was enhanced an average of 5.7-fold (Fig. 1A ). It is important that regulation of LIX has not previously been linked to IL-17 signaling and thus represents a novel connection between IL-17 and the chemokine network.



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  		Figure 1. LIX is up-regulated by combined stimulation with IL-17 and TNF-{alpha}. (A) Identification of LIX on Affymetrix microarrays. MC3T3 cells were grown to confluence in T175 flasks (~107 cells per sample). They were then incubated for 16 h in {alpha}MEM/0.3% FBS and stimulated with TNF-{alpha} (2 ng/ml) alone or TNF-{alpha} (2 ng/ml) together with IL-17 (200 ng/ml) for 2 h before harvesting for preparation of total cellular RNA. Relative mRNA levels were assessed using the Affymetrix murine gene chip U74v2. The increase of LIX induced in TNF-{alpha} + IL-17-induced samples as compared with TNF-{alpha}-induced samples is shown (averaged from two experiments). (B) Synergistic up-regulation of LIX. MC3T3 cells were stimulated for 2 h with no cytokines (U, lane 1), increasing concentrations of IL-17 (50–500 ng/ml, lanes 2–5), increasing concentrations of TNF-{alpha} (0.02–2 ng/ml, lanes 6–8), or increasing TNF-{alpha} (0.02–2 ng/ml) together with IL-17 (200 ng/ml, lanes 9–11). Total mRNA was prepared and separated on a denaturing agarose gel and visualized on Northern blots with 32P-labeled cDNA probes corresponding to LIX (upper) or GAPD (lower). (C) Quantitation of LIX expression. Scanning densitometry was performed on B, and the intensities of the LIX bands were normalized to the intensities of the corresponding GAPD band.

 
To confirm results from the microarray and to examine the effects of individual cytokines on LIX expression, we performed quantitative real-time PCR analysis of mRNA from MC3T3 cells stimulated for 2 h without cytokines or with TNF-{alpha} and/or IL-17 (Table 1 ). IL-17 and TNF-{alpha} alone induced considerable LIX mRNA over unstimulated levels (73-fold and 125-fold, respectively), and the combination showed an even greater enhancement (1686-fold over unstimulated). It is not surprising that the magnitude of LIX mRNA up-regulation was higher by real-time PCR analyses than by microarray (13.4-fold vs. 5.7-fold, respectively), as Affymetrix microarrays are well known to underestimate differences among samples.


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  		Table 1. Expression of Chemokines in MC3T3 Cells in Response to IL-17 and TNF-{alpha}

 
Next, we performed a Northern blot of mRNA prepared from MC3T3 cells treated with various concentrations of IL-17 and/or TNF-{alpha}. Consistent with the previous data, LIX mRNA was up-regulated in a dose-dependent manner following a 2-h stimulation of IL-17 and TNF-{alpha} (Fig. 1B) . Scanning densitometry of the X-ray film showed that the combination of TNF-{alpha} and IL-17 resulted in a synergistic induction of LIX mRNA (Fig. 1C) . This finding was consistent with the real-time PCR analysis (Table 1) , although the degree of synergy was lower than that observed by the latter method. Together, these data suggest the potential for amplification of the inflammatory response through the cooperation of the innate immune system (as represented by TNF-{alpha}) and the adaptive immune system (as represented by IL-17).

To determine the kinetics of LIX expression, we examined the time-course of LIX up-regulation by Northern blotting (Fig. 2A ). LIX mRNA was mildly enhanced by TNF-{alpha} alone as early as 30 min post-stimulation. In addition, a synergistic induction of LIX mRNA was observed following IL-17 + TNF-{alpha} stimulation at this time-point. The same trend was observed at the 2- and 4-h time-points, and maximal induction occurred by 2 h (Fig. 2B and data not shown). Thus, LIX mRNA is up-regulated rapidly following IL-17 + TNF-{alpha} stimulation.



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  		Figure 2. Time-course of LIX up-regulation. (A) LIX expression peaks at 2 h post-stimulation. MC3T3 cells were stimulated for 0.5, 1, 2, and 4 h without cytokines (U, lanes 1, 5, 9, 13) or with IL-17 (17; 200 ng/ml, lanes 2, 6, 10, 14), TNF-{alpha} (T; 2 ng/ml, lanes 3, 7, 11, 15), or TNF-{alpha} (T+17; 2 ng/ml) plus IL-17 (200 ng/ml, lanes 4, 8, 12, 16). Total mRNA analyzed by Northern blotting as described in Figure 1B . Note that all samples are derived from the same film. (B) Quantitation of LIX expression. Scanning densitometry was performed on A, and the intensities of the LIX bands were normalized to the intensities of the corresponding GAPD band.

 
Rapid induction of LIX gene expression could be a result of new gene transcription and/or enhancement of mRNA stability. Previous reports indicated that IL-17 signaling increases the stability of cytokine transcripts in a myofibroblast cell line, and chemokine mRNA stability is often regulated by proinflammatory cytokines such as TNF-{alpha} [10 , 44 ]. However, IL-17 and TNF-{alpha} did not promote a strong increase in the stability of LIX mRNA (Fig. 3A and 3B ). Specifically, the kinetics of LIX mRNA degradation in samples treated with the transcriptional inhibitor actinomycin D was only slightly different between cells treated continuously with IL-17 and TNF-{alpha} and those left untreated. We made very similar observations when performing this experiment using real-time PCR (data not shown). This finding suggests that costimulation of MC3T3 cells with IL-17 and TNF-{alpha} does not markedly enhance the stability of LIX mRNA, and thus, it is likely that new gene transcription is mainly responsible for the rapid enhancement of LIX.



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  		Figure 3. Mechanism of LIX induction by IL-17 and TNF-{alpha}. (A) IL-17 + TNF-{alpha} stimulation does not stabilize LIX mRNA. MC3T3 cells were stimulated for 2 h without cytokines (U, lane 1) or with TNF-{alpha} (T; 2 ng/ml) + IL-17 (17; 200 ng/ml; Pre, lanes 2–9). Following stimulation, cells were washed twice with PBS and treated with Actinomycin D for the durations indicated. Lanes 6–9 were incubated without cytokines, and lanes 2–5 were stimulated with TNF-{alpha} (2 ng/ml) plus IL-17 (200 ng/ml; Post). Total mRNA was visualized on Northern blots as described in Figure 1B . (B) Kinetics of LIX mRNA degradation. Scanning densitometry was performed on the gel in A. LIX expression in each sample from A is presented as a percentage of LIX message in the samples without Actinomycin D treatment (lanes 2 and 6, respectively). The slope of the best linear curve fit for each set of conditions is shown. The solid line indicates samples post-treated without cytokines (lanes 6–9), and the dashed line indicates samples post-treated with IL-17 + TNF-{alpha} (lanes 2–5). (C) Induction of LIX mRNA by IL-17 and TNF-{alpha} does not require IL-6 production. MC3T3 cells were pretreated for 2 h with neutralizing antibodies to IL-6 (lanes 5 and 6) and were then treated for 2 h without cytokines (U, lane 1), 200 ng/ml IL-17 (17; lane 2), 2 ng/ml TNF-{alpha} (T; lane 3), TNF-{alpha} plus IL-17 (T+17; lane 4), or TNF-{alpha} plus IL-17 and an IL-6-neutralizing antibody ({alpha}-IL-6; 0.5 and 5 µg/ml, lanes 5 and 6). Total mRNA was separated on a denaturing agarose gel and visualized on Northern blots as described in Figure 1B . (D) Quantitation of LIX expression. Scanning densitometry was performed on C, and the intensities of the LIX bands were normalized to the intensities of the corresponding GAPD band.

 
To define the mechanisms by which TNF and IL-17 up-regulate LIX mRNA, we examined whether protein synthesis is necessary for this event. First, as IL-17 and TNF-{alpha} induce IL-6 production in MC3T3 cells [6 , 45 ], we examined whether IL-6 was responsible for driving induction of LIX through an autocrine feedback mechanism. However, pretreating cells with a neutralizing antibody to IL-6 did not diminish expression of LIX following a 2-h stimulation with IL-17 and TNF-{alpha} (Fig. 3C , lanes 5 and 6). In addition, we attempted to determine whether new protein synthesis is required through the use of the protein synthesis inhibitor cycloheximide. However, cycloheximide treatment alone induced LIX mRNA in MC3T3 cells (data not shown), a finding that contrasts with observations in Swiss 3T3 fibroblasts [32 ]. A similar phenomenon of "superinduction" has been observed with other cytokines such as IL-6 and is predominantly a result of nonspecific stabilization of pre-existing mRNA by this drug [46 ]. Therefore, the requirement for new protein synthesis remains unresolved.

Next, we examined up-regulation of LIX protein expression. ELISA analysis of MC3T3 supernatants indicated that IL-17 and TNF-{alpha} drive synergistic production of LIX protein at time-points only slightly later than mRNA production (Fig. 4A ). This result is consistent with previous observations that IL-17 acts to amplify the inflammatory response and drive the synergistic production of mediators such as IL-6 and Gro{alpha} (reviewed in refs. [47 , 48 ]).



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  		Figure 4. Chemokine expression induced by IL-17 and TNF-{alpha}. (A) IL-17 + TNF-{alpha} stimulation synergistically up-regulates LIX. MC3T3 cells were incubated without cytokines (U) or with IL-17 (17; 200 ng/ml) and/or TNF-{alpha} (T) for the indicated time-points. (B) IL-17 + TNF-{alpha} stimulation synergistically up-regulates Gro{alpha}. The supernatants used in A were analyzed in triplicate for Gro{alpha} by ELISA. (C) IL-17 + TNF-{alpha} stimulation synergistically up-regulates MIP-2. The supernatants used in A were analyzed in triplicate for MIP-2 by ELISA.

 
Like other ELR+ CXC chemokines, LIX is a potent chemotactic factor for neutrophils [36 , 37 ]. To confirm that IL-17 + TNF-{alpha}-stimulated MC3T3 cells regulate neutrophil activity, we performed neutrophil migration assays (Fig. 5 ). Specifically, neutrophils isolated from mouse bone marrow by magnetic bead separation were added to the top wells of a transwell plate, and conditioned supernatants from MC3T3 cells were added to the lower chambers. After a timed incubation of 10–20 min, cells in the lower chambers were counted in multiple random microscope fields. As expected, supernatants from untreated MC3T3 cells triggered very little neutrophil migration to the lower chambers. However, neutrophil migration increased in response to media from cells treated with IL-17 or TNF-{alpha} alone and was synergistically enhanced by conditioned media from cells treated with both agents (Fig. 5A) . Importantly, the migration of neutrophils was partially blocked by coincubation with a LIX neutralizing antibody, demonstrating cytokine-specific induction of functional LIX (Fig. 5B) . However, as blocking LIX alone did not result in a complete inhibition of migration, we examined the effect of blocking Gro{alpha}, another chemokine identified in the microarray study (ref. [6 ] and Table 1 ). In most experiments, neutrophil migration was only marginally inhibited by incubation with a Gro{alpha}-neutralizing antibody, but coincubation with LIX and Gro{alpha} antibodies further reduced neutrophil migration as compared with the LIX antibodies alone in a statistically significant manner (Fig. 5B) , indicating that the effects of these chemokines are nonredundant. These results demonstrate that MC3T3 cells stimulated with IL-17 and/or TNF-{alpha} secrete factors that induce neutrophil migration and that LIX contributes significantly to this process. This result is also consistent with observations in vivo showing that IL-17 signaling is required for effective neutrophil recruitment [31 ].



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  		Figure 5. Neutrophil migration is induced by IL-17 and TNF-{alpha}. (A) MC3T3 cells induce neturophil migration. MC3T3 cells were incubated with IL-17 (200 ng/ml) and/or TNF-{alpha} for 24 h. Cell supernatants were added to the lower well of the transwell chamber, and bone marrow-derived neutrophils were added to the upper chamber. Neutrophils in the lower chamber were counted in a minimum of 10 randomly chosen microscope fields after 15 min. (B) LIX contributes to neutrophil migration. MC3T3 cells were stimulated as in A, and supernatants were added to the lower well of the transwell chamber together with neutralizing antibodies to LIX and/or Gro{alpha} as indicated. Neutrophils in the lower chamber were counted in a minimum of 10 randomly chosen microscope fields after 20 min. This value was converted to a percentage of total input neutrophils as a function of the relative area of the microscope field and the transwell. *, Statistically significant difference between the sample without anti-LIX antibodies and the sample with anti-LIX antibodies as determined by Student’s t-test followed by Mann-Whitney Rank Sum test (P=0.006). **, Statistically significant difference between the sample with anti-LIX antibody and the sample with anti-LIX plus anti-Gro{alpha} antibody (P=0.004).

 
As there was residual, chemotactic activity in the conditioned supernatants even after blocking LIX and Gro{alpha}, we examined the expression of other neutrophil-activating chemokines induced in MC3T3 cells by quantitative real-time PCR (Table 1) and ELISA (Fig. 4B and 4C) . Consistent with other findings, expression of MIP-2 and Gro{alpha} was synergistically enhanced by IL-17 and TNF-{alpha} stimulation in a pattern similar to LIX. In general, the magnitude of change was more dramatic in the RNA samples as compared with the protein samples. For example, LIX mRNA was enhanced 13.4-fold in the TNF-{alpha}/IL-17-stimulated samples as compared with the TNF-{alpha}-stimulated samples as measured by real-time PCR analysis, but LIX protein was only enhanced 2.4-fold by ELISA. Similarly, Gro{alpha} mRNA was up-regulated 10.7-fold in the IL-17/TNF-{alpha} samples compared with the TNF-{alpha} samples, but protein was enhanced only 7.5-fold. These differences may reflect different turnover rates for mRNA versus protein as well as inherent experimental variation. The chemokines RANTES and MCP-1 do not act primarily on neutrophils, and we found that TNF-{alpha}/IL-17 stimulation only exerted a mild cooperative effect on their up-regulation, although TNF-{alpha} induced both chemokines substantially over basal levels (Table 1) .

IL-17 and TNF-{alpha} are elevated at sites of infection. For example, IL-17 mRNA is significantly up-regulated in peripheral blood mononuclear cells stimulated with Porphyromonas gingivalis outer membrane protein derived from patients with periodontal disease [49 ]. However, little is known about whether IL-17 cooperates with oral microbial components to induce new gene expression. Therefore, we assessed LIX expression in MC3T3 cells following stimulation with various microbial components, alone or in conjunction with IL-17 (Fig. 6 ). First, as expected, purified LPS from E. coli induced LIX in a dose-dependent manner (Fig. 6A , lanes 6–8), and costimulation of LPS with IL-17 resulted in a further, approximately additive increase of LIX gene expression. BspA is a cell-surface molecule from the gram-negative anaerobe Bacteroides forsythus [43 ]. This microorganism is associated with periodontitis, and its presence correlates with the severity of disease [50 , 51 ]. Our data indicate that purified BspA is also capable of inducing LIX, and costimulation of cells with BspA and IL-17 additively enhances LIX expression (Fig. 6A , lanes 9–11). It should be noted that this is not a nonspecific effect of contaminating LPS in the BspA preparation, as the addition of PMB did not appreciably affect LIX expression (Fig. 6A , lane 11). Thus, IL-17 potentiates the ability of microbial components to induce LIX and may be important in the inflammatory response to infectious pathogens.



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  		Figure 6. IL-17 and microbial components cooperatively induce LIX mRNA. (A) MC3T3 cells were stimulated for 2 h without cytokines (U, lane 1) or with IL-17 (17; 200 ng/ml, lanes 2, 6–8, 10, and 11), increasing concentrations of Escherichia coli LPS (100, 500, and 1000 ng/ml, lanes 3–8) or purified BspA (10 µg/ml, lanes 9–11). PMB was added to ensure that effects were not a result of contaminating LPS (lane 11). Total mRNA was visualized on Northern blots as described in Figure 1B . (B) Quantitation of LIX expression. Scanning densitometry was performed on the gel in A, and the intensities of the LIX bands were normalized to the intensities of the corresponding GAPD band.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
IL-17 is a key mediator in inflammation and host defense against bacterial organisms. Proposed models suggest that during infection, bacterial products directly induce a subset of T cells to secrete IL-17, which then acts on the microenvironment of infected tissue and hematopoietic organs to release secondary mediators such as chemokines and G-CSF, which in turn activate and recruit neutrophils [52 ]. In addition, IL-17 works in conjunction with many innate immune system cytokines such as TNF-{alpha} and IL-1ß to amplify the immune response and the inflammatory process. However, the full spectrum of molecular mechanisms that controls inflammation and neutrophil recruitment and activation remains incompletely defined.

Previous studies have shown that IL-17 up-regulates numerous chemokines such as Gro{alpha}, MIP-2, MCP-1, and RANTES, which participate in the recruitment of neutrophils and other leukocytes (reviewed in ref. [3 ]). It is interesting that depending on the microenvironment, the accumulation of neutrophils can be pathological or protective to the host. For example, in severe acute asthma and chronic obstructive pulmonary disease, the infiltration of airway neutrophils leads to the release of proteases and reactive oxygen-free radicals, which contribute to mucus secretion, airway remodeling, and lung tissue destruction [53 ]. Although there is some evidence that neutrophils are pathological in certain types of aggressive periodontal disease (reviewed in ref. [54 ]), neutrophil infiltration is generally considered to be protective against periodontitis as a result of the ability of these cells to phagocytose and kill oral bacteria (reviewed in ref. [55 ]). To understand the complex role of the cytokine/chemokine network in dictating neutrophil function, it is important to define agonists of neutrophil recruitment and subsequently identify genes associated with chemotactic activity. In this study, we have begun to characterize mechanisms by which inflammatory cytokines and microbial components act in concert to regulate LIX, the murine homologue of the human neutrophil chemoattractant chemokines ENA-78 and GCP-2.

The expression of chemokines is mediated at numerous levels, including mRNA stabilization (reviewed in ref. [56 ]). Specific 3'-untranslated regions correlate with message destabilization and often involve AU-rich regions containing or adjacent to the pentamer AUUUA (reviewed in ref. [57 ]). It is interesting that the LIX mRNA contains five AUUUA sequences, which may be targets for specific RNA-binding proteins that affect transcript stabilization [32 ]. However, based on our studies, IL-17 and TNF-{alpha} to do not strongly affect LIX mRNA stability (Fig. 3) , and thus, LIX expression is more likely to be controlled at the level of transcription, at least in MC3T3 cells. In this regard, the transcription factors mediating LIX expression are not well defined. The NF-{kappa}B pathway has been implicated in TNF-{alpha}-driven LIX expression [37 ], and the 5'-flanking region of the lix gene contains conserved NF-{kappa}B and GATA-binding sites [39 ]. Although NF-{kappa}B may be important, it is unlikely to be sufficient for LIX regulation in MC3T3 cells, as we have previously shown that synergy between IL-17 and TNF-{alpha} appears not to be mediated by the NF-{kappa}B pathway [6 ]. To date, we have not examined the possible role of GATA-1 in LIX transcription. However, other transcription factors may also contribute to the synergistic induction of LIX. For instance, we have found that members of the CCAAT/enhancer-binding protein (C/EBP; NF-IL-6) family are important for synergistic production of IL-6 by IL-17 and TNF-{alpha} [6 ]. Moreover, at least one C/EBP site is present in the 5'-proximal region of the LIX gene, based on results from the AliBaba2 computer program for predicting transcription factor-binding sites (data not shown). Further work is clearly needed to determine how LIX expression is controlled by IL-17, TNF-{alpha}, and microbial stimuli such as LPS and BspA.

LIX expression is regulated in a tissue-specific manner, the cellular basis of which has only been partially explored [33 , 37 ]. The present work is the first report of LIX expression in bone cells, as represented by the preosteoblast cell line MC3T3. Expression of LIX in bone may impact inflammatory diseases such as RA and periodontitis [45 , 58 ]. First, the human LIX homologue ENA-78 has been implicated in human RA [42 ]. Second, LIX is overexpressed in two mouse models of RA: namely, the human T cell leukemia virus type I transgene (Tg) and IL-1R antagonist knockout Tg mouse models [59 , 60 ] (Yoichiro Iwakura, personal communication). Third, in a rat model of adjuvant-induced arthritis, expression of ENA-78 is enhanced, and blocking ENA-78 with specific antibodies reduces severity of disease [61 ]. Not surprisingly, ENA-78 has also been found in several infection settings [62 63 64 ]. Finally, our data indicate LIX expression can be triggered by microbial components such as LPS and BspA (Fig. 6) . The induction of LIX by oral microbes in periodontal disease may serve to initiate the innate immune response and thereby promote the infiltration of bone-protective neutrophils.

Our data show that IL-17 and TNF-{alpha} synergistically up-regulate expression of LIX mRNA and protein (Figs. 1 2 3 4) . It is interesting that IL-17 has been consistently shown to cooperate with innate cytokines such as TNF-{alpha} to promote the synthesis of factors involved in neutrophil chemotaxis (reviewed in ref. [47 ]). For example, cotreatment of human bronchial epithelial cells with IL-17 and TNF-{alpha} synergistically up-regulates IL-8, Gro{alpha}, and G-CSF production, suggesting a mechanism for neutrophil recruitment in respiratory diseases [65 ]. Similarly, synergistic induction of LIX by IL-17 and TNF-{alpha} in bone cells provides a potential mechanism by which neutrophils can be recruited efficiently to inflamed bone tissue. Accordingly, LIX may be a potential target of rational therapies aimed at abrogating neutrophil-mediated inflammation in diseases such as arthritis, asthma, and inflammatory bowel disease (reviewed in ref. [66 ]). Conversely, LIX activity may be beneficial in the context of periodontal diseases, where neutrophils are largely protective (reviewed in ref. [55 ]).

In conclusion, this study identifies a mechanism by which IL-17 and TNF-{alpha} may promote the infiltration of neutrophils to areas of inflammation through the up-regulation of the CXC chemokine LIX. These findings suggest that LIX and its human homologues ENA-78 and GCP-2 may play important roles in inflammatory bone diseases such as arthritis and periodontal disease. Further investigation of the regulation of these chemokines by IL-17 may eventually lead to new strategies for potential treatments.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the National Institutes of Health (NIH; AI49329), the Immune Deficiency Foundation, and the Arthritis Foundation to S. L. G. M. J. R. was supported by the NIH Training Grant AI07614 awarded to the Witebsky Center for Microbial Pathogenesis and Immunology of the State University of New York at Buffalo. J. B. S. was supported by NIH Grant HL57008 and A. S., by NIH Grant DE014749. We thank Dr. Robert Schifferle for kindly providing E. coli LPS. We are grateful to Drs. Jonathan Snow, Xin Lin, George Hajishengallis, and Lee Ann Garrett-Sinha and members of the Gaffen Laboratory for many helpful suggestions.

Received February 3, 2004; revised March 11, 2004; accepted March 13, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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