Thirty-three patients with BD, who met the International Study Group criteria for diagnosis of BD 27, were enrolled in the study. Their mean age was 47.7 ± 15.0 years, and 13 were male and 20 were female.

All of the patients were under the care of the Yokohama City University Hospital. As previously described 28, 13 patients with one or more lesions (including genital ulcers, uveitis, erythema nodosum, arthritis, gastrointestinal lesions, central nervous system lesions, and/or C-reactive protein >10 mg/l) were regarded to have active disease during the study.

The patients had been treated with a combination of the following agents: colchicines (17 patients), corticosteroids (13 patients), nonsteroidal anti-inflammatory drugs (14 patients), sulfasalazine (two patients), and cytotoxic drugs such as methotrexate (one patient), cyclosporine (four patients), tacrolimus (one patient) and cyclophosphamide (one patient). Thirty healthy age- and sex-matched individuals were also included as a control group. HLA-B type was determined by SRL Inc. (Tokyo, Japan) using lymphocyte cytotoxicity assay or a PCR reverse sequence specific oligonucleotides method. All experiments were conducted after written informed consent has been obtained, which was approved by the local institutional review board.

Reagents were obtained from the following manufactures: recombinant human TNF-α (R&D; Minneapolis, MN, USA), polymyxin B and LPS Escherichia coli O111: B4 (Calbiochem; La Jolla, CA, USA), low endotoxin recombinant human HSP60 (Stressgen; Victoria, Canada), and IgG₁κ (Serotech; Oxford, UK). Infliximab was kindly provided by Tanabe Seiyaku (Osaka, Japan).

PBMCs and polymorphonuclear leukocytes (PMNs) were isolated by centrifugation over two Ficoll-Hypaques gradients of specific gravities 1.077 (ICN; Aurora, OH, USA) and 1.119 (Nacalai; Kyoto, Japan). Purity of the separated neutrophils, which were determined by flow cytomeric scattergram, was typically above 97% 7. Monocytes were negatively selected by magnetic cell sorting (Miltenyi Biotec; Gladbach, Germany) using a monocyte isolation kit (Miltenyi Biotec). More than 95% of the obtained monocytes expressed CD14, based on flowcytomeric analysis 26.

The cells were incubated in hepes modified RPMI1640 (Sigma-Aldrich; Saint Louis, MO, USA) containing 10% fetal calf serum (Equitech-bio; Kerrville, TX, USA), 2 mmol/l L-glutamine (Sigma-Aldrich), 100 U/ml penicillin plus 100 μg/ml streptomycin (Sigma-Aldrich) in a 5% carbon dioxide in an air incubator at 37°C. To determine HO-1 expression at mRNA and protein levels, cells were cultured in the presence or absence of LPS (10 ng/ml) or HSP60 (3 μg/ml) for 6 to 24 hours.

Purified monocytes (1 × 10⁶) were transfected with 3 μg of human HO-1 expression vector or control vector by using Nucleofector (Amaxa Biosystems; Gaithersburg, MD, USA) and human monocyte Nucleofector kit (Amaxa Biosystems) 2526. Twenty four hours later, the cells were used for further experiments.

Total RNA was isolated from cells with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) 21242526. One microgram of total RNA served as a template for single-stranded cDNA synthesis in a reaction using oligo (dT) primers and SuperScript II (Invitrogen). For the reverse transcription PCR, 1 μl cDNA was incubated with 9.375 μl de-ionized distilled water, 2 μl dNTP, 2.5 μl 10 × PCR buffer, 0.125 μl Taq polymerase (Takara, Ohtsu, Japan), and primer pairs for target genes. The primers used in the study are summarized in Table 1.

Cycling conditions included 35 cycles of amplification for 30 seconds at 94°C, 30 seconds at 55°C, 1 minute at 72°C, and a final extension phase consisting of one cycle of 10 minutes at 72°C. The primers and probes for human HO-1, TLR2, TLR4, CD14, TNF-α, MD-2 (Myeloid differentiation factor-2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) used in the real-time PCR were purchased from PE Applied Biosystems (Foster City, CA, USA). Real-time PCR was performed using a BD Qtaq DNA polymerase (BD Bioscience), and the data were analyzed by the ABI prism 7700 sequence detection system (PE Applied Biosystems, Franklin Lakes, NJ, USA). Briefly, 1/50 of cDNA derived from 1 μg total RNA, 200 nmol/l probe, and 800 nmol/l primers were incubated in 25 μl at 50°C for 2 minutes and 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. The analysis system determined the number of cycles at which the amplified DNA in the sample exceeded the threshold during the PCR. Gene expression levels of the individual samples were calculated on standard curves of each cDNA generated by serial dilutions of the PCR amplified products. The data on HO-1, TLR2, TLR4, and TNF-α were standardized to the expression of GAPDH in the same samples, using multiplex PCR technique. Expression level of HO-1 mRNA in a sample is indicated as arbitrary units.

The expression of HO-1 protein was determined by immunoblotting as described previously 25. Briefly, cells were treated with lysis buffer (137 mmol/l NaCl, 20 mmol/l Tris-HCl, 50 mmol/l NaF, 1 mmol/l EDTA, and Triton-X), supplemented with a protease inhibitor cocktail (Sigma-Aldrich) for 30 minutes on ice, and the supernatants were recovered by centrifugation at 15,000 rpm for 30 minutes. For TLR2 and TLR4 immunoblotting, after addition of lysis buffer, cells were homogenized for 15 minutes by ultrasonifier (Branson Japan, Kanagawa, Japan). The samples were resolved electrophoretically on a 4% to 20% gradient of polyacrylamide gel (Daiichi Kagaku, Tokyo, Japan) and transferred onto a polyvinyldene difluoride membrane (Millipore, Billerica, MA, USA). After blocking with 5% skimmed milk/Tris-buffered saline overnight at 4°C, the membrane was incubated with optimally diluted anti-HO-1 monoclonal antibody (Stressgen), anti-TLR2 and anti-TLR4 (Imgenex, San Diego, CA, USA) monoclonal antibody, or anti-actin goat polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 hour at room temperature or overnight at 4°C, and subsequently for 45 minutes with horseradish peroxidase-conjugated anti-mouse secondary antibody (Amersham Life Sciences, Piscataway, NJ, USA) or rabbit anti-goat IgG horseradish peroxidase conjugate (Zymed, South San Francisco, CA, USA). The signals were developed by using the enhanced chemiluminescence detection system (Amersham Life Sciences). The amount of blotted protein was measured densitometrically by using Scion image analysis and image processing software (NIH Image Engineering, Bethesda, MD, USA).

Mann-Whitney U-test, Kruskal-Wallis test with post-hoc Scheffe's test, paired t-test, and regression analysis were used to test for differences. P values less than 0.05 were considered statistically significant.

HO-1 mRNA expression level was determined in circulating leukocytes from BD patients by using a real-time PCR technique (Figure 1). A good correlation between HO-1 mRNA and protein levels has been demonstrated 26. Consistent with previous findings 24, we found no significant difference in HO-1 mRNA expression in PBMCs between BD patients (including both patients with active and those with inactive disease) and healthy control individuals (data not shown). A more detailed analysis based on disease activity, however, revealed that PBMCs from patients with active BD, but not those with inactive disease, expressed significantly lower HO-1 mRNA levels than did PBMCs from healthy control individuals (Figure 1a). Because HO-1 is preferentially expressed by monocytes among PBMCs, amounts of HO-1 mRNA may depend on the proportion of monocytes detected 26. CD14 mRNA levels determined by real-time PCR were comparable between BD and healthy control individuals, indicating that there was no difference between groups in the proportion of monocytes among circulating leukocytes (data not shown). Moreover, no significant difference was found in absolute counts of monocytes between patients with active BD and those with inactive disease (active 497.9 ± 218.8/μl versus inactive 462.7 ± 182.4/μl; P = 0.77, by Mann-Whitney U-test), indicating that HO-1 expression was reduced in individual cells from patients with active disease. As shown in Figure 1b, HO-1 mRNA levels in PMNs were not significantly different between BD patients and control individuals (Figure 1b).

No particular clinical manifestations, including ocular lesions (Table 2) and treatments (data not shown), were associated with the reduction in HO-1 mRNA expression in PBMCs. There were no differences in mRNA expression levels of HO-1 and TLRs between HLA-B51-positive and -negative patients (Table 2). Levels of HO-1 mRNA expression were not altered by treatment with colchicine or prednisolone in the patients (data not shown).

Because HSP60 has been implicated in the pathogenesis of BD 17, levels of mRNA for TLR2 and TLR4 (both of which recognize HSP60 as a ligand) were examined in PBMCs and PMNs from patients with BD (Figure 2). In preliminary experiments, the relationship between levels of TLR mRNA and protein in circulating leukocytes was examined. Briefly, after fractionating PBMCs into CD14-positive cells and CD14-depleted cells by means of magnetic cell sorting, mRNA and protein levels of TLRs were compared by using real-time PCR and immunoblotting techniques, respectively. TLR4 was preferentially expressed on CD14-positive cells, but not CD14-depleted cells, at both mRNA and protein levels (Additional file 1). Moreover, TLR4 and TLR mRNA levels correlated well with protein levels.

No significant differences were found in levels of TLR2 mRNA expression in PBMCs between patients with active BD, patients with inactive BD, and healthy control individuals (Figure 2a). On the other hand, TLR4 mRNA expression levels were elevated in PBMCs from patients, irrespective of disease activity (Figure 2b) and HLA-B51 phenotype (data not shown). However, no significant differences in levels of mRNA expression for CD14 and MD-2, which are critically involved in LPS-mediated signal transduction of TLR, were found between patients and control individuals (data not shown). There was no abnormality in mRNA expression of TLR2 and TLR4 in PMNs (Figure 2c,d). The results indicate that TLR4 mRNA expression is constitutively increased in PBMCs from BD patients.

TLR4 signaling triggers activation of the innate immune system, whereas HO-1 plays a regulatory role in inflammatory response. Analysis of the relationship between the two molecules showed that TLR4 mRNA was inversely correlated with HO-1 mRNA in PBMCs from BD patients (Additional file 2; P < 0.05, r = -0.42, by regression analysis). Because LPS (a TLR4 ligand) has been shown to suppress interleukin-10-dependent HO-1 expression in human PBMCs 29, it is plausible that excessively expressed TLR4 contributes to defective HO-1 expression in PBMCs from BD patients. As expected, the immunoblotting study revealed that stimulation with LPS reduced HO-1 expression in PBMCs from patients with BD, irrespective of the presence or absence of interleukin-10 (Figure 3a). The suppressive effect on HO-1 expression was completely abrogated by a LPS neutrizer, namely polymyxin B (Figure 3a). Similarly, real-time PCR analysis revealed a reduction in HO-1 mRNA levels in LPS-stimulated PBMCs (Figure 4a) when TNF mRNA expression was elevated (Figure 4b). The magnitude of LPS-induced HO-1 suppression (calculated as the gap in HO-1 mRNA between PBMCs subjected to 6 hours of LPS treatment and untreated PBMCs [ΔHO-1]) was significantly correlated with TLR4 mRNA expression levels in untreated PBMCs (Figure 4c).

In our previous study 26 we demonstrated that TNF enhances HO-1 mRNA degradation, resulting in a reduction in HO-1 expression in human monocytes. Because TLR4 signaling leads to synthesis of TNF, which may be involved in the reduction in HO-1 expression in PBMCs from patients with BD. However, levels of TNF mRNA did not correlate with those of HO-1 in PBMCs from patients with BD (data not shown). Moreover, although the preliminary experiments confirmed that 10 ng/ml LPS efficiently stimulated PBMCs to produce substantial amounts of TNF protein, anti-TNF-α antibody infliximab did not eliminate the suppressive effect of LPS on HO-1 expression in monocytes in vitro (Additional file 3). The findings indicated that the effect is not solely dependent on TNF (Figure 3d).

Because our previous study demonstrated bidirectory interactions between HO-1 and TNF 26, we also examined effects of HO-1 upregulation on TLR levels in monocytes. Over-expression of HO-1 protein was confirmed by immunoblotting analysis 24 hours after transfection with pHO-1 (human HO-1 expression vector) into monocytes. Under these conditions, no differences were found in expression levels of TLR2 and TLR4 between HO-1 cDNA transfected monocytes and controls (Figure 5). Taken together, our findings implicate the involvement of excessive TLR4 expression in low levels of HO-1 mRNA expression in PBMCs from patients with BD.