Dr A. Gratchev, Klinik für Dermatologie, Venerologie und Allergologie, Universitätsklinikum Mannheim gGmbH, Ruprecht-Karls-Universität Heidelberg, Theodor-Kutzer Ufer 1–3, 68167, Mannheim, Germany. E-mail: email@example.com
Alternative activation of macrophages, induced by Th2 cytokines and glucocorticoids, is essential for the proper functioning of anti-inflammatory immune reactions. To this end, alternatively activated macrophages (aaMΦ) express a not yet fully unravelled set of genes including cytokines such as alternative macrophage activation-associated CC-chemokine (AMAC)-1 and pattern recognition molecules such as the scavenger receptor CD163. In order to further characterize the molecular repertoire of aaMΦ, differential gene expression was analyzed by combining subtractive suppression cloning and differential hybridization. We show here that aaMΦ induced by interleukin (IL)-4 overexpress the prototype extracellular matrix (ECM) protein fibronectin on the mRNA and protein level. This overall increase is accompanied by a shift in fibronectin splice variants from an embryonic to a mature pattern. In addition, the expression of another ECM protein, βIG-H3, is also upregulated by IL-4 in aaMΦ. In contrast to IL-4 and in line with its inhibitory effect on wound healing, dexamethasone exerts a strongly suppressive effect on fibronectin and βIG-H3 expression. In conclusion, overexpression of ECM proteins induced by IL-4 in macrophages suggests that aaMΦ may be involved in ECM deposition and tissue remodelling during the healing phase of acute inflammatory reactions and in chronic inflammatory diseases.
In contrast to classical activation of macrophages by interferon (IFN)-γ or lipopolysaccharide (LPS), activation of macrophages by IL-4 or glucocorticoids may be classified as alternative [1, 2]. Two phenotypic markers, MS-1 high molecular weight protein (MS-1-HMWP) [3, 4] and RM3/1 antigen/CD163 [5–7] especially characterize the alternative pathway of macrophage activation. The expression of these molecules is induced by IL-4 and, vice versa, suppressed by IFN-γ[3, 8]. In addition, we have recently cloned a novel β-chemokine (AMAC)-1, whose expression is restricted to IL-4-treated macrophages and dendritic cells (DCs) . The AMAC-1 expression is strongly downregulated in macrophages by IFN-γ in a dose-dependent manner. Promoter analysis showed that an antagonistic regulation of the AMAC-1 expression by IL-4 and IFN-γ is mediated by a combined STAT-6/STAT-1 binding element . Apart from AMAC-1, MS-1-HMWP and RM3/1 antigen/CD163, there are several other genes with a less restricted expression pattern on which IL-4 and IFN-γ also exert antagonistic effects. Expression of the three species of Fcγ receptors [11, 12], for example, is induced by IFN-γ and inhibited by IL-4. Vice versa, macrophage mannose receptor [2, 13] and 15-lipoxygenase  are induced by IL-4 and inhibited by IFN-γ. Most importantly, the expression and synthesis of proinflammatory cytokines such as IL-1 [15, 16], IL-6 , and tumour necrosis factor (TNF)-α[15, 16] are also inhibited by IL-4 in macrophages. In contrast to these findings, IL-4 and IFN-γ may sometimes act synergistically, for example in the accumulation of cytoplasmic CD23 .
These differences in molecular repertoire suggest that there are also differences in terms of function between classically and alternatively activated macrophages . Alternatively activated macrophages are found during the healing phase of acute inflammatory reactions , in chronic inflammatory diseases, such as rheumatoid arthritis  and psoriasis , and in wound-healing tissue .
In this study, a combination of subtractive suppression cloning and differential hybridization was used for the identification of further genes overexpressed in alternatively activated macrophages in comparison to classically activated macrophages. We here demonstrate that the IL-4 stimulation of macrophages induces an increased expression of two extracellular matrix proteins, i.e. fibronectin and βIG-H3, that are important in cell attachment and during postinflammatory and wound-healing processes.
Materials and methods
Cells The isolation and cultivation of human monocytes/macrophages was done as described . The cells were purified from pooled buffy coats. A total of 35 ml blood were layered on top of 15 ml Ficoll-Paque (Biochrom, Berlin, Germany) in a 50-ml Leucosep tube (Greiner, Fickenhausen, Germany). After 40 min centrifugation in a swing out rotor (Beckman Coulter, Fullerton, CA, USA) at 650 × g peripheral blood mononuclear cells (PBMC) were collected from the Ficoll-Serum interphase. PBMCs were washed three times with Ca2+ and Mg2+ free phosphate-buffered saline (PBS) (Biochrom). The Percoll gradient was preformed by centrifugation of freshly prepared Percoll (13.5 ml Percoll (Pharmacia, Freiburg, Germany) 1.5 ml 10 × Earle's Minimal Essential Medium (MEM), 15 ml Spinner's medium supplemented with penicillin/streptomycin, glutamine and nonessential amino acids (all from Biochrom)) at 12000 × g for 12 min at 20 °C without breaks in a SS-34 rotor (Sorvall, Dusseldorf, Germany). Five to 8 × 108 PBMCs were layered on top of the Percoll gradient and centrifuged at 650 × g for 40 min at 20 °C without breaks. The upper layer, containing 80–90% monocytes was collected and washed three times with PBS. For the culture monocytes were resuspended in McCoy's medium (Biochrom) supplemented with 15% fetal calf serum (FCS) (Biochrom) and appropriate concentrations of penicillin/streptomycin, glutamine and nonessential amino acids at a concentration of 1.3 × 106 cells/ml. The cell suspension was supplemented with appropriate mediators and transferred to UV irradiated, Teflon-coated plastic bags. The bags were incubated in the presence of 7.5% CO2. In order to detach the cells from the plastic, the bags were incubated on ice for at least 30 min before harvesting. The bags were cut open and the cell suspension was collected. The cells were washed twice with PBS and used for RNA isolation, or protein lysate preparation. For cytospin preparation 1 × 104 cells were centrifuged in cytocentrifuge at × 100 gfor 4 min.
Mediators Human IFN-γ, from TEBU Peprotech (Frankfurt am Main, Germany), was used at a concentration of 1000 U/ml. IL-4 was from PromoCell (Heidelberg, Germany), and was used at a concentration of 15 ng/ml. Dexamethasone was used at 5 × 10−7 M.
Subtractive hybridization Total RNA was isolated using RNAclean (Hybaid-AGS, Heidelberg, Germany) reagent according to the recommendations of the manufacturer. Briefly, 3 × 106 cells were resuspended in 1 ml of the reagent. The lysate was extracted with 100 µl of chloroform. After 10 min of incubation on ice and 10 min of centrifugation at × 17000 g4 °C the aqueous phase was recovered. RNA was precipitated with isopropanol, washed twice with 70% ethanol and dissolved in DEPC-treated water. Concentration was determined spectrophotometrically. Total RNA was used for Northern blot analysis, or for mRNA isolation. mRNA was isolated from 200 µg of total RNA using DynaBeads (Dynal, Oslo, Norway). RNA was denatured at 65 °C for 5 min. Poly(A) + RNA was bound to oligo-d(T)25 magnetic beads by incubation at RT with continuous mixing for 5 min. After a triple wash, mRNA was eluted with DEPC-treated water by incubation for 5 min at 65 °C. The resulting amount of mRNA was estimated on an agarose gel. Subtractive hybridization was performed using the PCR-SelectTM kit (Clontech, Palo Alto, CA, USA) according to the recommendations of the manufacturer. The mRNA population that contains the differentially expressed transcripts of interest will hereafter be called ‘tester’ and the reference mRNA population will be referred to as ‘driver’. Tester and driver mRNA (2–5 µg each) were converted to cDNA's by reverse transcription with AMV reverse transcriptase. The obtained cDNAs were digested with RsaI restriction endonuclease, generating blunt ended fragments. Tester cDNA was split into two parts and each was ligated to an adapter. Two hybridizations were performed. Initially each sample of adapter ligated tester was mixed with an excessive amount of driver cDNA, heat denatured, and hybridized. During the second hybridization, the two primary hybridization samples were mixed together without denaturing. The obtained population of molecules was then subjected to polymerase chain reaction (PCR) with adapter primers to amplify the differentially expressed sequences. The obtained PCR product was digested with RsaI and gel purified using QIAEX II kit (Qiagen, Hilden, Germany) and ligated into pBSSK (Stratagene, La Jolla, CA, USA) vector digested with EcoRV restriction enzyme (MBI Fermentas, Vilnius, Lithuania). Alternatively gel purified fragments were used as a probe for hybridization.
Clone analysis Ligation reaction of subtracted fragments was transformed into TOP10F′E. coli (Invitrogen, Groningen, the Netherlands), and plated on agar plates supplemented with appropriate concentrations of ampicillin, X-gal and Isopropyl-β-D-thiogalactopyranoside (IPTG). After overnight culturing at 37 °C white bacterial colonies were transferred in 96-well plates, containing 200 µl/well Luna Broth (LB) medium supplemented with ampicillin. After overnight growth replicas of the plates were prepared: 50 µl of medium were transferred onto a nylon membrane GeneScreen Plus (NEN) using a DotBlot apparatus (BioRad, München, Germany). The membrane was then denatured in 1 N NaOH for 5 min and neutralized in 2 × Saline Sodium Cytrate (SSC) for 5 min. After fixing by incubation for 2 h at 80 °C, the membrane was hybridized with an appropriate probe. Positive clones were selected and plasmids were isolated from 3 ml of overnight culture using r.p.m. kit (BIO101). Sequencing was performed with BigDyeTM terminator cycle sequencing kit (PE Applied Biosystems, Weiterstadt, Germany).
Northern blot hybridization Twenty micrograms of total RNA was electrophorezed in a denaturing agarose gel as described  and blotted using the capillary method with 20 × SSC on Nylon membrane GeneScreen Plus (NEN). The RNA was fixed on the membrane by incubation for 2 h at 80 °C. Probes for hybridization were labelled with the Ready-to-GoTM random primer labelling kit (Pharmacia) with 50 µCi [α-32P]dCTP with specific activity 6000 Ci/mmol (NEN). Membranes were prehybridized for 30 min at 60 °C in an ExpressHyb hybridization solution (Clontech) and hybridized with denatured probe and salmon sperm DNA (100 µg/ml) for 3 h at 60 °C. After hybridization the membrane was washed with 2 × SSC, 0.5% SDS for 15 min at RT, and twice with 0.2 × SSC, 0.1% SDS at 60 °C until the background became sufficiently low. Membranes were dried, wrapped in SaranWrap, and exposed to X-omat AR film (Kodak, New Haven, CT, USA).
Probes for hybridization Probe for fibronectin detection was a subcloned fragment of fibronectin cDNA (bp 6222–6958). Probe for βIG-H3 was a subcloned fragment of βIG-H3 cDNA (bp 1390–2690). Probe for GAPDH gene was generated in PCR with primers GAPDH F021 (5′-ATA TTG TTG CCA TCA ATG ACC CCT TCA, pos 146–172) and GAPDH R021 (5′-TTG ACA AAG TGG TCG TTG AGG GCA, pos 986–963).
RT-PCR Reverse Transcription (RT) was done with Ready-To-GoTM Your Primer First Strand synthesis beads (Pharmacia). The total RNA (5 µg) was mixed with 40 pmol of gene specific primer and incubated for 40 min at 65 °C in order to remove secondary structures of RNA. Denatured RNA was transferred to the tube containing cDNA synthesis bead and incubated for 1 h at 37 °C. First strand cDNA was used directly for PCR. Alternative splicing of fibronectin (Acc.# X02761) was analyzed with primers FNF011 (5′-AGA GGA GCA CCA CCC CAG ACA TTA, pos 3507–3530) and FNR011 (5′-GTG AGT AAC GCA CCA GGA AGT TGG, pos 3830–3807) for the EDB region of alternative splicing, and FNF009 (5′-CAA ACA GAA ATG ACT ATT GAA GGC, pos 4700–4723) and FNR009 (5′-TGA GTG AAC TTC AGG TCA GTT GGT, pos 5115–5092) for the EDA region. The program for amplification was in both cases: denaturing at 94 °C for 45 s, annealing at 60 °C for 1 min and extension at 72 °C for 1 min for 30 cycles.
Protein detection The fibronectin expression on the protein level was analyzed by immunohistochemistry using the standard APAAP method with monoclonal αFN antibody (Sigma, Diesenhofen, Germany, F7387) used in 1 : 500 dilution. For Western blot, cells were lysed in buffer containing 20 m m Tris HCl pH 8.0, 50 m m NaCl, 1 m m EDTA and 1% NP40. Proteins (10 µg) were denatured by boiling and separated on a 4–12% NuPAGE precast gel (Novex, Groningen, the Netherlands). Wet blotting on Immobilon-P membrane (Millipore, Eschborn, Germany) according to the recommendations of the manufacturer. Fibronectin was detected with αFN antibody (Sigma) at a dilution of 1 : 1000. The membrane was developed with Enchanced Chemiluminiscence (ECL) system (Amersham).
Detection of differentially expressed genes
In order to find genes differentially expressed in alternatively activated macrophages, a combination of subtractive hybridization and differential hybridization was used . Monocytes, isolated from human pooled buffy coats were stimulated with IL-4 or IFΝ-γ. On day 6 of stimulation, the RNA was isolated. Subtractive hybridization was performed with the PCR select (Clontech) kit. The total number of clones obtained after cloning of subtracted cDNA was about 20000. Owing to the presence of an amplification step in the subtraction protocol it is to be assumed that the actual number of independent clones is much lower. Therefore, only 384 clones were randomly picked for further analysis. Subtracted cDNA obtained from the second stimulation experiment was used as a probe for hybridization of the membranes containing spotted clones. Only about half of the selected clones gave a significant hybridization signal. Sequencing of the first 10 clones revealed prevalence of human fibronectin fragments, therefore clones were also selected not to contain the most frequent fibronectin fragment (bp 6222–6958).
Out of a total of 55 sequenced clones, 23 appeared to be fibronectin, six represented AMAC1, a gene previously described to be specifically expressed in alternatively activated macrophages , and two clones corresponded to the βIG-H3 gene. The remaining single clones were either known to be overexpressed in macrophages by IL-4 stimulation such as cytochrome b, or were not confirmed by Northern blot analysis such as Caspase 3. Only three clones had no homology in the database. However, none of them was confirmed by Northern analysis.
Detailed analysis of fibronectin and βIG-H3 expression in alternatively versus classically activated macrophages
For further analysis of the expression pattern of fibronectin and ‘βIG-H3 in activated macrophages, macrophages were stimulated with IFN-γ, IL-4, dexamethasone and IL-4+dexamethasone. Non-activated cells served as a control. Cells were harvested after 3 and 6 days after activation. Hybridization with the fibronectin probe revealed a strong overexpression of the gene induced by IL-4 already on the third day of activation (Fig. 1A, lane 3); no expression was observed in the control, IFN-γ- or dexamethasone-activated cells (Fig. 1A, lanes 1, 2 and 4). In combination with IL-4, the dexamethasone strongly inhibited fibronectin expression (Fig. 1A, lane 5). No significant differences in the expression pattern of fibronectin were observed when comparing days 6 and 3 of activation (Fig. 1A, lanes 6–10).
The pattern of βIG-H3 expression differs from that of fibronectin in a higher baseline level of expression that is suppressed by dexamethasone, but not IFN-γ(Fig. 1B, lanes 1,2,4,6,7,9). On day 3 the expression of βIG-H3 in dexamethasone-treated macrophages was much lower than in the control cells (Fig. 1B, lane 4), and on day 6 the βIG-H3 expression was hardly detectable after the glucocorticoid treatment (Fig. 1B, lane 9). The βIG-H3 was strongly superinduced by IL-4 on day 3 as well as on day 6 and this IL-4-induced overexpression was strongly suppressed by dexamethasone (Fig. 1B, lanes 3,5,8,10).
Analysis of fibronectin splicing
It has been well described in the literature that fibronectin can be alternatively spliced under certain in vivo and experimental conditions (reviewed in ). In order to test whether this is also the case during alternative activation of macrophages, the PCR amplification of the fibronectin fragments with primers flanking the alternative splice sites EDA and EDB was performed (Fig. 2A,B). The amplification with primers flanking the EDA region revealed two bands: a 146-bp band corresponding to the EDA-negative splice variant, and a 416-bp band corresponding to the EDA-positive splice variant. Treatment of macrophages with IL-4 caused an increase in the EDA-negative splice variant on day 3 (Fig. 2A, lanes 3,5) as well as on day 6 (Fig. 2A, lane 8) as compared to nonstimulated or IFN-γ-stimulated macrophages (Fig. 2A, lanes 1,2,6,7). Dexamethasone did not have a clear-cut effect on the ratio of the EDA-positive/EDA-negative splice variants (Fig. 2A, lanes 4,8). Amplification with primers, flanking EDB region showed the presence of EDB-negative splice variant only (Fig. 2B).
In order to confirm the data obtained by Northern analysis, fibronectin protein was analyzed by immunohistochemistry and Western blot. Immunohistochemical staining of cytospins obtained from the same cell preparation as the RNA for the Northern blot showed no positive staining in the control, IFNγ- and dexamethasone-activated macrophages on day 3 and day 6. In the case of activation by IL-4, many, but not all, cells were stained and the activation by IL-4 together with dexamethasone produced only single positive cells (Fig. 3).
Western blot analysis revealed a similar expression pattern on the protein level (Fig. 4) as had been detected on the RNA level, indicating that the fibronectin expression is not modified on the postranscriptional level.
Alternatively activated macrophages play a major role in the healing phase of acute inflammatory reactions, in chronic inflammatory diseases such as rheumatoid arthritis and in wound-healing processes [3, 19–21]. Apart from IL-4, the differentiation of alternatively activated macrophages may be induced by other mediators, such as transforming growth factor (TGF)-β and glucocorticoids . While IL-4 is strictly confined to an immunologically mediated downregulation of inflammation by Th2 cells, the TGF-β shows a broader expression and predominates in the wound healing. Healing processes in inflammatory reactions and in wound healing, however, share many cellular events such as clearance of tissue debris and tissue remodelling . In this respect, alternatively activated macrophages are intimately involved in angiogenesis: they preferentially occur in diseased tissues with strong vascularization and they strongly induce endothelial cells to proliferate by the expression of a special set of angiogenic factors [26–28].
The induction of angiogenesis, however, is not the only role of alternatively activated macrophages during these healing processes, but they are also involved in extracellular matrix deposition. In line with previous findings in unstimulated macrophages and fibroblasts [29, 30], we show here that alternatively activated macrophages are induced to differentially express high levels of fibronectin mRNA and protein by IL-4. A fibronectin expression in monocytes may also be induced by stimulation with such cytokines as IL-1α, IL-6 or TNF-α. Surprisingly, IL-4 had a suppressive effect on the fibronectin expression when used in combination with these cytokines . As this complex experimental system is not comparable to the plain stimulation shown here and as Kitamura et al.  did not analyze the effects of IL-4 alone, these data do not contradict the results presented here. In addition, we demonstrate that the IL-4 stimulation induces alterations of a fibronectin splice variant distribution, increasing the fraction of EDA-negative versus EDA-positive species. Fibronectin splice variants containing EDA and EDB preferentially occur during embryogenesis, while the fibronectin lacking EDA and EDB represents the mature adult form also known as plasma fibronectin. In fibroblasts, embryonic EDA+/EDB+ fibronectin is induced by TGF-β[32, 33]. Macrophages have been shown to express an embryonic fibronectin during the early stages of wound healing while the expression of the mature adult forms occurs during later phases . Thus, the IL-4 induced shift towards the expression of more mature adult fibronectin splice variants (EDA-, EDB-) by alternatively activated macrophages fits well with the notion that IL-4 exerts its regulatory functions late in the inflammatory processes, i.e. in the healing phase of acute inflammatory reactions and in chronic inflammatory diseases, while wound healing per se is dominated by TGF-β-induced effects.
In addition to fibronectin, several other ECM proteins have been ascribed as a functional commitment in the healing processes, among them the fasciclin-domain containing protein βIG-H3. The βIG-H3 was identified as a protein induced by TGF-β in the lung adenocarcinoma cell line A549 . It was supposed to be an ECM protein owing to the presence of a secretory domain and of an integrin recognition site . The βIG-H3 is expressed in many tissues, and was shown to promote the adhesion and spreading of dermal fibroblasts . The experiments presented here demonstrate a strong superinduction of the βIG-H3 expression in alternatively activated macrophages by IL-4. The similarity in the expression patterns of the βIG-H3 and fibronectin in alternatively activated macrophages provides some evidence that the βIG-H3 may also be involved in postinflammatory healing processes.
In contrast to IL-4, dexamethasone had a strongly suppressive effect on the expression of fibronectin and βIG-H3 by alternatively activated macrophages. While these data are in good agreement with the well-known inhibitory effect of glucocorticoids on wound healing , they contrast with the influence of glucocorticoids on other functions and phenotypic traits of alternatively activated macrophages. Regarding the macrophage activation, glucocorticoids act synergistically with IL-4 as to the induction of CD163 and to the suppression of proinflammatory mediators while they only exert a minor effect on the expression of MS-1-HMWP and AMAC-1 by alternatively activated macrophages. Thus, alternatively activated macrophages may not be a static, terminally differentiated subpopulation of cells, but they may rather represent versatile players adapting to different needs in a continuum of in vivo situations ranging from tolerance induction to downregulation of inflammation, removal of debris and tissue regeneration.
In conclusion, the overexpression of ECM proteins in IL-4-stimulated macrophages suggests that alternatively activated macrophages are involved in generating a special type of extracellular matrix mediating attachment and spreading of fibroblasts and guidance of endothelials during angiogenesis and postinflammatory healing.
The work presented here was supported by a grant of the Deutsche Forschungsgemeinschaft to S.G. (Go 470–4/1)