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Clin Exp Immunol. Jul 2001; 125(1): 48–55.
PMCID: PMC1906098

Subtractive screening reveals up-regulation of NADPH oxidase expression in Crohn's disease intestinal macrophages

Abstract

Macrophages play a central role during the pathogenesis of inflammation. In normal intestinal mucosa surface expression of typical macrophage markers such as CD14, CD16, CD11b or T-cell co-stimulatory molecules such as CD80 or CD86 is low indicating anergy and low pro-inflammatory activity of these cells. During inflammatory bowel disease (IBD) the mucosa is invaded by a population of macrophages displaying these markers, secreting higher cytokine levels and representing an activated cell population. CD33+ cells (macrophages) were isolated from normal and Crohn's disease mucosa and mRNA was isolated by polyT magnetic beads. A subtractive screening was performed subtracting mRNA from normal macrophages from those of Crohn's disease macrophages. Oxidative burst activity was determined by flow cytometry. Seventy clones were obtained by the subtractive mRNA screening. Sequencing showed >99% homology to mRNA of monocyte chemoattractant protein-1 (MCP-1) for three clones. Five clones obtained by subtraction revealed >99% homology to mRNA of cytochrome b (subunit gp91). Differential expression of the cytochrome b subunit gp91 and the cytosolic NADPH oxidase subunit p67 was confirmed by RT-PCR and ‘virtual’ Northern blots. The fluorescence ratio of stimulated versus unstimulated cells was 0·9 ± 0·16 in control macrophages indicating a lack of oxidative burst activity. In Crohn's disease this ratio was significantly increased to 1·80 ± 0·8 (P = 0·004) confirming the molecular data. In conclusion NADPH oxidase mRNA is down-regulated or absent in macrophages from normal mucosa correlating with a lack of oxidative burst activity. In IBD macrophage-oxidative burst activity is increased and NADPH oxidase mRNA induced. Inhibition of NADPH oxidase could be a new therapeutical target in IBD and reduce mucosal tissue damage in active IBD.

Keywords: cytochrome b, inflammatory bowel disease, macrophages, NADPH oxidase, oxidative burst, subtractive screening

Introduction

Intestinal macrophages represent one of the largest compartments of the mononuclear phagocyte system in the body [1,2]. They are localized preferentially at the sites of antigen entry, e.g. in the peri-epithelial region of the small intestine and in the subepithelial domes of Peyer's patches [38]. Macrophages constitute 10–20% of the mononuclear cells in the lamina propria, as determined by immunohistochemistry and tissue disaggregation experiments [25,9].

Recently we and others analysed the phenotype of colonic macrophages from normal and inflamed intestinal mucosa. Intestinal macrophages express CD44 and CD68, acid phosphatase and non-specific esterase [1013]. CD33, a member of the sialoadhesin family of sialic acid-dependent cell adhesion molecules, could be identified as a useful recognition marker for intestinal macrophages in flow cytometrical analysis [12,13]. Only very few macrophages from normal colonic mucosa express the typical monocyte/macrophage-specific surface markers CD14, CD16, CD11b and CD11c [13]. Also the expression of the T cell co-stimulatory molecules B7-1 (CD80) and B7-2 (CD86) on intestinal macrophages is low [13].

During acute flares of inflammatory bowel disease (IBD) the heterogeneity of the intestinal macrophage population is strongly increased [10]. The expression of ICAM-1 is up-regulated from 7% in controls to 70% in ulcerative colitis and 46% in Crohn's disease [14]. Interleukin-2 receptor (CD25) is expressed in intestinal macrophages of patients with ulcerative colitis, but not in controls [15]. In our own studies in IBD mucosa there was not only an increase in expression of CD14 (LPS-receptor) compared to control mucosa, but also of CD16 (FcγIII-receptor), HLA-DR, CD11b and CD11c (members of the integrin family) [13]. In addition, on macrophages from IBD colon we found a significant increase in the expression of T cell co-stimulatory molecules CD80 and CD86 [16]. While in normal mucosa only 10·6% ± 4·9% of the macrophages expressed CD14, more than 90% of the CD86/CD80 positive cells of the inflamed mucosa were positive for CD14 [16].

Despite these differences in phenotype, detailed data on functional differences of intestinal macrophages from normal and inflamed mucosa are lacking. To elucidate possible functional differences we performed a subtractive screening of mRNA expression in intestinal macrophages from normal and inflamed mucosa.

We purified intestinal macrophages from normal and Crohn's disease mucosa according to a protocol recently established in our laboratory [13]. We performed a subtractive screening method to study mRNA expression in macrophages from Crohn's disease patients. Here we report the subtraction of mRNA obtained from control macrophages from that of Crohn's disease macrophages. We obtained 70 gene products. Three of the differentially expressed clones had homology to monocyte chemoattractant protein-1 (MCP-1). Five of them had homology to the cytochrome b subunit gp91. This led us to investigate differences in oxidative burst activity in isolated macrophages from control and inflamed mucosa.

Materials and methods

Isolation of human lamina propria mononuclear cells (LPMNC)

Biopsy specimens from inflamed and normal mucosa were obtained by endoscopy. The study was approved by the University of Regensburg Ethics Committee. Biopsy specimens were obtained from 10 patients with Crohn's disease, 10 patients with ulcerative colitis and one patient with diverticulitis. Control biopsy specimens were taken from 10 patients who underwent endoscopy because of other reasons (screening for colonic adenomas and irritable bowel syndrome). Biopsies obtained by endoscopy or small pieces of intestinal mucosa from surgical specimen were kept in sterile RPMI at 4°C supplemented with PenStrep and 10% fetal calf serum for transportation. Specimens were then incubated in calcium- and magnesium-free Hanks's balanced salt solution (HBSS) with 1 mmol/l ethylenediaminetetraacetic acid (EDTA) for 30 min at 37°C under gentle shaking to remove the intestinal epithelial cells. Human lamina propria mononuclear cells were isolated according to a modification of the method of Bull and Bookman, as described recently [13]. Specimens were incubated for 30 min in 2 ml phosphate-buffered saline (PBS) with 1 mg/ml collagenase type I (= 336 U/ml, Sigma Aldrich Chemie, Deisenhofen, Germany), 0·3 mg/ml deoxyribonuclease I (DNase I, Boehringer, Mannheim, Germany) and 0·2 mg/ml hyaluronidase (Sigma Aldrich Chemie, Deisenhofen, Germany) without fetal calf serum (FCS) at 37°C. Cells were washed in 15 ml PBS with 500 µl FCS and finally submitted to Ficoll density gradient centrifugation for 20 min at 2000 r.p.m. (≈690 g, without brake) in a Heraeus centrifuge for the isolation of mononuclear cells. The interphase was carefully removed and washed with PBS.

Antibodies for identification of macrophages

For the immunomagnetic isolation of macrophages mouse antihuman CD33 MicroBeads (isotype: mouse IgG1, clone: P67·6, Miltenyi Biotec, Bergisch Gladbach, Germany) were used. For the identification of the intestinal macrophage population in isolated LPMNCs the mouse antihuman CD33 antibody (isotype: mouse IgG2b, clone: 906(MY9), Immunotech, Hamburg, Germany) and a TRI-COLOR goat antimouse (isotype: IgG-2b, clone: M32406, Caltag, Burlingame, CA) were used.

Isolation of intestinal macrophages

LPMNC were isolated from normal and Crohn's disease mucosa specimens. Macrophages were labelled with immunomagnetic MicroBeads armed with CD33 antibody and purified twice with the help of type AS separation columns (Miltenyi Biotec) as described recently [13]. Briefly, LPMNC with magnetically labelled macrophages were passed through an AS separation column which was placed in the permanent magnet SuperMACS. The magnetically labelled cells were retained in the column and separated from the unlabelled cells, which pass through. After removal of the column from the magnetic field, the retained fraction could be eluted. Eluted cells were passed through a second AS separation column to increase purification of macrophages to a final purity of >95% as determined by flow cytometry.

Isolation and amplification of mRNA

mRNA was isolated by polyT magnetic beads (Dynal, Oslo, Norway) from CD33+ mononuclear cells according to the manufacturers protocol. Integrity of the mRNA was verified by Gene Checker™ kit (Invitrogen, Carlsbad, CA, USA). mRNA was reverse transcribed and amplified with the SMART PCR cDNA synthesis kit (Clontech, Palo Alto, USA). Briefly, to obtain amplified cDNA, first-strand synthesis was performed with a modified oligo(dT) primer with an additional 5′ sequence and a second primer with an oligo(dG) sequence at its 3′ end. When reverse transcriptase (RT) reaches the 5′end of the mRNA, the enzyme's terminal transferase activity adds a few deoxycytidine nucleotides to the 3′ end of the cDNA. The second primer pairs with the (dC)stretch. RT then switches templates and continues replicating to the end of the oligonucleotide. cDNA was then amplified with the modified oligo(dT)- and oligo(dG) primers.

Subtractive screening

A subtractive screening of the cDNA populations was performed by subtracting cDNA from normal macrophages from those of Crohn's disease macrophages with the Clontech PCR-Select cDNA subtraction kit. cDNA from Crohn's disease macrophages was subdivided into two portions, which were ligated with different adaptors. Two hybridizations were performed. In the first, an 10-fold excess of cDNA from normal macrophages was added to each sample of cDNA from Crohn's disease macrophages. The samples were then heat denatured and allowed to anneal, generating all different kinds of complementary molecules in each sample. Single-stranded cDNA-molecules from Crohn's disease macrophages were significantly enriched for differentially expressed sequences, as cDNAs that were not differentially expressed form hybrids with cDNAs from normal macrophages. During the second hybridization, the two primary hybridization samples were mixed together without denaturation. Only the remaining single stranded molecules from Crohn's disease macrophages could re-associate and form new hybrids. The new hybrids were double-stranded molecules with different ends, which correspond to the sequences of the two ligated adaptors. The entire population of molecules was subjected to PCR to amplify the desired differentially expressed sequences. Only molecules with two different adaptors were amplified exponentially. A secondary PCR amplification was performed using nested primers to further reduce any background PCR products and enrich for differentially expressed sequences.

Polyacrylamide gel electrophoresis and single strand conformation polymorphism (SSCP) gel electrophoresis were performed with the subtracted cDNA. Products were cloned with TOPO™-TA Cloning Kit (Invitrogen, Carlsbad, CA, USA), sequenced and a similarity search was performed. The World Wide Web address to access the nucleic acid sequences is http://www.ncbi.nlm.nih.gov/index.html. The blast program was used to perform DNA database-searches for sequence similarities. During a blast run the query sequence for every search is filtered to eliminate low complexity regions. Each comparison is given a score reflecting the degree of similarity between the query and the sequence being compared. The result of the blast records was given together with a score (arbitrary units), considering length of the alignment, mismatches and gaps. A higher score therefore means a greater degree of similarity. In this work only hits with an arbitrary score >700 bits were selected for further analysis. Several products were verified by dot blot analysis, semiquantitative PCR and ‘virtual’ Northern blots with mRNA not set for subtractive screening.

‘Virtual’ Northern blots

For ‘virtual’ Northern blotting isolated mRNA from both Crohn's disease and normal macrophages was reverse transcribed and amplified with the SMART PCR cDNA synthesis kit as mentioned above. The yield of RNA that could be isolated from purified intestinal macrophages was to low to perform conventional Northern Blots. Linear amplification was controlled by running the reaction on a 1·2% agarose gel after 12, 15 and 18 cycles. Occurrence of distinct bands indicated over-amplification of certain cDNAs. Only SMART PCR samples without signs of over-amplification were used for further analysis; 1–5 μg of amplified cDNA were run on 1% agarose gels and transferred to MAGNA nylon membranes (msi, Westborough, MA, USA). Thus, blotting was performed with cDNA instead of RNA due to the limited quantity of the starting material. Therefore this method is referred to as ‘virtual Northern Blot’.

cDNA was hybridized with oligonucleotide probes complementary to bp 1538 to bp 1781 of gp91 and bp 541 to 1201 bp of actin derived from the published sequences (NM_000397 and XM_004814 at http://www.ncbi.nlm.nih.gov/index.html). These probes were labelled with Klenow DNA polymerase (Boehringer, Mannheim, Germany) and α(32P) adenosine triphosphate (5000 Ci/mmol, Amersham, Freiburg, Germany). Hybridization conditions were: 50% formamide, 5 × sodium phosphate (pH 7·4), 0·1% sodium dodecyl sulphate (SDS), 1 mmol/l EDTA and 150 µg/ml tRNA at 65°C overnight. Washing conditions were: 2 × SSC, 0·1% SDS at 42°C and 0·1 × SSC, 1% SDS at 65°C 15–30 min each. The probes were subsequently hybridized to the same membranes after stripping of the previous probes with 0·1 × SSC, 1% SDS at 70°C for 1 h.

X-ray films (Biomax™ ML, Eastman Kodak Company, Rochester, USA) were exposed to the membranes for 1–7 days with an intensifying screen (Biomax™ TranScreen HE, Eastman Kodak Company, Rochester, USA). The intensity of the radioactive signals was determined by laser densitometry (Personal Densitometer, Molecular Dynamics, Sunnyvale, USA) of the X-ray films.

Antibody staining for FACS analysis

Biopsy specimens from inflamed and normal mucosa were obtained by endoscopy and LPMNC were isolated as described above but without further immunomagnetic purification of the macrophages. For flow cytometry analysis of intestinal macrophages, the LPMNC were resuspended in PBS. For each staining 80 µl of cell suspension were placed into 1·5 ml polypropylene tubes and 10 µl of each antibody solution were added. LPMNC suspensions were stained with a mouse antihuman macrophage CD33 antibody (isotype: mouse IgG2b, clone: 906(MY9), Immunotech, Hamburg, Germany) and with a TRI-COLOR-conjugated goat antimouse IgG-2b (isotype: IgG-2b, clone: M32406, Caltag, Burlingame, CA, USA). Incubation was performed in the dark for 15 min on ice to ensure specific staining. Cells were washed with PBS and resuspended in 1 ml PBS for further dihydrorhodamine staining (DHR, Molecular Probes, Leiden, Netherlands, stock 1 mm in N,N-dimethylformamide, DMF) and if required stimulated with phorbol 12-myristate 13-acetate (PMA, Sigma, Deisenhofen, Germany, stock 1 mm in DMF).

DHR staining and PMA stimulation

Antibody-stained LPMNC were resuspended in 1 ml PBS and prewarmed to 37°C for 10 min. Samples were incubated with 10 µl DHR, stimulated with 10 µl PMA for 25 min at 37°C, and placed on crushed ice for a minimum of 3 min to terminate the reaction [17]. Cells were washed immediately with PBS, fixed with 1% formaldehyde and resuspended in PBS. Tubes remained on ice until analysis. The cell solution was transferred to fluorescence activated cell sorter (FACS) tubes just before FACS analysis. Flow cytometry was performed within 30 min after DHR staining. The production of reactive oxygen intermediates was measured by the oxidation of the membrane-permeable, non-fluorescent DHR to the positively charged, membrane-impermeable, and green fluorescent rhodamine 123.

Flow cytometry

Flow cytometry was performed using a FACScan (Becton Dickinson, San Jose, CA, USA) equipped with an argon ion laser with an excitation power of 15 mW at 488 nm. The fluorescence of 10 000 cells was collected on a linear scale through right angle scatter (side scatter), fluorescence for FITC was collected at 530 nm (FL1), TRI-COLOR emission was defined by 600 nm (FL3).

Analysis gates were set around debris, and intact cells on a forward scatter versus side scatter dot plot. The fluorescence dot plots were generated using the gated data. Acquisition was performed on fixed cells. Data acquisition and analysis were performed automatically using lysis TM II Software, Version 1·1 (Becton Dickinson, San Jose, CA, USA) or WIN-MDI Software.

Statistical analysis

The degree of inflammation of the specimens was graded endoscopically [18]: +, low degree of inflammation (increased granularity and friability of the mucosa in ulcerative colitis and single small aphthous lesions in Crohn's disease); + +, moderate inflammation (mucous membranes, spontaneous bleeding and small ulcers in ulcerative colitis, multiple aphtous lesions and small ulcers in Crohn's disease) and ++ +, severe inflammation (large ulcers in ulcerative colitis and large ulcerous lesions in Crohn's disease).

Data are expressed as mean (± s.d.). Statistical analyses were performed using the Mann–Whitney U rank sum test. Differences were considered significant at P-value of <0,05.

Results

Subtractive screening

To elucidate possible functional differences of intestinal macrophages from normal and inflamed mucosa, we performed a subtractive screening of mRNA expression in intestinal macrophages from normal and inflamed mucosa as described in Materials and methods. To avoid individual differences responsible for subtraction results macrophages from two inflamed specimens and from three normal specimens were pooled. Figure 1a shows an agarose gel of un-subtracted cDNA with a smear of cDNA without clear bands and of subtracted cDNA with a pattern of distinct bands. This indicated successful subtraction with selective amplification of mRNA species present only in inflammation associated macrophages.

Fig. 1
Subtractive screening with macrophage mRNA isolated from mucosa of patients with Crohn's disease and patients with non-inflamed mucosa. (a) Subtracted and amplified cDNA with prominent bands (sub.) and not subtracted control (‘uns.’); ...

The subtracted cDNAs were further separated with a SSCP-gel. Overall 70 different products were obtained. All cDNAs were cloned and sequenced. Table 1 gives the results of a similarity search of differentially expressed genes not including the subtracted products mentioned below. Some genes were obtained several times since sequenced products of the subtraction contained different parts of the same gene. As genes obtained several times were considered more likely to be truly up-regulated we further focused on these clones.

Table 1
List of genes identified to be up-regulated in macrophages from colonic mucosa of patients with Crohn's disease using the technique of subtractive screening as described. Sequence similarity search was performed with the blast program as described in ...

A search of the expressed sequence tag database showed that 3 of these 70 subtracted products had > 99% homology to mRNA of monocyte chemoattractant protein-1 (MCP-1). MCP-1 already had been shown to be significantly up-regulated during mucosal inflammation [19]. To demonstrate reliability of the subtractive screening we performed semiquantitative RT-PCR for MCP-1 with macrophage RNAs from Crohn's disease and control specimen. RT-PCR was repeated twice with mRNA not set for subtractive screening procedure. Whereas no specific PCR-product could be amplified from control macrophages with 32 cycles (Fig. 1b, right lane) a strong signal could be obtained with RNA from Crohn's disease macrophages (Fig. 1b, left lane). PCR for both GAPDH (data not shown) and actin proved integrity of the RNA from normal and inflamed mucosa. In addition two ‘virtual’ Northern blots were performed as described. ‘Virtual’ Northern blots from normal and inflamed Crohn's disease specimen, different from those used for subtractive screening, supported the data obtained by subtraction (Fig. 1c). Whereas no specific product could be obtained from control mucosa (Fig. 1c, right lane) a strong signal was visible after hybridization with linearly amplified cDNA from Crohn's disease macrophages (Fig. 1c, left lane). Densitometry revealed 0 versus 763 relative density units.

A further search of the expressed sequence tag database revealed that five of the 70 subtracted products had > 99% homology to mRNA of the cytochrome b subunit gp91-phox. As mentioned above the frequency of appearance of a certain RNA after a subtractive screening should reflect the initial difference in expression. Cytochrome b is the functional subunit of the NADPH oxidase, responsible for production of reactive oxidants in phagocytic cells during respiratory burst. As performed for MCP-1 semiquantitative RT-PCR was used for confirmation of the screening result. No specific product was obtained in RT-PCR with RNA from control macrophages (Fig. 2a, upper right lane) whereas a strong signal was visible with RNA from inflamed mucosa (Fig. 2a, upper left lane). Integrity of the mRNA was verified by Gene Checker™ kit. RT-PCR for five different housekeeping genes was performed and proved integrity of the RNA (only actin shown). Two ‘virtual’ Northern blots with cDNA of macrophages purified from normal mucosa and inflamed Crohn's disease mucosa specimens, which were different from those used for the subtraction, proved up-regulation of gp91 inflammation (0 versus 583 relative density units) (Fig. 2b). As NADPH oxidase is composed of six subunits which are expressed in a 1:1:1:1:1:1 stoichiometry we investigated the expression of another NADPH subunit, p67, by semiquantitative PCR. We could demonstrate an up-regulation of p67-mRNA in colonic macrophages during Crohn's disease supporting the relevance shown for gp91. Again no signal could be obtained with RNA from control macrophages (Fig. 2a) whereas a strong signal was seen with RNA from Crohn's disease macrophages (Fig. 2a). Integrity of RNA was verified by Gene Checker™ kit by amplification of five different housekeeping genes (only actin shown).

Fig. 2
RT‐PCR and ‘virtual’ Northern blot analysis for gp91 and p67. (a) RT‐PCR with macrophage mRNA isolated from mucosa of patients with Crohn's disease (CD, left) and patients with not inflamed mucosa (NI, right) for the large ...

Flow cytometric determination of oxidative burst reaction by intestinal macrophages

As NADPH oxidase is the central enzyme in cellular oxidative burst reaction the differential up-regulation of mRNA expression of NADPH oxidase subunits led us to investigate differences in oxidative burst activity in isolated macrophages from control and inflamed mucosa. For this purpose the intercellular formation of the reactive oxidants superoxide anion and hydrogen peroxide during the oxidative burst reaction was measured indirectly by fluorochrome formation.

Consequently, the data obtained by subtractive screening of mRNA could be confirmed on a functional level. LPMNC were isolated from biopsies of colonic mucosa as described (Fig. 3a). Macrophages were stained with mouse antihuman CD33 primary antibody and TRI-COLOR-conjugated goat antimouse IgG-2b (gamma) secondary antibody. LPMNC were incubated with DHR and stimulated with PMA. Flow cytometrical analysis allowed an easy discrimination of the CD33 positive population (Fig. 3b). The flow cytometric assay measures the respiratory burst activity by quantifying the intracellular oxidation of the membrane-permeable and non-fluorescent DHR to the cationic and intracellularly trapped, green fluorescent rhodamine 123 in single viable cells [17,20]. DHR is oxidized to rhodamine 123 in the simultaneous presence of H2O2 and peroxidase but not by O2 or H2O2 alone, as shown in cell-free assays [20]. The oxidation of DHR in phagocytes specifically measures the NADPH oxidase-dependent intracellular accumulation of reactive oxygen intermediates, i.e. those metabolites that are formed during the respiratory burst. The intracellular oxidation of nonfluorescent DHR to green fluorescent rhodamine 123 was determined for the gated CD33 positive intestinal macrophages in the LPMNC population for unstimulated (Fig. 3c) and PMA-stimulated cells (Fig. 3d). Macrophage populations isolated from biopsy specimens from 10 patients with Crohn's disease and 10 patients without IBD were analysed. There was a minor, though not statistically significant difference between PMA stimulated and unstimulated macrophages from normal colon (ratio stimulated vs. unstimulated: 0·91) (Fig. 4). In many cases even a slightly lower mean fluorescence was obtained for the oxidative burst activity of PMA-stimulated control macrophages, due possibly to a quench effect of PMA. In contrast to normal colon, significant differences in the ability to form of reactive oxidants were observed in the macrophage population obtained from 10 biopsy specimens of patients with Crohn's disease. CD33+ cells showed an increased oxidative burst response upon PMA-stimulation (ratio stimulated vs. unstimulated 1·80 ± 0·8, P = 0·004) compared to the macrophage-response in normal colonic mucosa (Fig. 4). However, there was no clear correlation of the oxidative burst response from macrophages with the macroscopic (endoscopic) degree of inflammation in patients with Crohn's disease (data not shown).

Fig. 3
Gating of CD33 positive cells (macrophages) and production of reactive oxygen intermediates in CD33+ cells isolated from mucosa of an inflammatory bowel disease patient as measured by the production of green fluorescent rhodamine 123. Dot plot of (a) ...
Fig. 4
Oxidative burst response from macrophages from 10 patients with Crohn's disease, 10 patients with ulcerative colitis and 10 patients without intestinal inflammation. The ratio of the rhodamine 123-fluorescence from PMA-stimulated divided by that of unstimulated ...

In an experiment with LPMNC from a patient with diverticulitis, macrophages showed only low oxidative burst response upon PMA-stimulation (ratio stimulated by unstimulated 1·2) compared to the macrophage-response in mucosa from patients with ulcerative colitis or Crohn's disease patients (Fig. 4).

To investigate whether the up-regulation of NADPH oxidase was specific for Crohn's disease or a feature common to other autoimmune incited inflammatory responses we further studied oxidative burst reaction in LPMNCs isolated from patients with ulcerative colitis (UC) (n = 10). The oxidative burst response of PMA-stimulated UC-macrophages was significantly higher (ratio of stimulated to unstimulated: 1·81 ± 0·6, P = 0·006) (Fig. 4) compared to macrophages from normal colonic mucosa. This indicated that the up-regulation of oxidative burst reaction is not specific for Crohn's disease.

Discussion

Several techniques are currently utilized to identify differentially expressed genes from two cell populations grown under different conditions. Most approaches require considerable amounts of nucleic acid as starting material. In this study we performed subtractive screening/hybridization to identify genes expressed differentially in macrophages of normal colonic mucosa and macrophages present in Crohn's disease mucosa. Only highly over-represented mRNA species are amplified by this method.

Subtraction and mRNA analysis revealed an up-regulation of MCP-1 in macrophages from Crohn's disease mucosa. Since Reinecker and coworkers already demonstrated an up-regulation of MCP-1 gene expression in inflammatory bowel disease mucosa [19], we considered the performed subtractive screening as highly specific.

Furthermore, we identified a low abundance of mRNA for cytochrome b-subunit gp91 in macrophages from normal mucosa versus a high abundance in Crohn's disease using a ‘virtual’ Northern blotting approach. Rugtveit and coworkers already described an increased respiratory burst associated with adherent LPMNC from patients with IBD [21]. Secretion of superoxide was elevated (median 30%) in macrophages from inflamed intestinal mucosa, which were believed to be newly recruited. In addition to these data, we could demonstrate mRNA for an additional subunit of the NADPH oxidase, which is responsible for superoxide production. Functional analysis as measured by flow cytometry confirmed increased intracellular reactive oxidants (elevated ratio stimulated vs. unstimulated Crohn's disease macrophages, median 98%). We determined increased oxidative burst activity in macrophages from patients with Crohn's disease but also in ulcerative colitis macrophages indicating that our finding was not specific for Crohn's disease.

The number of sequenced products from subtractive screening was limited. Further subtraction could have revealed evidence of up-regulation of other NADPH oxidase subunits as, for example, for the second NADPH oxidase subunit p67. However, the increased specificity of RDA in comparison with complementary strategies as for example random primed PCR (RAP-PCR) is associated with reduced sensitivity. Therefore only a fraction of the differentially expressed genes can be found by this technique.

The production of superoxide and other reactive oxygen intermediates responsible for microbicidal, tumoricidal and inflammatory activities in phagocytes is mediated by a membrane-associated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase [22,23]. This generation of toxic oxygen species by phagocytes is instrumental to the tissue damage of diverse conditions, including infection, ischemic injury, arthritis and other chronic inflammatory and autoimmune disorders [24], and may also be contributory to mutation and carcinogenesis [25]. The enzyme system responsible for superoxide generation forms a small transmembrane electron transport system that results in the oxidation of NADPH on the cytoplasmic surface and the generation of superoxide on the outer surface of the membrane. The terminal electron donor to oxygen is a flavocytochrome b [26,27] located primarily in the plasma membrane [28]. It is a heterodimer composed of a 91- kDa glycoprotein (termed gp91-phox, for glycoprotein (91 kDa) of phagocyte oxidase) and a 22- kDa polypeptide (p22-phox) [29]. In addition to the cytochrome b heterodimer, four cytosolic factors (p47-phox, p67-phox, p40-phox and p21 Rac) are required for NADPH oxidase activity [3032]. The enzyme is dormant in resting cells and becomes active upon stimulation. PMA initiates translocation of the cytosolic factors to the plasma membrane where they associate with cytochrome b. Superoxide anion is the starting point for the formation of other reactive oxidants such as hydrogen peroxide, which is further converted by a lysosomal myeloperoxidase to hypochlorous acid. Secretion of these oxidants can be associated with inflammatory tissue destruction in diseases such as ulcerative colitis or Crohn's disease [33].

Oxidants may be important contributors to the tissue damage seen in inflammatory bowel disease. In relation to the oxidative stress the colon may be subjected to by the production of reactive oxygen species from phagocytic leucocytes during inflammation, the colonic mucosa contains relatively small amounts of antioxidant enzymes [34]. The colon may be overwhelmed with reactive oxygen during times of active inflammation, which could result in intestinal injury.

Antioxidants have been shown to reduce mucosal inflammation in IBD and could become part of anti-inflammatory therapy in the future. It was suggested that nutritional antioxidants have a potential as therapeutic and chemopreventive agents [35] to settle the imbalance in the formation of reactive oxygen species and antioxidant micronutrients in the pathogenesis and/or perpetuation of the tissue injury in IBD [36]. Yamada and coworkers suggested that the reason that the sulfasalazine metabolite 5-ASA is so effective in vivo is due to its antioxidant properties and that more potent antioxidants may prove beneficial in the treatment of IBD [34]. However, antioxidants cannot prevent the formation of oxygen radicals but are able to neutralize them after formation. Glucocorticoids have been proven to inhibit superoxide production by phagocytes in a number of experimental models [3744]. However, antioxidants cannot prevent the formation of oxygen radicals but are able to neutralize them after formation [3844]. Dexamethasone is able to directly down-regulate the expression of gp91-phox and gp-47-phox [45]. Also non-steroidal anti-inflammatory drugs (NSAIDs) interfere with phagocyte NADPH oxidase activity in several experimental models [4649]. These studies indicate that a part of the treatment efficacy of drugs used in the treatment of inflammatory bowel disease could be mediated by the reduction of reactive oxygen metabolites.

Our data support the hypothesis that the up-regulation of enzymes responsible for oxidative burst activity in macrophages may be a major pathogenetic event in the tissue destruction during Crohn's disease. Therapeutic intervention reducing the oxidative burst activity may become a new alternative in IBD therapy. Specific inhibitors of cytochrome b have been described [5052]. Their efficacy in animal models of IBD is presently investigated.

Acknowledgments

This study was supported by the Deutsche Forschungsgemeinschaft (Ro 1236/3–1) and by the BMBF Kompetenznetz – CED (H.H., G.R.). We thank the endoscopists and nurses of the endoscopy department for providing colonic specimens. In addition, we thank Dr Cynthia Snyder for careful reading and helpful critic on this manuscript.

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