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Inhibition of Nitric Oxide Can Ameliorate Apoptosis and Modulate Matrix Protein Gene Expression in Bacteria Infected Chondrocytes in Vitro

Posted on: Sunday, 20 March 2005, 03:00 CST

Abstract

Bacterial infection stimulates nitric oxide (NO) production in chondrocytes. However, the role of NO in chondrocyte apoptosis after infection remains unclear. The purpose of the study was to test if inhibition of NO could ameliorate apoptosis and modulate matrix protein gene expression in bacteria-infected chondrocytes. It was shown that pre-treating chondrocytes with L-NAME (1 mM) significantly decreased the release of NO (from 72 to 14 M) and the extent of apoptosis (from 52.9% to 18.9%). Pre-treatment with L- NAME also counteracted the bacteria-induced downregulation of Type II collagen (from 26% to 79%) and aggrecan (from 63% to 105%) mRNA levels. Inhibition of NO after the induction of infection could not decrease the extent of apoptosis and modulate matrix protein gene expression. The results of this study support the hypothesis that NO has an important role in bacteria-induced chondrocyte apoptosis. Pre- treatment but not post-treatment could ameliorate the extent of apoptosis and reestablish the cartilage matrix protein gene expression. This study suggests that in addition to NO, other mechanisms may be responsible for the sustained destruction of articular cartilage in the post-infectious arthropathy.

2004 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved.

Keywords: Nitric oxide; Apoptosis; Bacterial infection; Chondrocyte

Introduction

Septic arthritis is a serious medical problem where there is no adequate treatment to prevent the prolonged destruction of articular cartilage. Within 48 h of joint infection, 40% of the glycosaminoglycans can be lost from the articular cartilage and by three weeks 50'Xi of the collagen [18,21]. Although secreted bacterial toxins and the host inflammatory response account for early processes of cartilage destruction [2,5,6,22,25,26], the precise mechanisms causing the early degenerative change after septic arthritis remain unclear. In our previous study, we have demonstrated that bacteriainfected chondrocytes are susceptible to infection and undergo apoptosis [11,12]. Because the apoptotic process cannot be abolished by bacterial removal using antibiotic alone [12], a reasonable explanation is that other factors, co- expressed in or induced by bacteria, participate in the pathogenesis of chondrocyte apoptosis.

Nitric oxide (NO) is a free radical synthesized from the amino acid L-arginine by a family of enzymes, the nitric oxide synthase (NOSs). NO is a multifunctional molecule that coordinates diverse physiological processes including inflammation and cytotoxicity [4,15]. NO has been implicated as a mediator of inflammatory arthritis in several in vitro and in vivo studies [10,14,20,23]. Smith et al. in 1995 showed that a purified staph factor is a potent inducer for NO release from articular cartilage [2O]. However, the role of NO on chondrocyte apoptosis in bacterial infection is still unclear. The purpose of this study was to investigate the hypothesis that inhibition of NOS could ameliorate the extent of apoptosis and modulate matrix protein gene expression in bacteria-infected chondrocytes.

Methods

Chondrocyte isolation and culture

Cartilage was obtained from patients receiving total joint replacement for osteoarthritis after approval by the local Institutional Review Board and Research Committee. Cartilage slices were taken from sites without subchondral bone exposure. Chondrocytes were harvested by overnight digestion of cartilage slices in a digestion buffer containing DMEM (Dulbecco's Modified Eagle Medium). 10% fetal bovine serum (FBS), antibiotics, and I mg/ ml clostridial collagenase (Sigma Chemical Co., St. Louis, MO. USA) at 37 C. After serial washings with phosphate buffered saline, the cells were then resuspended in DMEM/10% FBS and cultured at 37 C in 951X, air and 5% CO2.

Study design

Chondrocytes were plated at high density (2 10^sup 4^ cm^sup - 2^) in DMEM without FBS and were divided into five groups in triplicates. All groups, except the control group, were subjected to the induction of bacteria infection using Sliiphylococciis aiireus ATCC 2921.3 in a dosage of one CFU (colony forming unit) of bacteria per one cell as described previously [12]. In the control group, chondrocytes were cultured in DMEM and not infected by bacteria. In the pre-treatment group, cells were pre-treated with a NOS inhibitor. Nω-nitro-L-arginine methyl ester (L-NAME. 1 mM; Sigma), 2 h prior to the induction of infection. In the pre- treatment/vancomycin group, vancomycin (1 mg/ml) was added 2 h after the induction of infection to the group of cells that were pre- treated with L-NAME. In the post-treatment group, both L-NAME and vancomycin were added 2 h after the induction of infection. To determine whether NO mediates chondrocyte responses to bacterial infection, dose response experiments using LNAME at 5, 10, and 20 m M were performed in the post-treatment group. In the infection group, chondrocytes were challenged with bacteria without any treatment. All groups were harvested 24 h after the induction of infection.

Analysis for apoptosis

Apoptosis was analyzed by two methods. With flow cytometric analysis of apoptotic nuclei, nonattached cells collected by centrifugation and attached cells collected by trypsinization were harvested and washed three times with PBS. The cell pellets were then resuspended in 1.5 ml hypotonie lluorochrome solution (propidium iodide 50 g/ml in 0.1% sodium citrate plus 0.1% Triton X- IOO) were subjected to flow cytometric analysis after an overnight incubation at 4 C [16]. The red fluorescence of individual nuclei traversing the light beam of a 488 nm argon laser was registered on a logarithmic scale. The forwardand right angle light scatter signals were gated to avoid registering unwanted signals such as degraded bacterial nucleic acids. All data were recorded in a Hewlett Packard computer using FACScan research software (Lysis II) and were expressed as the percentage of apoptotic nuclei. Apoptosis was further analyzed by quantifying amount of nucleosomal cleavage of DNA using a Nucleosome ELISA kit (Oncogene). In brief, cell lysates were added to a precoated ELISA plate containing DNA binding protein. Anti-histone biotin-labeled antibodies then recognized the historic component of captured nucleosome.s and were detected following incubation with a streptavidin-linked horseradish peroxidase conjugate and chromagen. tetramethylbenzidine (TMB).

Quantification of NO release

The concentration of nitrite, the stable end product of NO oxidation, was used as an indicator of NO synthesis. Nitrite in the culture medium was measured spectrophotomelrically using the Griess reaction with sodium nitrite as the standard [1O]. An aliquot of 100 l collected culture medium were incubated with 50 l of 0.1% sulfanilamide in 5% phosphoric acid and 50 l of 0.1% N-1-naphthyl- ethylenediamine dihydrochloride (Sigmal for 10 min for measurement of absorbance at 550 nm.

Analysis of mRNA expression

Total RNA was extracted with Tri-Reagent (Sigma). To investigate the mRNA expression, 800 ng of total RNA were reverse-transcribed using Omniscript reverse transcriptase (Qiagen, Valencia. CA, USA) in a reaction buffer containing 1 M random primer (GIBCO). 0.5 mM each dNTP, and 10 units ribonuclease inhibitor (GIBCO). After incubation at 37 C for 60 min. the reaction was stopped by heating at 93 C for 5 min. Multiplex polymerase chain reaction (PCR) amplification was performed in a thermocycler (PTC-K)O, MJ Research, Inc.. Waltham. MA, USA) using hotStarTaq DNA polymerase (Qiagen). The PCR profile was: initial denaturation at 95 C for 15 min. followed by 30 cycles of denaturation at 94 C (for 1 min), subsequent annealing at 58 C (for 40 s). and extension at 72 C (for 40 s). The final cycle (at 72 C) included 10 min for extension. PCR products were visualized on ethidium bromide-stained 1.2% agarose gels and the images were captured using Bio-Capt V.99 (Vilber Lourmat, Cedex. France). The signals were quantified on an imaging analysis software (Bio-ID V.99, Vilber Lourmat) and were normalized to the expression of a constitutively expressed gene, β-actin. In some experiments, another constitutively expressed housekeeping gene, GAPDH, was used as the reference for normalization of signal levels and it correlated well with the expression of β-actin. The PCR primer pairs are summarized in Table 1.

Statistical analysis

One-way analysis of variance (ANOVA) with post-hoc Bonferroni multiple comparisons was used for statistical comparisons with p < 0.05 considered to be significant.

Results

NO and NOS mRNA expression

In the control group, the basal level of the nitrite in the culture medium was 9.7 3.2 m. In this experimental setting, osteoarthritic chondrocytes increased NO release (71.721.7 M) at 24 h after the induction of bacterial infection (Fig. 1). In chondrocytes that were prc-trcated with L-NAME, infection-induced NO levels decreased to the levels in the control group. (14 2.9 M in pre-treatment; 14.8 2.6 M in pre-treatment/ vancomycin) In contrast, the nitrite levels in the posttreatment (1 mM L-NAME) group were significantly elevated compared to the pre-treatment groups (ANOVA p<0.001). However, they were stilllower than in the infection group (ANOVA p = 0.002). With increasing dose of L-NAME, the nitrite levels decreased to 12 1.3, 13.5 3.1, and 12.6 4.6 M, at 5, 10, and 20 mM of L-NAME, respectively.

Table 1

PCR Primer Pairs

Fig. 1. The release of NO after bacteria] infection in osteoarthritic chondrocytes. Ctrl = Control, Pre-treat = Pre- treatment with L-NAME, Pre-treat/ V = Pre-treatment with L-NAME/ Vancomycin. Post-treat = Post-treatment with L-NAME at the concentration of 1. 5, 10 and 20 inM. * ANOVA p<0.001 comparing "Ctrl, Pre-treat, Pre-treat/V, Post-treat (5 mM), Post-treat (10 niM). and Post-treat (20 niM)" to "Post-treat (1 mM) or Infection".

Fig. 2. Representative RT-PCR results for aggrecan, type II collagen, MMP-2, iNOS, and β-actin mRNAs. Ctrl = Control, P = Pre-treatment. P/V = Pre-treatment/Vancomycin, Pl = Post-treatment with 1 mM L-NAME, P5 = Post-treatment with 5 mM L-NAME, Inf= Infection.

In the control group, the iNOS mRNA could not be detected (Fig. 2). But interestingly, the iNOS mRNA levels were upregulated in all groups of cells that were infected by bacteria. Pre-treatment of cells with the NOS inhibitor, L-NAME, did not abolish the iNOS mRNA expression but decreased slightly.

Extent of apoptosis

The extent of apoptosis (Fig. 3A) was significantly increased from 1 0.3% in the control group to 52.9 1.31%) in the infection group as analyzed by flow cytometry study (ANOVA p < 0.001). Pre- treatment with L-NAME with or without the addition of vancomycin significantly decreased the extent of apoptosis to 18.9 7.4% and 13.1 4.31%, respectively, as compared to the infection group (ANOVA p < 0.005). The addition of L-NAME and vancomycin 2 h after the infection induction (post-treatment group) did not decrease significantly the extent of apoptosis as compared to the infection group (ANOVA p = 0.07). The extent of apoptosis analyzed by nucleosome ELISA showed similar results that pre-trcatment with L- NAME (1 mM) plus vancomycin (1 mg/mL) decreased the extent of apoptosis marginally as compared to the infection group (ANOVA p = 0.58) (Fig. 3B). All post-treatment groups with 1 mM, 5 mM, 10 mM, or 20 mM of L-NAME did not decrease the extent of apoptosis as compared to the infection group.

Fig. 3. The extent of apoptosis alter bacterial infection. (A) Flow cytometry analysis for apoptotic nuclei. * ANOVA p = 0.008 comparing the "Pretreat" to the "Ctrl". ** ANOVA p < 0.005 comparing "Ctrl, Pre-treat, Pre-treat/V" to "Post-treat and Infection". (B) Nucleosomal DNA ELISA. * ANOVA p < 0.02 comparing the "Ctrl" to other groups except the "Pre-treat/V" group. * ANOVA p = 0.058 comparing the "Pre-treat/V" to the "Infection" group.

Extracellular matrix protein gene modulation

The relative mRNA expression levels of type II collagen were 26 10% in the infection group, 79 15% in the pre-treatment group, 62 32% in the pre-treatment/vancomycin group, 47 23% in the post- treatment 1 mM L-NAME group, and 54 1% in the post-treatment 5 mM L- NAME group (Fig. 4). The relative mRNA expression levels of aggrecan were 63 18% in the infection group, 105 28% in the pre-treatment group, 93 31% in the pre-treatment/ vancomycin group, 84 21% in the post-treatment 1 mM L-NAME group, and 63 18% in the post-treatment 5 mM group (Fig. 4). The MMP-2 mRNA expression was upregulated by bacterial infection in all groups of chondrocytes with or without L- NAME or vancomycin treatment (Fig. 4).

Discussion

Even though the general responses to the current treatment of septic arthritis arc good, the post-infectious arthropathy remains to be a serious medical condition. In joints with inflammation and infection, chondrocytes release high concentrations of NO [18,20,23], Bacterial toxins such as staphylococcal proteoglycan releasing factor and lipopolysaccharide arc also known stimulators for NO synthesis in chondrocytes [20,21,23]. Since NO has been reported as the primary induccr of chondrocyte apoptosis [1] and bacterial infection could also induce apoptosis in chondrocytes [11,12], it was of interest to investigate the role of NO in bacteria-infected chondrocytes. In this study, bacterial infection increased the release of NO through the uprcgulation of iNOS mRNA. The role of NO in bacteria-induced apoptosis was demonstrated in this study since pre-treatment of chondrocytes by L-NAME could significantly decrease the release of NO and the extent of apoptosis in vancomycin treated chondrocytes infected by bacteria. But in clinical practice, it is not possible to give NOS inhibitor or antibiotic prior to the infection. In this study, the NO levels could be decreased by increasing doses of a NOS inhibitor and vancomycin added 2 h after the induction of infection. However, the post-treatment with a NOS inhibitor and vancomycin could not decrease the extent of apoptosis significantly as compared to the infection group. Apoptosis in bacteria-infected chondrocytes has been reported to involve the activation of intracellular signaling pathways such as stress-activated protein kinase [12]. Staphylococcal alpha-toxin has also been reported to induce apoptosis through the formation of membranous pores [9]. The results of this study imply that multiple pathways, including bacteria- induced NO, are involved in the triggering of apoptosis in bacteria- infected chondrocytes.

Fig. 4. Relative mRNA expression of type II collagen, aggrecan, and MMP-2. * ANOVA p < 0.05 comparing the "Ctrl. Pre-lreat" to Infection. ** ANOVA p < 0.05 comparing the "Ctrl" to "Pre-treat" and "Infection".

The impacts of NO on cartilage metabolism are pleiotropic. NO inhibits glycosaminoglycan synthesis in IL-1β treated chondrocytes [8,13], but inhibition of NO synthesis exacerbates proteoglycan catabolism by increasing levels of matrix metalloproteinases [24]. In this study, pre-treatment of bacteria- infected chondrocytes with L-NAME could ameliorate the extent of apoptosis and could modulate the extracellular matrix gene expression. It was also found that post-treatment with L-NAME and vancomycin could not affect the bacterial infection mediated downregulation of type Il collagen, aggrecan mRNA, and upregulation of MMP-2 mRNA. Cloning of the human iNOS gene showed that the 5'l- flanking region contains consensus sequences for NFκB and γ-interferon responsive clement (γ-IRE) sites, reported to be involved in LPS and γ-interferon-induced gene expression [3,17]. In that study, it was demonstrated that the upregulated NOS and MMP-2 mRNA by bacterial infection could not be abolished by L- NAME pre-treatment. It is possible that bacterial infection elicits a complex response that can not be manipulated by modification of a single pathway. Other bacterial toxins such as staphylococcal proteoglycanreleasing factor [25,26] and reactive oxygen species might also participate in the triggering of various cellular responses in bacteria-infected chondrocytes since both would be upregulated after infection and inhibition of their production could lessen the severity of arthritis [14,19]. However, it is important to emphasize that this study is a simplified in vitro model without consideration of extracellular matrix, synovial tissue, and other inflammatory cells. In addition, we used osteoarthritic chondrocytes that might be more susceptible to stresses and could release higher levels of NO under stress conditions [7,1O].

In conclusion, the results of this study support the hypothesis that bacterial infection-induced NO has an important role in the pathogenesis of chondrocyte apoptosis. Inhibition of NO release by pre-treating chondrocytes with L-NAME could ameliorate the extent of apoptosis and reestablish the cartilage matrix protein gene expression. However, neither the extent of apoptosis nor the matrix protein gene expression could be affected by adding L-NAME after the induction of infection. It is therefore suggested that in addition to NO, other mechanisms may be involved in the sustained responses of articular cartilage in the post-infectious arthropathy and need further study.

Acknowledgement

This study was supported by the National Science Council (NMRPG0057) and the CGMH intramural funding (CMRP835).

References

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M.S. Lee(a), Y.K. Tu(a), C.C.K. Chao(b), S.C. Chen(a), C.Y. Chen(a), Y.S. Chan(a), W.L. Yeh(a), S.W.N. Ueng(a,*)

(a) Departemtn of Orthopaedic Surgerv. Chang Gung Memorial Hospital, No. 222, Mai-chin Road, Keelimg, Taiwan

(b) Department of Biochemistry, Chang Gang University, Taiwan

Received 11 June 2003; accepted 18 June 2004

* Corresponding author. Tel./lax: +886 3327 8113.

E-mail address: wenneng@adm.cgmh.org.tw (S.W.N. Ueng).

Copyright Journal of Bone and Joint Surgery, Inc. Mar 2005

Source: Journal of Orthopaedic Research

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