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NDT Advance Access originally published online on September 27, 2006
Nephrology Dialysis Transplantation 2007 22(1):77-87; doi:10.1093/ndt/gfl555
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© The Author [2006]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Up-regulation of adhesion molecule expression in glomerular endothelial cells by anti-myeloperoxidase antibody

Tomokazu Nagao1, Mimiko Matsumura1,2, Ayako Mabuchi3, Akiko Ishida-Okawara1, Osamu Koshio4, Toshinori Nakayama5, Haruyuki Minamitani2 and Kazuo Suzuki1

1Department of Bioactive Molecules, National Institute of Infectious Diseases, Tokyo, Japan, 2Graduate School of Science and Technology, Keio University, Yokohama, Japan, 3Department of Physiology, University of Otago, Dunedin, New Zealand, 4Department of Medicine, Teikyo University, Tokyo, Japan and 5Graduate School of Medicine, Chiba University, Chiba, Japan.

Correspondence and offprint requests to: Kazuo Suzuki PhD, Chief of Biodefense Laboratory, National Institute of Infectious Diseases (NIID-NIH), Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan. Email: ksuzuki{at}nih.go.jp



   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Background. Anti-neutrophil cytoplasmic antibody directed against myeloperoxidase (MPO-ANCA) has been implicated in pauci-immune crescentic glomerulonephritis. It stimulates primed neutrophils to adhere to glomerular endothelial cells (GECs), thereby releasing reactive oxygen and other toxic substances and ultimately damaging the GECs. Though, a pathogenic role for MPO-ANCA is not fully understood, we hypothesized that MPO-ANCA modulates GEC functions by the increases in expression of adhesion molecules.

Methods. A polyclonal rabbit anti-recombinant mouse MPO antibody (anti-rmMPO IgG) was evaluated in mouse GEC (mGEC) for its effect on adhesion molecule expression. The primary culture of mGEC was incubated with anti-rmMPO IgG or isotype control and the expression of intercellular adhesion molecules-1 (ICAM-1) was evaluated by real-time reverse transcription–polymerase chain reaction (RT–PCR) analysis and ICAM-1 cell ELISA.

Results. The real-time RT–PCR analysis showed that a treatment with 100 µg/ml anti-rmMPO IgG increased the expression of mRNAs for ICAM-1, vascular cell adhesion molecule-1 and E-selectin by approximately 12.5, 7.5 and 10.5-fold, respectively. ICAM-1 cell ELISA also substantiated increased expression of ICAM-1. This enhancement of ICAM-1 expression was mediated by the antigen specificity of anti-rmMPO IgG. In addition, there were several proteins in mGEC specifically immunoprecipitated with anti-rmMPO IgG.

Conclusions. These results showed that anti-MPO antibody activates not only neutrophils, but also GEC, indicating that anti-rmMPO IgG-induced direct activation of GEC contributes to neutrophil adhesion to GEC, thereby increasing glomerular neutrophil infiltration in initiation and progression of pauci-immune glomerulonephritis.

Keywords: adhesion molecules; crescentic glomerulonephritis; glomerular endothelial cells; MPO-ANCA



   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Anti-neutrophil cytoplasmic antibody directed against myeloperoxidase (MPO-ANCA) are involved in the development of small vessel vasculitis such as idiopathic crescentic glomerulonephritis and microscopic polyangiitis [1–3]. MPO-ANCA has been used as a specific marker for these vasculitides, as evident from clinical observations that MPO-ANCA titres generally correlate with disease activity [4–6], although pathogenicity of the autoantibodies has not been clearly understood. Neutrophils have been reported to be a primary target of MPO-ANCA due to localization of MPO in the azurophilic granules of neutrophils and their translocation to plasma membrane upon priming with cytokines such as tumour necrosis factor-{alpha} (TNF-{alpha}) [7,8]. Therefore, previous studies have mainly focused on the signalling pathway in respiratory burst and degranulation of neutrophils stimulated with MPO-ANCA and initiation mechanisms of small vessel vasculitis by MPO-ANCA through neutrophil activation [9–12].

Adhesion of neutrophils to endothelial cells is an important step for neutrophil infiltration into glomeruli which is a histological feature of crescentic glomerulonephritis. Endothelial cells express cell surface counter receptors for integrins to be adhered with neutrophils in circulating blood and glomerular endothelial cells express intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). An elevated level of soluble ICAM-1 in sera of vasculitis patients has been reported [13–15]. Expression of the adhesion molecules on the surface of the endothelial cells is increased by stimulation with a wide variety of cytokines [16]. Similarly, elevated cytokine levels in the patient's blood seem to be a trigger for the enhancement of the expression of adhesion molecules on the surface, resulting in the development of vasculitis. However, a question remains whether ANCA also have direct effects on endothelial cells. Therefore, the direct effects of MPO-ANCA on the activation of glomerular endothelial cells must be elucidated in addition to the activation of neutrophils.

There are various studies available on the effects of sera or immunoglobulins from patients of autoimmune disease on endothelial cell activation. Johnson et al. [17] reported that autoantibody-positive serum samples from patients with vasculitis up-regulated ICAM-1 on human umbilical vein endothelial cells (HUVEC), although the molecular target of the autoantibody still remains unclear. De Bandt et al. [18] and Mayet et al. [19] demonstrated that anti-proteinase-3 antibodies from patients with Wegener's granulomatosis mediate ICAM-1 and VCAM-1 expression, respectively. Antibodies directed against endothelial cells from Scleroderma and Behçet's disease patients also activate HUVEC and up-regulated adhesion molecule expression [20,21]. However, the direct effect of MPO-ANCA on endothelial cells has not been investigated thus far. And, most research in this area has been done in HUVEC that can differ substantially from the cells in microvasculature, especially glomerular endothelial cells [22].

Here, we report the direct effect of polyclonal rabbit anti-recombinant mouse MPO antibody (anti-rmMPO) on expression of adhesion molecules in mouse glomerular endothelial cells (mGEC) and the mechanism of action.



   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Acknowledgements
 References
 
Primary culture of mouse glomerular endothelial cells and mesangial cells
We isolated mGEC from male C57BL/6 mice, which were maintained under specific pathogen-free conditions, according to the guidelines approved by the National Institute of Infectious Diseases Animal Care and Use Committee. Kidneys were obtained from freshly sacrificed mice perfused through the heart with cold Hank's balanced salt solution (HBSS) and glomeruli were prepared by a serial sieving method. Minced renal cortex tissue was serially passed through 150, 106 and 75 µm mesh stainless steel screens and glomeruli were collected using 53 µm mesh. The glomeruli, which contain small debris of renal tubule, were suspended in HBSS (Gibco-BRL Life Technologies, Gaithersburg, MD), washed twice by brief centrifugation (800 x g, 1 min) and digested in 1 mg/ml collagenase (Sigma, St Louis, MO) for 30 min at 37°C with occasional vortexing. Undigested glomeruli were pelleted by brief centrifugation and the supernatant containing single cell suspension of mGEC was suspended in growth medium [RPMI-1640 (Sigma) supplemented with 20% heat-inactivated foetal bovine serum (FBS, Gibco-BRL), 5 ng/ml vascular endothelial growth factor (VEGF) (Peprotech, Rocky Hill, NJ), 10 ng/ml bFGF (Sigma), 10 ng/ml epidermal growth factor (EGF) (Sigma), 20 U/ml heparin, 1 µg/ml hydrocortisone (Sigma), 50 U/ml penicillin and 50 µg/ml streptomycin (Gibco-BRL)] and plated on fibronectin-coated 35 mm dishes (Becton Dickinson, Franklin Lakes, NJ). Small colonies of mGEC were observed within 1 week after plating. To remove contaminating mesangial and epithelial cells, brief trypsinization was performed until a culture with purity over 90% of endothelial cells was achieved. The cells were maintained in collagen-coated 100 mm culture dish (Iwaki, Tokyo, Japan).

Mesangial cells were isolated from the outgrowth of the undigested glomeruli according to the method described by Deocharan et al. [23] and maintained in RPMI-1640 supplemented with 20% heat-inactivated FBS, insulin-transferrin-selenium supplement (Gibco-BRL), 50 U/ml penicillin and 50 µg/ml streptomycin.

Rabbit anti-recombinant mouse myeloperoxidase antibody
Anti-rmMPO antibody was prepared as described previously [24]. Briefly, rmMPO was prepared from Escherichia coli transfected with a plasmid containing MPO cDNA of mouse origin (C57BL/6). The expressed recombinant protein consisted of His-tag-L-chain-H-chain of mouse MPO. Anti-rmMPO IgG was raised by immunization of rabbit with purified rmMPO and IgG fraction of the polyclonal antibody was isolated from the serum using protein A (Amersham Biosciences Co., Piscataway, NJ). The reactivity to purified native mouse MPO was confirmed as shown previously [24]. Normal rabbit IgG was prepared by the same procedure except for non-immunization with rmMPO.

Antibody titre to rmMPO was measured as described previously [24]. Briefly, rmMPO was coated onto an ELISA plate (Toyoshima Co., Tokyo, Japan) overnight at 4°C. The plate was blocked with 1% bovine serum albumin (BSA) (Sigma) and then incubated with anti-rmMPO IgG for 1.5 h at room temperature. The bound anti-rmMPO IgG was detected by 2 h incubation with alkaline phosphatase-labelled anti-rabbit IgG antibody (Bio-Rad, Hercules, CA). The bound secondary antibodies were subsequently quantified by changes in the absorbance at 405 nm after incubation with 1 mg/ml p-nitrophenyl phosphate (Sigma).

Modification of antibody
To deplete IgG from the anti-rmMPO antibody, 1 ml of anti-rmMPO IgG varying the concentration from 0 to 200 µg/ml was incubated with or without protein A Sepharose [100 µl slurry in phosphate-buffered saline (PBS), Amersham Biosciences Co.] for 1 h at 4°C. The resultant supernatants after centrifugation (3000 x g, 5 min) were used for experiments. To adsorb MPO-specific antibody, we have taken advantage of the insolubility of the rmMPO in the absence of a high concentration of urea and used it to remove specific binding antibodies from anti-rmMPO IgG by adsorption [25]. Aggregated rmMPO was prepared by mixing and incubation with PBS for 1 h at 4°C. The aggregates were intensively washed with PBS until protein was undetectable in supernatant by UV absorption and incubated with 10 mg/ml anti-rmMPO IgG for 1 h at 4°C with agitation. As a control, the same amount of anti-rmMPO IgG was incubated in the absence of aggregated rmMPO. The mixture was centrifuged and supernatant was used for experiments. F(ab')2 fragments were prepared using an ImmunoPure F(ab')2 preparation kit (Pierce, Rockford, IL) according to the manufacturer's instructions.

Detection of ICAM-1 in mGEC (Cell ELISA)
The mGEC were plated on collagen-coated 96-well plates (Iwaki) at a density of 3 x 103 cells/well. At confluency, the cells were washed once with warmed HBSS and incubated for 1 h in RPMI-1640 medium containing 1% heat-inactivated FBS (assay medium). Anti-rmMPO IgG or control rabbit IgG diluted in assay medium was added to the wells and incubated for indicated periods, then the cells were washed with warmed PBS three times and fixed in 0.2% glutaraldehyde for 5 min at 4°C. For the blocking experiment, a neutralizing rat monoclonal antibody to TNF-{alpha} (BD Pharmingen, San Diego, CA) was included in assay medium throughout the incubation period. Non-specific binding was blocked by incubation with 1% BSA in PBS overnight at 4°C, followed by incubation for 1.5 h at room temperature with 0.5 µg/ml rat anti-mouse ICAM-1 monoclonal antibody (KAT-1; Chemicon, Temecula, CA). The primary antibody was detected by incubation for 1.5 h at room temperature with an alkaline phosphatase-conjugated anti-rat IgG antibody (1:2000, Bio-Rad). The bound secondary IgG was measured by the same procedure as aforementioned.

Detection of anti-endothelial cell antibodies
Anti-endothelial activity of antibodies used in this study was measured by the methods previously described by Carvalho et al. [20] with modification. mGEC were seeded onto a collagen-coated 96-well plates (Iwaki) at a density of 3 x 103 cells/well. The cells were fixed with 0.2% glutaraldehyde for 5 min at 4°C. The plates were blocked overnight with 1% BSA in PBS at 4°C and then incubated for 2 h with various concentrations of anti-rmMPO IgG or control IgG. The bound IgG was detected by an alkaline phosphatase-conjugated anti-rabbit IgG antibody (1:4000, Bio-Rad) with subsequent quantification of phosphatase using p-nitrophenyl phosphate as aforementioned. To measure the reactivity of anti-rmMPO IgG to live mGEC, the antibodies were first incubated with the live mGEC in the assay medium and then fixed with 0.2% glutaraldehyde. The bound IgG was measured by the same procedure as described before.

Immunoprecipitation
The anti-rmMPO or control rabbit IgG were bound to protein A Sepharose beads in lysis buffer [20 mM Tris–HCl, 150 mM NaCl, 1% NP-40, 0.5 mM phenylmethylsulphonyl fluoride (PMSF), pH 7.5] containing 0.1% BSA for 2 h at 4°C and washed several times with lysis buffer. Cells were lysed with lysis buffer and incubated with the antibody-bound protein A sepharose beads for 2 h at 4°C. The sepharose beads were washed three times with lysis buffer and bound proteins were solubilized by boiling in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. The protein samples were electrophoresed on 5–20% gradient gel and visualized by SYPRO Ruby staining (Molecular Probes, Eugene, OR).

Reverse transcript polymerase chain reaction (RT–PCR)
Total RNA was extracted using an ISOGEN (Nippongene, Tokyo, Japan), according to the manufacturer's instructions and quantified by UV absorption. One microgram of total RNA was reverse transcribed using ReverTra Ace-alpha (TOYOBO, Osaka, Japan). A semi-quantitative RT–PCR was used for the estimation of TNF-{alpha} mRNA expression. One microlitre of the resulting cDNA was used for PCR in 20 µl of Ex Taq buffer containing 0.2 mM dNTP mix, 0.5 µM of each primer and 25 U/ml Ex Taq polymerase (all reagents were purchased from TaKaRa Bio Inc.). Primer sequences for specific cDNA fragments were TNF-{alpha}: forward 5'-ctactgaacttcggggtgatcg-3', reverse 5'-aagtctaagtacttgggcagattgac-3', ß-actin: forward 5'-atctggcaccacaccttctacaatgagctgcg-3', reverse 5'-catcgtactcctgcttgctgatccacatctgc-3'. The amplification reactions were allowed to proceed for 30 cycles for TNF-{alpha} or 20 cycles for ß-actin consisting of 94°C for 30 s, 55°C for 30 s and 72°C for 60 s. PCR products (10 µl) were electrophoresed on 2% agarose gels and stained with ethidium bromide. mRNA expression of ICAM-1, VCAM-1 and E-selectin was quantified by using a real-time RT–PCR. First-strand cDNA was synthesized as described above. One microlitre of cDNA samples was used for the PCR reaction and analysed by the ABI Prism 7000 Sequence Detection System (Applied Biosystems) using Taqman 2X Universal PCR Master Mix and Applied Biosystems Assays-on-Demand primers and Taqman probe sets specific for mouse ICAM-1, VCAM-1 and E-selectin, according to the manufacturer's instructions. A non-template control was included for each target analysed. Relative quantification of all targets was calculated by using the comparative cycle threshold method [26]. The levels of gene expression were standardized with those of the glyceraldehyde 3-phosphate dehydrogenase.

Immunofluorescence microscopy
Both mGEC and MC were plated on collagen-coated or non-coated glass coverslips (22 x 22 mm) in 35 mm culture dishes, respectively. The confluent monolayer was fixed in ice-cold methanol and blocked with 1% BSA in PBS overnight at 4°C. Rat monoclonal antibody to CD31 (MEC13.3, BD Pharmingen), or mouse monoclonal antibody to desmin (Sigma) diluted 1:100 in 1% BSA in PBS, were added onto each coverslip and the samples were incubated for 1 h at room temperature. The coverslips were washed three times with PBS and further incubated for 1 h at room temperature in FITC-conjugated goat anti-rat IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for CD31 or Alexa Fluor 488-conjugated anti-mouse IgG antibody (Molecular Probes) for desmin. The coverslips were washed and observed by fluorescence microscopy. For the detection of ICAM-1, mGEC were incubated for 6 h with either 100 µg/ml anti-rmMPO or control IgG, fixed and immunostained for ICAM-1 using primary rat anti-mouse ICAM-1 monoclonal antibody and secondary FITC-conjugated goat anti-rat IgG antibody.

Statistical analysis
All data are expressed as mean ± SD. For individual comparisons, Student's t-test or Welch's corrected t-test, where appropriate, were used and differences with P < 0.05 were considered significant.



   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Acknowledgements
 References
 
Characterization of the primary mGEC
The population of the cells isolated from mice was characterized as endothelial cells by homogeneous monolayer of phase contrast image (Figure 1A) and positive staining with anti-CD31 (Figure 1B) in the junctional area and negative with anti-desmin (Figure 1C). The negative staining with anti-desmin indicates that there was little contamination of mesangial cells in the primary culture. In addition, RT–PCR analysis confirmed that mRNA of CD31 was expressed in the mGEC, but not in the mesangial cells (data not shown).


Figure 1
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  		Fig. 1. Primary culture of mouse glomerular endothelial cells. Isolated mGEC were cultured on coverslips and phase contrast image (A) and immunofluorescence image for CD31 (B) were obtained in the same field. The mGEC were negatively stained by anti-desmin (C). Mesangial cells stained for desmin (D) served as a positive control. Bar = 20 µm.

 
Up-regulation of adhesion molecule expression by anti-rmMPO IgG
To determine the direct effect of anti-rmMPO IgG on endothelial cell function, transcription levels of adhesion molecules in anti-rmMPO IgG-treated mGEC were examined. As shown in Figure 2A, quantitative RT–PCR revealed increases in mRNA levels of ICAM-1, VCAM-1 and E-selectin upon treatment with anti-rmMPO IgG in a dose-dependent fashion. The presence of 100 µg/ml of anti-rmMPO IgG in the culture medium increased ICAM-1, VCAM-1 and E-selectin transcripts in mGEC by 12.5, 7.5 and 10.5-fold, respectively. The control cells treated with normal rabbit IgG did not show any significant changes in the mRNAs of these adhesion molecules. Since ICAM-1, showed the highest increase in mRNA expression by anti-rmMPO IgG and, is a counter receptor for CD11b/CD18 integrins expressed abundantly in neutrophils, the following experiments were particularly focused on this molecule. The dose-dependent increase in ICAM-1 protein level in the anti-rmMPO IgG-treated mGEC was further confirmed by ELISA (Figure 2B). The mGEC treated with 100 µg/ml anti-rmMPO IgG for 6 h doubled the cellular ICAM-1 level and this condition was used for the following studies. In this condition, the increased expression of ICAM-1 by anti-rmMPO IgG was evident as shown by immunofluorescence microscopy for ICAM-1 (Figure 2C). In terms of cell morphology, there was no significant difference between anti-rmMPO IgG-treated cells and control normal IgG-treated cells (data not shown).


Figure 2
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  		Fig. 2. Up-regulation of adhesion molecule expression in mGEC induced by anti-rmMPO IgG. (A) Total RNA was extracted from the mGEC treated with either anti-rmMPO IgG or control rabbit IgG for 4 h. These RNA samples were subjected to real-time RT–PCR analysis as described in Materials and methods. The mRNA levels were normalized by that of non-treated control cells and shown as fold increase. * P < 0.05 vs control IgG at same concentration. (B) The mGEC were incubated for 6 h with either anti-rmMPO IgG or control rabbit IgG with increasing concentrations. The cells were fixed and ICAM-1 expression level was evaluated by cell ELISA. Data are expressed by optical density at 405 nm. * P < 0.05 vs control IgG at same concentration. (C) The mGEC were treated with either 100 µg/ml anti-rmMPO or control IgG and then immunostained for ICAM-1 as described in Materials and methods. TNF-{alpha}-treated cells served as a positive control. Bar = 20 µm.

 
Involvement of rmMPO-specific antibodies in up-regulation of ICAM-1 expression in mGEC
To rule out the possibility of enhanced adhesion molecule expression by some contaminated substances, various concentrations of anti-rmMPO IgG were incubated with or without protein A sepharose beads for 1 h at 4°C and then the supernatants were used for assays. Anti-rmMPO activity was almost diminished at any concentration by incubation with protein A sepharose beads (Figure 3A). Similarly, the expression of ICAM-1 returned to control level by the depletion of IgG (Figure 3B), indicating that the enhancement of the ICAM-1 expression was induced only by the IgG molecules in anti-rmMPO IgG.


Figure 3
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  		Fig. 3. Up-regulation of ICAM-1 is induced only by IgG molecules and is not attributable to contaminated substances. Anti-rmMPO IgG was incubated with or without protein A sepharose for 1 h at 4°C. After incubation, sepharose beads were spun down and the supernatants were used for the experiments. (A) Anti-rmMPO titre of the supernatants was evaluated by ELISA. The anti-rmMPO titre was expressed by optical density at 405 nm. (B) mGEC were incubated with the supernatants for 6 h and ICAM-1 expression levels of the mGEC were measured by cell ELISA. Data are expressed by optical density at 405 nm. * P < 0.05 vs IgG before depletion at the same concentration.

 
Since the polyclonal antibodies to rmMPO were used in this study, to rule out the up-regulation by non-specific IgGs, the experiment was conducted to confirm that the effects were induced by rmMPO-specific IgG. Recombinant mMPO-specific IgG was adsorbed by incubating with aggregated rmMPO protein, and titre of the resultant supernatant was determined by ELISA. Figure 4A shows the anti-rmMPO reactivity before and after incubation with aggregated rmMPO. The titre in adsorbed antibody decreased to approximately half of the non-adsorbed control. The degree of up-regulation of ICAM-1 expression was also decreased in the cells treated by the adsorbed antibody as compared with the original anti-rmMPO IgG (Figure 4B). Therefore, the increased expression of ICAM-1 seemed to be mediated by the rmMPO-specific IgG molecules and the molecular specificity of the antibody is one of the important factors in the activation.


Figure 4
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  		Fig. 4. Antibodies specific to rmMPO induce endothelial activation. MPO-specific IgG was adsorbed by incubating with aggregated rmMPO protein by batch method-like procedure as described in Materials and methods. Protein concentration of the adsorbed IgG was measured and then the IgG was used to measure antibody titre (A) and inducibility of ICAM-1 up-regulation (B). Both anti-rmMPO titres and ICAM-1 expression levels are expressed by optical density at 405 nm. * P < 0.05 vs IgG before adsorption at the same concentration.

 
To investigate the importance of molecular specificity, anti-rmMPO IgG was cleaved into F(ab')2 and Fc fragments by incubating with pepsin (Figure 5A) and the F(ab')2 portion was tested for its ability to up-regulate ICAM-1 expression. As shown in Figure 5B, since there was a significant decrease in the activity of the antibody even after the incubation in sodium acetate buffer without pepsin, the concentration of antibody tested was increased up to 1000 µg/ml. The loss of activity seemed to be due to low-pH-induced denaturation of IgG molecules in the process of digestion in sodium acetate buffer (pH 4.5). The F(ab')2 portion of anti-rmMPO IgG induced a significant increase in ICAM-1 expression in a dose-dependent manner, although it had less activity than the antibody incubated in acetate buffer without pepsin. The results demonstrate that the antigen specificity of the F(ab')2 portion of anti-rmMPO IgG mediates the enhanced ICAM-1 expression in mGEC.


Figure 5
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  		Fig. 5. Up-regulation of ICAM-1 expression in mGEC is associated with antigen specificity of F(ab')2 portion of anti-rmMPO IgG. Anti-rmMPO IgG was digested into Fc and F(ab')2 portions by incubating with pepsin in sodium acetate buffer. (A) The original IgG and the isolated F(ab')2 were subjected to SDS-PAGE and stained with Coomassie Blue. (B) The F(ab')2 fragments of anti-rmMPO were tested for the inducibility of enhanced ICAM-1 expression. For positive control, antibodies prepared by the same procedure except for incubation without pepsin were included in this experiment. Original anti-rmMPO incubated in PBS without pepsin was also prepared to check the effect of sodium acetate buffer. The ICAM-1 expression levels are expressed by optical density at 405 nm. * P < 0.05 vs IgG incubated in sodium acetate buffer without pepsin at the same concentration.

 
It has been reported that anti-endothelial cell antibodies from scleroderma or Behçet's disease patients induced adhesion molecule expression [20,21]. To check whether anti-endothelial activity of the anti-rmMPO IgG mediates the activation of the mGEC, anti-endothelial cell activity of anti-rmMPO IgG was compared with control normal rabbit IgG. As shown in Figure 6A, both anti-rmMPO and control IgG showed slight dose-dependent increases in binding to mGEC, which seemed to result mostly from non-specific binding of the antibodies. Contrary to ICAM-1 expression, the difference in the binding activity between control and anti-rmMPO antibodies was undetectable by ELISA. To examine whether antibody binding is specific for living cells, mGEC were treated with anti-rmMPO IgG for 4 h before fixation and bound anti-rmMPO IgG was quantified. As a result, the live mGEC showed binding kinetics similar to fixed mGEC (data not shown). Considering that antigen specificity of anti-rmMPO IgG was necessary to induce the enhanced ICAM-1 expression, a target molecule must be present in mGEC. To test whether there is a target antigen for anti-rmMPO IgG in mGEC, an immunoprecipitation study was performed with anti-rmMPO and control IgGs. As shown in Figure 6B, there were several bands on SYPRO Ruby stain of endothelial proteins specifically immunoprecipitated with anti-rmMPO IgG. The molecular weights of these proteins did not correspond to those of precursor (90 kDa), heavy chain (57.5 kDa) or light chain (14 kDa) of MPO.


Figure 6
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  		Fig. 6. Anti-endothelial activity of anti-rmMPO IgG. (A) Binding of anti-rmMPO IgG to fixed mGEC was evaluated by cell ELISA as described in Materials and methods. The anti-endothelial activities are expressed by optical density at 405 nm. (B) Immunoprecipitates from whole-cell lysate of mGEC either with anti-rmMPO or with control IgG were analysed by SDS-PAGE and SYPRO Ruby staining. The anti-rmMPO IgG-specific bands are magnified.

 
Up-regulation of TNF-{alpha} expression by anti-rmMPO IgG and its relation to adhesion molecule expression
In activated endothelial cells, a wide variety of cytokine genes are expressed other than adhesion molecules [27,28]. Inflammatory cytokines are the most important molecules expressed and secreted from the activated endothelial cells. Especially TNF-{alpha} is one such cytokine which contributes to the development of glomerulonephritis [13,24,29]. To examine whether the expression of TNF-{alpha} is involved in the activation of mGEC by anti-rmMPO IgG, TNF-{alpha} mRNA was measured by semi-quantitative RT–PCR. As shown in Figure 7A, the cells incubated for 6 h with various concentrations of anti-rmMPO IgG exhibited an increase in mRNA expression of TNF-{alpha} in a dose-dependent manner, whereas the control rabbit IgG failed to do so. Since TNF-{alpha} is one of the most potent endothelial activators and induces expression of adhesion molecules including ICAM-1, we examined the effect of neutralizing anti-TNF-{alpha} antibody on the enhanced expression of ICAM-1 in mGEC. The mGEC were treated with 100 µg/ml anti-rmMPO IgG in the presence of 0–20 µg/ml neutralizing anti-TNF-{alpha} antibody and the expression of ICAM-1 was evaluated. As shown in Figure 7B, there was no inhibition of the enhanced expression of ICAM-1 after 6 h. However, the endothelial activation was partially suppressed by neutralizing anti-TNF-{alpha} antibody in a dose-dependent manner if the cells were treated for 18 h.


Figure 7
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  		Fig. 7. Involvement of TNF-{alpha} expression in up-regulation of ICAM-1. (A) The mGEC were incubated with indicated concentrations of anti-rmMPO IgG or control rabbit IgG for 6 h and then mRNA expression of TNF-{alpha} was determined by semi-quantitative RT–PCR method. RT–PCR products of ß-actin of the same samples were included as internal control. (B) Inhibition of up-regulation of ICAM-1 expression by neutralizing anti-TNF-{alpha} antibody. The mGEC were incubated for 6 or 18 h with 100 µg/ml anti-rmMPO IgG in the presence of the indicated concentrations of neutralizing anti-TNF-{alpha} antibodies. ICAM-1 expression levels of the cells were measured by cell ELISA and were normalized by that of the cells treated with 100 µg/ml anti-rmMPO IgG in the absence of neutralizing antibody. * P < 0.05 vs cells treated with anti-rmMPO IgG in the absence of neutralizing antibody.

 


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Acknowledgements
 References
 
We have demonstrated that anti-rmMPO IgG induced an up-regulation of adhesion molecules. Johnson et al. [17] reported an enhanced expression of ICAM-1 in HUVEC with sera or purified IgG from patients with autoimmune vasculitis. However, the relationship between antigen recognition of the antibodies and the up-regulation of ICAM-1 has not been clear. In the present study, activating effects of ‘MPO-specific’ antibody on endothelial cells have been demonstrated. Since the F(ab')2 fragment of anti-rmMPO IgG could also up-regulate the adhesion molecule, it was concluded that antigen recognition was involved in this process. However, we were unable to demonstrate whether cross-linking of the target antigen was involved because digestion and purification of Fab fragment led to loss of activity.

It has been reported earlier that MPO is not expressed in HUVEC [30] and we also confirmed by RT–PCR analysis that the isolated mGEC also do not express murine MPO (data not shown). The difference in anti-endothelial activity between anti-rmMPO and control rabbit IgG was undetectable by ELISA, presumably because non-specific binding (e.g. binding to Fc receptors) was dominant. However, since we successfully detected several bands on SYPRO Ruby stain of immunoprecipitates specific for anti-rmMPO IgG, we believe that only subtle binding of anti-rmMPO IgG to a certain molecule existing on mGEC can trigger the activation of mGEC. Therefore, we speculate that certain molecules expressed in mGEC (probably on plasma membrane) may share the epitope of anti-rmMPO IgG and transduce signals leading to the enhanced expressions of adhesion molecules. Identification of the molecules bound with anti-rmMPO IgG will be analysed in a future study.

Although it has been reported earlier that monoclonal antibodies against MPO do not induce endothelial activation [17], yet the present study shows its induction of adhesion molecules. We, however, here utilized the polyclonal anti-rmMPO IgG containing antibodies with many different binding sites (epitopes) to MPO. The reports, on epitope mapping of MPO-ANCA using recombinant deletion mutants of MPO, have demonstrated that MPO-ANCA in sera of the patients recognizes different epitope sites of MPO with a restricted number of epitopes located on heavy chain of MPO [31–33]. Furthermore, the epitope recognition profiles are also related to clinical features. In other words, only a few clones targeting the risk epitopes can initiate and progress MPO-ANCA-associated glomerulonephritis. Therefore, the polyclonal antibody used in this study seems to contain the antibodies against the specific risk epitopes. An epitope mapping of the anti-rmMPO IgG which activated mGEC in the present study may help to elucidate the cross-reactivity of anti-rmMPO IgG and the mechanism of endothelial activation related to pathogenicity of MPO-ANCA.

The results of the neutralizing anti-TNF-{alpha} antibody experiment show that although TNF-{alpha} was not likely to be a primary activator in the initial period (6 h), endothelial ICAM-1 expression was enhanced by TNF-{alpha} secreted from activated mGEC after 18 h. The production of TNF-{alpha} induced by anti-rmMPO IgG may in part contribute to the up-regulation of adhesion molecules in an autocrine fashion. However, it still remains controversial whether anti-rmMPO IgG directly induces the up-regulation of adhesion molecules because the other inflammatory mediators might be released from mGEC, and subsequently activate mGEC even just after the binding of anti-rmMPO IgG. Besides TNF-{alpha}, a wide variety of inflammatory cytokines or chemokines such as interleukin (IL)-1, IL-8 and monocyte chemoattractant protein-1 (MCP-1) are released from endothelial cells in response to stimuli [34,35]. These cytokines/chemokines activate endothelial cells and up-regulate ICAM-1 expression [36–38] and have been further reported to correlate with clinical presentation of MPO-ANCA-associated glomerulonephritis. Adhesion molecules, investigated in this study as well as TNF-{alpha} are mainly expressed through NF-{kappa}B activation [39–42]. Therefore, it is expected that anti-rmMPO IgG activated NF-{kappa}B which subsequently induced up-regulation of adhesion molecules and inflammatory cytokines/chemokines. Blocking antibodies to these cytokines/chemokines or inhibitor of NF-{kappa}B may further reveal the role of these mediators in up-regulation of adhesion molecules.

Sensitivity of endothelial cells to cytokine exposure or injury mediated by activated neutrophils differs with their origin [22,43]. We also examined the change in ICAM-1 expression by anti-rmMPO IgG using other microvascular endothelial cells such as primary mouse lung endothelial cells (mLEC) and pancreatic islet endothelial cell line (MS1). These cells also showed an increased mRNA expression of ICAM-1 (see ‘Supplementary’ data), indicating that these effects were commonly seen in microvascular endothelium and not limited in the kidney. MPO-ANCA is believed to be associated with the development of small vessel vasculitis. Therefore, the comparison of adhesion molecule expression by anti-rmMPO IgG between endothelial cells from small and large vessels might help to understand the pathogenesis of ANCA-associated vasculitis.

In conclusion, the present results clearly indicate that the anti-MPO antibody up-regulates the expression of ICAM-1, VCAM-1 and E-selectin in mGEC, suggesting that not only neutrophils but also glomerular endothelial cells are activated by MPO-ANCA and contribute to neutrophil adhesion to GEC, thereby increasing glomerular neutrophil infiltration in initiation and progression of pauci-immune glomerulonephritis. It is still unknown how anti-rmMPO IgG stimulates the expression of adhesion molecules in mGEC, although our study revealed that antigen recognition of anti-rmMPO IgG is necessary for the stimulation. Future in vitro studies on the aforementioned issues will provide a more precise mechanism for endothelial activation in pauci-immune glomerulonephritis. Furthermore, using the animal model of MPO-ANCA-associated glomerulonephritis [44], we will investigate the role of MPO-ANCA-induced expression of adhesion molecules and inflammatory cytokines/chemokines in mGEC in vivo.



   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
References
 
This study was in part supported by grants from Japan Human Science Foundation and Ministry of Health, Labour and Welfare, Japan.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 

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Received for publication: 14. 4.06
Accepted in revised form: 18. 8.06


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