Systemic Anti-TNFa Treatment Restores Diabetes-Impaired Skin Repair in ob/ob Mice by Inactivation of Macrophages

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1 ORIGINAL ARTICLE Systemic Anti-TNF Tretment Restores Dietes-Impired Skin Repir in o/o Mice y Inctivtion of Mcrophges Itmr Goren 1, Elke Müller 1, Dn Schiefelein 1, Urs Christen 1, Josef Pfeilschifter 1, Heiko Mühl 1 nd Stefn Frnk 1 To dte, dietes-ssocited skin ulcertions represent therpeutic prolem of clinicl importnce. The insulin-resistnt type II dietic phenotype is functionlly connected to oesity in rodent models of metolic syndrome through the relese of inflmmtory meditors from dipose tissue. Here, we used the impired wound-heling process in oese/oese (o/o) mice to investigte the impct of oesity-medited systemic inflmmtion on cutneous wound-heling processes. Systemic dministrtion of neutrlizing monoclonl ntiodies ginst tumor necrosis fctor (TNF) () or monocyte/mcrophge-expressed EGF-like modulecontining mucin-like hormone receptor-like (Emr)-1 () into wounded o/o mice t the end of cute wound inflmmtion initited rpid nd complete neo-epiderml coverge of impired wound tissue in the presence of persisting dietic phenotype. Wound closure in ntiody-treted mice ws prlleled y mrked ttenution of wound inflmmtion. Remrkly, nti-tnf- nd nti--treted mice exhiited strong reduction in circulting monocytic cells nd reduced numers of vile mcrophges t the wound site. Our dt provide strong evidence tht nti-tnf therpy, widely used in chronic inflmmtory diseses in humns, might lso exert effects y trgeting ctivted TNF-expressing mcrophge susets, nd tht inctivtion or depletion of misehving mcrophges from impired wounds might e novel therpeutic clue to improve heling of skin ulcers. Journl of Investigtive Dermtology (7) 127, ; doi: /sj.jid ; pulished online 26 April 7 INTRODUCTION The lst two decdes hve seen n explosive glol increse in people dignosed with dietes mellitus (Zimmet et l., 1). The dietic skin ulcertion represents n unmet medicl prolem in mny dietic individuls. Dietic ulcers still hve poor prognosis (Apelqvist et l., 1995) nd the 3-yer survivl rtes fter lim mputtions re etween 50 nd 59%, s ssessed for Itly nd Sweden, respectively (Apelqvist et l., 1993; Fgli et l., 1). By contrst, the efforts to identify novel phrmcologicl pproches to improve significntly severe dietes-impired heling conditions hve filed nd only recominnt pltelet-derived growth fctor is now ville for tretment of foot ulcers (Wiemnn et l., 1998). 1 phrmzentrum frnkfurt/zafes, Institut für Allgemeine Phrmkologie und Toxikologie, Klinikum der Johnn Wolfgng Goethe-Universität, Frnkfurt m Min, Germny Correspondence: Dr Stefn Frnk, phrmzentrum frnkfurt, Institut für Allgemeine Phrmkologie und Toxikologie, Klinikum der JW Goethe-Universität Frnkfurt/M., Theodor-Stern-Ki 7, D Frnkfurt/M., Germny. E-mil: S.Frnk@em.uni-frnkfurt.de Arevition: TNF, tumor necrosis fctor Received 19 Jnury 7; revised 15 Ferury 7; ccepted 19 Ferury 7; pulished online 26 April 7 In the pst decde, rodent models of oesity nd dietes hve een extensively used to unrvel the functionl connection etween n expnding dipose tissue nd n incresing insulin resistnce. Initil experiments evidenced the pro-inflmmtory tumor necrosis fctor (TNF) s n dipose tissue-derived meditor tht might directly contriute to insulin resistnce in oese rodents (Hotmisligil et l., 1993). However, recent studies now implicte one mrrowrecruited mcrophges locted within dipose tissue rther thn resident dipocytes to represent the cellulr source of pro-inflmmtory meditors in oesity (Weiserg et l., 3). Interestingly, plsm TNF levels were lso dependent on dipose tissue mss in humns (Kern et l., 1995; Mntzoros et l., 1997), nd clinicl studies confirmed tht the presence of inflmmtory meditors predicts the development of type II dietes mellitus (Schmidt et l., 1999; Prdhn et l., 1). Here, we used the oese/oese (o/o) mouse s model system of impired wound heling. These mice re chrcterized y severe dietes nd oesity syndromes (Colemn, 1978). The disesed phenotype is medited y the functionl loss of the o gene product: the 16 kd cytokine leptin (Zhng et l., 1994). tin is now well estlished s n dipocytokine, which is secreted from dipocytes nd functions in the regultion of neuroendocrine circuits, energy & 7 The Society for Investigtive Dermtology

2 homeostsis, nd s n importnt meditor of immunemedited diseses nd inflmmtory processes (Tilg nd Moschen, 6). Systemic leptin dministrtion into wounded o/o mice led to the resolution of the dietic phenotype nd improved the severely distured heling conditions y cting s mitogen for wound kertinocytes, ttenuting the inflmmtory wound response nd restoring insulin signling in wound tissue (Frnk et l., 0; Goren et l., 3, 6). Interestingly, lso systemic ppliction of n nti-tnf ntiody mrkedly incresed insulin receptor expression in wound tissue (Goren et l., 6). These recent findings strongly suggest possile functionl connection etween dipose tissue-derived TNF nd wound-heling disorders in o/o mice, lthough the mechnism still remins unknown. TNF is mjor product of clssiclly ctivted mcrophges (Gordon, 2; Mosser, 3); however, it must not necessrily e secreted. TNF is synthesized s memrne protein, which cn e cleved y TNF-converting enzyme to llow the susequent relese of the cytokine from ctivted mcrophges (Solomon et l., 1999). In this study, we provide strong evidence tht improved skin repir in dietic nd oese mice fter systemic dministrtion of TNF-neutrlizing ntiody ws functionlly connected to mrkedly reduced monocyte/mcrophge numers in o/o mice. Thus, our dt strongly suggest tht impired wound-heling conditions in o/o mice were driven y TNF-expressing mcrophges, which occurred s consequence of metolic disorders in the nimls. Moreover, our findings prove eneficil effect of TNF neutrliztion strtegy in impired wound heling, which ws medited y yet unknown induction of cell deth of ctivted mcrophges in distured wound tissue, nd thus extend its therpeutic potentil eyond the tretment of chronic inflmmtory diseses. RESULTS Short-term nti-tnf () nd nti-mcrophge () tretments did not improve metolic syndrome in wounded o/o mice Adipose tissue-derived TNF is considered to e functionlly involved in oesity-ssocited insulin resistnce (Hotmisligil et l., 1993; Wellen nd Hotmisligil, 5). To elucidte the potentil mechnism of oesity-relted TNF interference with skin repir in o/o mice (Goren et l., 6), we first nlyzed circulting levels of TNF in serum of the nimls. To this end, we injected monoclonl ntiody ginst murine TNF () (Echtencher et l., 1990) into wounded o/o mice. However, we hd to void ny interference with physiologic functions of normlly expressed TNF during the cute phse of skin repir (Figure 1), s we intended to restrictively interfere only with potentil pthophysiologicl role of TNF during dietes-impired wound-heling conditions. To rech this gol, we dministered the rt monoclonl ntiody t dys 7, 9, nd 11 upon injury y intrperitonel injection. Specific ntiody tretment ws controlled y intrperitonel dministrtion of nonspecific rt IgG t dys 7, 9, nd 11 fter wounding. Consistent with Proe Ctrl skin 1d wound 3d wound 5d wound 7d wound 13d wound Proe Ctrl skin 1d wound 3d wound 5d wound 7d wound 13d wound Serum TNF-α (pg/ml) d Blood glucose (mg/dl) o/o c Serum insulin (ng/ml) Body weight (g) C TNF-α GAPDH pulished dt (Hotmisligil et l., 1993), we found low levels of TNF in the ser of o/o mice nd tretment did not reduce circulting TNF (Figure 1). Even 16-dy systemic leptin tretment of the mice, which served s control for correction of the metoliclly distured phenotype (Figure 1c e) nd impired heling in o/o mice (Pelleymounter et l., 1995; Frnk et l., 0; Ring et l., 0; Goren et l., 3, 6), did not reduce serum TNF levels (Figure 1). In cler contrst to unltered TNF levels upon ntiody or leptin regimens, we could oserve drmtic improvement of wound morphology in leptin- nd TNFtreted nimls compred to the IgG-dministered control group (Figure 2). One possiility to explin the discrepncy etween unchnged circulting TNF levels nd improved skin repir might rise from inding of to memrneound TNF in monocytes/mcrophges (Solomon et l., 1999; Lügering et l., 1) with susequent depletion of the cells y induction of poptosis nd/or complement ctivtion (Nut et l., 2). To experimentlly mimic this condition in the mice, we included the monocyte/mcrophge-specific rt monoclonl ntiody (Austyn nd Gordon, 1981; Hirsch et l., 1981) into our experimentl e Figure 1. Anti-TNF () nd nti-mcrophge () tretments did not improve the metolic syndrome in o/o mice. () RNse protection ssy demonstrting TNF mrna expression in non-wounded ck skin (ctrl skin) nd wound tissue isolted from C57Bl/6 nd o/o mice. The time fter injury is indicted for ech lne. Levels of TNF () nd insulin (c) in serum, lood glucose (d), nd ody weight (e) in o/o mice t dy 13 fter wounding. Mice hd een treted with leptin (strting t 2 dys efore wounding, one ppliction per dy), - nd -specific rt monoclonl ntiodies or nonspecific rt IgG (injections t dys 7, 9, nd 11 post-wounding for the ntiody tretment). Po0.01; Po0.05 vs IgG-treted nimls. Brs indicte the men7sd otined from four individul nimls (n ¼ 4) Journl of Investigtive Dermtology (7), Volume 127

3 Sc on wounds (% of totl wounds) Wound re (mm 2 ) setup y systemic ppliction t dys 7, 9, nd 11 upon wounding. Both the nd ntiodies did not significntly improve the distured metolic phenotype of o/o mice, s the nimls remined severely hyperinsulinemic (Figure 1c), hyperglycemic (Figure 1d), nd oese (Figure 1e). Short-term systemic nti-tnf () nd nti-mcrophge () tretments strongly improved heling of chronic wounds in o/o mice with persisting metolic syndrome Most remrkly, we oserved profound improvement of impired wound tissue in response to the dministrtion of the or ntiodies. Here, it is importnt to note tht chnges in chronic wounds towrd properly heling wound re occurred within 6 dys, s we pplied the,, or the nonspecific control ntiodies only three times from dy 7 fter wounding nd susequently nlyzed wound morphology s erly s dy 13 fter wounding. As shown in Figure 2, nti-tnf () or nti-monocyte/-mcrophge (F4/ 80) tretments resulted in generl resolution of chronic wound conditions in the nimls. We found significnt reduction of persisting scs on the cks of ntiody-treted c Figure 2. Anlysis of wound morphology. () Presence of sc-covered wounds nd wound re of 13 dy wounds in IgG-, leptin-, -, nd -treted o/o mice. Po0.01 versus IgG-treted nimls. Po0.01 s indicted y the rckets. Brs indicte the men7sd from 24 wounds (n ¼ 24) from four individul nimls (n ¼ 4). () photogrphs of 13-dy ck skin wounds in IgG-, leptin-, -, nd -treted o/o mice. (c) Representtive histologicl nlyses of 13 dy wound tissue isolted from individul IgG-, leptin-, -, nd -treted o/o mice s indicted. Formlin-fixed, prffin-emedded 6 mm sections were counterstined using eosin/hemtoxylin. Arevitions: t, dipose tissue; gt, grnultion tissue; gt, trophied gt; nd, neo-dermis; ne, neo-epidermis, sc, sc. Br ¼ 1mm. nimls (Figure 2, left pnel). This prmeter served s n initil redout for wound epitheliliztion, s scs re lost fter formtion of roust neo-epiderml coverge of the wound site. In prllel, wound res were lso significntly reduced y ntiody tretment (Figure 2, right pnel). tin dministrtion of nimls, strting t dy 2 efore wounding, is well estlished to medite roust improvement of impired wounds in o/o mice (Frnk et l., 0; Ring et l., 0; Goren et l., 3, 6). The ove-mentioned improved wound-heling conditions upon leptin,, nd pplictions (Figure 2) re documented y photogrphs of representtive nimls shown in Figure 2. As next step, we performed histologicl nlysis of wound tissue from the different experimentl settings. First, we oserved poor tissue regenertion in IgG-treted control mice nd wound res were chrcterized y trophied wound mrgin epitheli. Additionlly, the grnultion tissue ppered to e reduced (Figure 2c, IgG pnel). By contrst, systemic leptin dministrtion, which exerts its ctions in functionl connection to the resolution of the dietic phenotype (Figure 1c e), medited n improvement of wound morphology s ssessed y the formtion of oth roust multilyered neo-epithelium nd dense neo-dermis (Figure 2c, lep pnel). Although effects on wound morphology ppered to e not s pronounced compred to leptin tretment, oth nd ntiody dministrtions medited the formtion of n epithelil coverge of the wound re, which ws prlleled y the formtion of dense neo-dermis. Thus, tretment strtegies using the ntiodies were le to drive rpid formtion of n epithelil coverge onto chronic wounds in the presence of persisting hyperglycemi nd, thus, dietic phenotype in the mice (Figure 1c e). Short-term nti-tnf () or nti-mcrophge () tretments mrkedly ttenuted wound inflmmtory conditions tin-driven skin repir in o/o mice ws ssocited with mrked decline in wound inflmmtion following the cute phse of repir, wheres chronic heling conditions exhiited sustined inflmmtory response in o/o s well s d/d mice (Wetzler et l., 0; Goren et l., 3). Thus, it ws interesting to investigte the inflmmtory sttus of - or -improved wound tissue in o/o mice. We oserved strong ttenution of the inflmmtory response t the wound site upon trgeting oth the TNF or mcrophge-specific ntigens in the mice (Figure 3). IL-, TNF, or cyclooxygense (COX)-2 mrna levels were significntly reduced upon systemic ntiody ppliction. However, chemokine (C-C motif) lignd 2 (CCL2) mrna expression t the wound site ws much less influenced y the tretments (Figure 3). Nevertheless, we found cler reduction of IL-1, TNF, nd CCL2 proteins in heling-improved wound tissue isolted from leptin-, -, nd -treted mice (Figure 3). These dt provide strong evidence tht the eneficil effects of oth ntiodies in distured heling re functionlly linked to the reduction of excerted wound inflmmtory conditions in oese nd dietic mice

4 tin #1 #2 #3 #1 #2#3 #1#2 #3 #1#2 #3 Proe IL-1β protein (pg/25 μg wound protein) IL-1β TNF-α COX-2 CCL2 GAPDH TNF-α protein (pg/25 μg wound protein) IL-1β mrna (PSL units per 25 μg RNA) COX-2 mrna (PSL units per 25 μg RNA) 1,250 1, # d13 fter wounding d13 fter wounding TNF-α mrna (PSL units per 25 μg RNA) 5,000 4,000 3,000 2,000 1, n.s. 300 CCL2 protein (pg/25 μg wound protein) # n.s. CCL2 mrna (PSL units per 25 μg RNA) d13 fter wounding d13 fter wounding Figure 3. nd ttenute wound inflmmtion in impired wounds of o/o mice. Mice hd een treted with nonspecific IgG or leptin (strting t 2 dys efore wounding, one ppliction per dy), or nd (injections t dys 7, 9, nd 11 fter wounding). () IL-1, TNF, COX-2, nd CCL2 mrna expression in 13-dy wound tissue isolted from IgG-, leptin-, -, nd -treted o/o mice s indicted. #1 3 represent individul nimls. For the RNse protection ssy (left pnels), every experimentl time point depicts three wounds (n ¼ 3) isolted from single individul mouse. A glycerldehyde-3-phosphte dehydrogense hyridiztion is shown s loding control. 1,000 cpm of the hyridiztion proe were used s size mrker. Sttisticl nlysis of IL-1, TNF, COX-2 nd CCL2 mrna expression is shown in the right pnels. Po0.01; Po0.05 vs IgG-treted nimls. # Po0.05 s indicted y the rckets. Brs indicte the men7sd from 12 wounds (n ¼ 12) from four individul nimls (n ¼ 4). () IL-1-, TNF-, nd CCL2-specific ELISA nlyses from 13-dy wound lystes isolted from IgG-, leptin-, -, nd -treted o/o mice s indicted. Po0.01; Po0.05 versus IgG-treted nimls. Po0.01; NS, not significnt s indicted y the rckets. Brs indicte the men7sd from eight wounds (n ¼ 8) from four individul nimls (n ¼ 4). Short-term nti-tnf () or nti-mcrophge () tretments reduced circulting monocytes nd resident wound mcrophge numers Here, we hypothesized similr mode of ction for nd in o/o mice in vivo vi potentil depletion of mcrophges y complement ctivtion. Indeed, oth ntiodies medited n ccelerted skin repir nd n ttenuted wound inflmmtion in o/o mice in the presence of severe dietic phenotype. To strengthen our hypothesis, we focused our investigtions on the monocyte/mcrophge cell type s the potentil trget cell to explin the effects of oth pplied ntiody strtegies. First, we determined the impct of systemic ppliction of leptin,, nd on circulting monocyte numers. FACS nlysis using PEcoupled ntiody determined circulting monocytes to represent % frction s compred to the totl leukocyte count (Figure 4). Interestingly, leptin did not significntly chnge numers of circulting monocytes. By contrst, FACS nlysis reveled drmtic loss (out 80%) of monocytic cell numers in the circultion of o/o mice upon tretment with either or ntiodies (Figure 4). Loss of monocytic cells ws profound nd resulted in remining monocytic frctions of % for or % for tretment, respectively. These dt provided strong experimentl evidence tht oth ntiodies, Leukocyte count 13.4+/ / 8.0 P= / 1.0 P<0.01 F4/ / 0.3 P<0.01 Circulting + cells (in % of totl lymphocytes) which were not functionlly relted with respect to their ntigenic trget molecule, exhiited their therpeutic potentil y trgeting the identicl cell type. Moreover, susequent nlysis of wound mcrophge numers trnslted our ntiody-medited effects from the circultion into the poorly regenerting peripherl wound tissue. As shown in Figure 5, improved wound tissue isolted from - nd -treted o/o mice exhiited reduction in wound mcrophge P=0.701 P=0.117 Figure 4. Determintion of circulting -positive monocytes in o/o mice. Mice hd een treted with nonspecific IgG or leptin (strting t 2 dys efore wounding, one ppliction per dy), or nd (injections t dys 7, 9, nd 11 fter wounding). At dy 13 fter wounding, fresh lood from IgG-, leptin-, -, nd -treted o/o mice ws nlyzed for -positive monocytes y FACS nlysis s indicted. Po0.01 versus IgG-treted nimls. Brs indicte the men7sd from four individul nimls (n ¼ 4) Journl of Investigtive Dermtology (7), Volume 127

5 Proe tin #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 tin #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 Proe LysoM Emr-1 GAPDH GAPDH Lysozyme M mrna (PSL units per 25 μg RNA) P= Emr-1 mrna (PSL units per 25 μg RNA) 50 P=0.975 P=0.605 Mφ ( + cells) (per totl wound section) 1, Figure 5. nd reduce of mcrophge numers in impired wounds of o/o mice. Mice hd een treted with nonspecific IgG or leptin (strting t 2 dys efore wounding, one ppliction per dy), or nd (injections t dys 7, 9, nd 11 fter wounding). () Lysozyme M nd Emr-1 mrna expression in 13-dy wound tissue isolted from IgG-, leptin-, -, nd -treted o/o mice s indicted. #1 3 represent individul nimls. For the RNse protection ssy (upper pnels), every experimentl time point depicts three wounds (n ¼ 3) isolted from single individul mouse. A glycerldehyde-3- phosphte dehydrogense hyridiztion is shown s loding control. 1,000 cpm of the hyridiztion proe were used s size mrker. A sttisticl nlysis of lysozyme M nd Emr-1 mrna expression is shown in the lower pnels. Po0.01; Po0.05 versus IgG-treted nimls. Brs indicte the men7sd from 12 wounds (n ¼ 12) from four individul nimls (n ¼ 4). () Immunohistologicl determintion of wound mcrophge numers in situ. Sections (6 mm) were stined for mcrophge-specific ntigen. Representtive higher mgnifiction sections isolted from IgG-, leptin-, -, nd -treted o/o mice re shown in the left pnels. Br ¼ 50 mm. Asolute numers of -positive wound mcrophges from totl wound sections re given in the right pnel. Po0.01 versus IgG-treted nimls. Po0.01 s indicted y the rckets. Brs indicte the men7sd from four wounds from four individul nimls (n ¼ 4). numers. A quntittive nlysis of constitutively expressed mcrophge-specific lysozyme M nd Emr-1 mrna species in wound tissue (Figure 5) nd the immunohistologicl stining for -positive cells (Figure 5) oth demonstrted n pproximtely 50 60% reduction of wound mcrophges in - nd -improved heling conditions. In contrst to oth ntiody tretments, these dt suggested different mode of ction for leptin to improve repir in o/o mice, s the remining high numers of wound mcrophges did not drive n excerted wound inflmmtion, ut ppered to e silenced s direct consequence of leptin ction (Figure 3). Short-term nti-tnf () or nti-mcrophge () tretments triggered poptosis of wound mcrophges As we hd oserved strong depletion of monocytes from the circultion fter nd tretments, which were relted to reduced numers of wound mcrophges, we were finlly interested in infiltrting mcrophge conditions in the context of the pplied diverse tretment regimens nd tissue environments. To this end, we performed terminl deoxyrionucleotidyl trnsferse-medited iotin-16-dutp nick-end leling (TUNEL) ssy in doule-leling experiment, which llowed us simultneous detection of - nd TUNEL-positive poptotic cells within different wound tissues. Interestingly, we oserved the lowest percentge of TUNEL-positive nd thus poptotic mcrophges in wounds isolted from phosphte-uffered sline, s well s leptin-treted o/o mice (Figure 6 c). Impired wounds in IgG-treted nimls were thus chrcterized y high numers of vile mcrophges (Figure 6 c), which most proly triggered n ugmented nd sustined inflmmtory response t the wound site (Figure 3) tht oviously interfered with repir (Figure 2). By contrst, high numers of vile wound mcrophges upon leptin dministrtion here indeed suggested tht leptin did not exert its eneficil effects on skin repir y interfering with monocyte viility (Figures 4 nd 6) ut through downregultion of wound mcrophge inflmmtory responses (Figure 3). More importntly, wound tissue from - nd -treted o/o mice ws chrcterized

6 / TUNEL / DAPI Apoptotic mcrophges (% TUNEL-positive cells of totl mcrophge numers) Vile mcrophges (clculted numer of TUNELnegtive mcrophges per section) c d Figure 6. Wound mcrophges in - nd -treted o/o mice re in n poptotic stte. () Prffin-fixed sections (6 mm) of 13-dy wound grnultion tissue isolted from IgG-, leptin-, -, nd -treted o/o mice s indicted were simultneously stined for mcrophge-specific nd poptotic cells y TUNEL using fluorescence-sed technique. Nuclei were visulized y 4,6-dimidino-2-phenylindole stining. 4,6-Dimidino-2-phenylindole/ doule-positive cells hve een indicted y circles in the upper pnels. As TUNEL lels only nucler structures, we restricted our counting of mcrophge/ TUNEL doule-positive cell numers to 4,6-dimidino-2-phenylindole/ doule-positive cells, which hve lso een indicted y circles in the lower pnels. Note tht we hve chosen res with higher numers of -stined mcrophges within the grnultion tissues for the presented figure to visulize our quntittive nlysis. The comprle numers of -stined mcrophges for ll four tretment groups presented here does not reflect totl numers throughout the wound. Br ¼ 50 mm. A sttisticl nlysis is given in pnel (left pnel). Po0.01; Po0.05 versus IgG-treted nimls. Po0.01 s indicted y the rckets. Brs indicte the men7sd from three wounds from three individul nimls (n ¼ 3). The right pnel comines dt from () nd Figure 5, showing the clculted numer of vile mcrophges in 13-dy wound sections from IgG-, leptin-, -, nd -treted o/o mice s indicted. (c) Prffin-fixed sections (6 mm) of 13-dy wound grnultion tissue isolted from IgG-, leptin-, -, nd -treted o/o mice s indicted were simultneously stined for mcrophge-specific (red) nd poptotic cells y TUNEL (rown) using peroxidse-sed technique. TUNEL-positive mcrophges re indicted. Br ¼ 40 mm. (d) Grnultion tissue (13-dy wounds) from IgG-, -, nd -treted o/o mice s indicted ws nlyzed for mcrophge-specific (red) nd processed ctive cspse-3 (rown). nd ctive cspse-3 doule-positive mcrophges re indicted. Br¼ 30 mm. y highest numers of poptotic nd thus non-vile mcrophges (Figure 6 c). Interestingly, immunohistologicl stining for processed nd thus ctive cspse-3 strongly suggested tht systemic ntiody tretments exerted its ctions vi induction of controlled mcrophge cell deth. These dt show tht nerly ll (more thn 80%) infiltrting mcrophges tht were reduced in numer upon nd pplictions dditionlly ppered to e in n 2264 Journl of Investigtive Dermtology (7), Volume 127

7 poptotic stte, nd thus were no longer le to interfere with skin repir even under severe dietic conditions in the nimls. DISCUSSION A series of wound-heling experiments performed y Leiovich nd Ross (1975) in the 1970s estlished prime role of ctivted mcrophges in skin repir. These clssicl studies using guine pig wound model demonstrted tht depletion of mcrophges under sterile conditions resulted in the filure of nimls to cler wound tissue of mtrix, ded nd dmged cells, firin, nd deris (Leiovich nd Ross, 1975). In recent review, Leiovich questioned his erly sttement tht it hs lrgely een considered too to tinker too hevily with the inflmmtory response t the wound site for fer of orting this process (Mrtin nd Leiovich, 5). Indeed, oservtions from chronic woundheling conditions in humns, s well s mice, strongly support the wound mcrophge to interfere with the norml repir process under conditions of impired heling. It is known tht mcrophge numers re mrkedly elevted in humn chronic leg ulcers (Rosner et l., 1995; Loots et l., 1998) nd in chronic wounds of geneticlly dietic nd oese d/d nd o/o mouse models (Wetzler et l., 0; Goren et l., 3). Nevertheless, lthough the presence of elevted mcrophge numers t chronic wound sites strongly suggested key role of this cell type in the control nd orchestrtion of pthoiology in impired wound tissue, most of the experimentl work in the field restrictively focused on the role of solule meditors. Not unexpected, n errnt expression of diverse protein-type meditors hs een reported in distured heling conditions in series of niml studies (for review see Werner nd Grose, 3). Unfortuntely, however, therpeutic pproches to improve distured wound heling in humns nd mice y exogenous dministrtion of missing or dysregulted fctors y ppliction of single recominnt protein meditors hve filed (Jeffcote nd Hrding, 3). Oviously, resident cells in dietic ulcers re phenotypiclly ltered with respect to their sensitivity towrd exogenous signls (Flng, 5; Goren et l., 6). Thus, it is resonle to rgue tht yet poorly understood mechnism drives complex dysregultion of inter- nd intrcellulr signling mchineries tht normlly enle the proper nd ccurte communiction of resident cells emedded into heling wound tissue nd thus cuses the filure to phrmcologiclly correct wound-heling disorders through ppliction of recominnt meditors. Interestingly, one such underlying mechnism might emerge from studies using oese nd dietic rodents. TNF is now discussed s pivotl meditor connecting oesity nd insulin resistnce (Hotmisligil et l., 1993), ut low levels of free-circulting serum TNF even in oese mice suggested different mode of ction for the therpeuticlly pplied nti-tnf ntiody. Recent studies implicted mcrophges rther thn dipocytes s the cellulr source of oesity-ssocited TNF tht is functionlly connected to conditions of insulin resistnce. Adipose tissue mcrophges were one mrrow-derived, recruited vi chemokine (C-C motif) receptor 2 ctivtion, nd most likely responsile for dipose tissue-derived TNF production in oese mice (Weiserg et l., 3, 6). Mcrophges must e in n ctivted stte to produce TNF (Gordon, 2; Mosser, 3). As TNF did not pper in high levels in the circultion of oese mice (Hotmisligil et l., 1993; this study), it is tempting to rgue tht TNF might not e relesed from ctivted mcrophges during developing oesity to medite conditions of incresing insulin resistnce. This notion is strongly supported y oservtions tht inflmmtory stimuli induced memrne-ound form of TNF in monocytes or mcrophges, which might e susequently relesed y TNF-converting enzyme clevge under inflmmtory conditions (Solomon et l., 1999). Moreover, dt from our study provide evidence tht oesity-relted disturnces might e medited y centrl pool of circulting TNF-positive monocytes from which mononucler cells enter diverse orgn systems. Oviously, nti-tnf tretment of o/o mice depleted these ctivted monocytes from the circultion nd cused mrked reduction of mcrophge numers in distured wound sites in the nimls. More importntly, depletion of circulting nd wound monocytic cells in o/o mice resulted in rpid nd profound trnsformtion of chronic wounds into heling skin tissue. Additionlly, our dt demonstrte tht the oesitychnged mcrophge cell type itself nd not the ction of n oesity-induced TNF molecule is in the center of distured skin repir in dietes. Thus, TNF neutrliztion using systemiclly pplied ntiodies hs to e regrded s n epiphenomenon with respect to the oserved eneficil effects of the nti-tnf therpy on impired wound heling, s we otined comprle effect with respect to circulting monocyte numers nd improvl of skin repir using n ntiody directed ginst the monocyte/mcrophge-specific memrne protein (Austyn nd Gordon, 1981; Hirsch et l., 1981). Therefore, the mode of ction of inflixim, TNF-neutrlizing ntiody used for therpy of chronic inflmmtory diseses in humns, might e more diverse with respect to its TNF-inding properties thn initilly expected. This hypothesis is strongly supported y the work of Lügering et l. (1), who reported mrked induction of poptosis in circulting monocytes y inflixim in ptients with Crohn s disese. In ccordnce, our nd tretments of wounded dietic mice lso trgeted oesity-ctivted monocytes in the circultion y reducing their totl numers. Moreover, the remining monocytes tht hd ctully escped this process nd reched the chronified wound tissue were oviously in n poptotic stte nd could no longer interfere with the heling process. These findings re strongly supported y the potency of the nti-tnf ntiodies inflixim nd dlumim, ut not the solule receptor etnercept, to selectively medite cspse-dependent poptosis in cultured humn monocytes, process tht is prlleled y mrked decrese in cytokine relese from the cells (Shen et l., 5, 6). In summry, our oservtions indicte pivotl role for ctivted mcrophges in the development of dietes-impired wound

8 heling nd strongly suggest phrmcologic trgeting of those ctivted mcrophges s promising therpeutic pproch to improve impired wound-heling conditions in generl. MATERIALS AND METHODS Animls Femle C57Bl/6 nd C57BL/6J-o/o mice were otined from The Jckson Lortories (Br Hror, ME) nd mintined under 12 h light/12 h drk cycle t 221C until they were 8 weeks of ge. At this time, they were cged individully, monitored for ody weight nd wounded s descried elow. Tretment of mice Murine recominnt leptin (Cliochem, Bd Soden, Germny) ws injected intrperitonelly (2 mg/g ody weight, one injection per dy, from 2 dys efore wounding until dy 13 fter wounding) nd purified rt monoclonl ntiodies directed ginst TNF () (Echtencher et l., 1990) (Acm Ltd, Cmridge, UK), (Austyn nd Gordon, 1981; Hirsch et l., 1981) (SeroTec, Düsseldorf, Germny), or nonspecific rt IgG (Snt Cruz, Heidelerg, Germny) were injected intrperitonelly (1 mg/g ody weight) in 0.5 ml phosphte-uffered sline t dys 7, 9, nd 11 fter injury. Wounding of mice Wounding of mice ws performed s descried previously (Frnk et l., 1999; Stllmeyer et l., 1999). Briefly, mice were nesthetized nd six full-thickness wounds (5 mm in dimeter, 3 4 mm prt) were mde on the ck of ech mouse. An re of 7 8 mm in dimeter ws excised t the indicted time points for nlysis. As control, similr mount of skin ws tken from the cks of non-wounded mice. For ech experimentl time point, tissue from four wounds ech from four nimls (n ¼ 16 wounds, RNA nlysis) nd from two wounds ech from four nimls (n ¼ 8 wounds, protein nlysis) were comined nd used for RNA nd protein preprtion. All niml experiments were crried out ccording to the guidelines nd were pproved y the locl Ethics Animl Review Bord. RNA isoltion nd RNse protection nlysis RNA isoltion nd RNse protection ssys were crried out s descried previously (Chomczynski nd Scchi, 1987; Frnk et l., 1999). The cdna proes were cloned using RT-PCR. The proes corresponded to nt (for IL-1, NM008361), nt (for TNF, NM013693), nt (for COX-2, M64291), nt (for CCL2, NM011333), nt (for lysozyme M, BC002069), nt (for Emr-1, X93328), nd nt (for glycerldehyde-3-phosphte dehydrogense, NM002046) of the pulished sequences. Immunohistochemistry Mice were wounded s descried ove. Animls were killed t dy 13 fter injury. Complete wounds were isolted from the ck nd fixed in formlin. Bisected wounds were emedded in prffin. Immunohistochemistry from 6 mm deprffinized sections ws performed s descried (Stllmeyer et l., 1999). The monoclonl ntiody directed ginst mcrophge-specific ntigen ws from Serotec, Düsseldorf, Germny. The nti-ctive cspse-3 ntiody ws otined from Innovtive Dignostik Systeme (Hmurg, Germny). Determintion of poptotic mcrophges in wound tissue y TUNEL stining Isolted 13 dy wound tissue from o/o mice ws fixed ccording to the HOPE (Hepes glutmic cid uffer medited orgnic solvent protection effect) method nd susequently emedded in lowmelting prffin (Innovtive Dignostik Systeme, Hmurg, Germny). Sections (6 mm) of HOPE-fixed wound tissue ws stined for poptotic cells using the fluorescence-sed ded end clorimetric TUNEL system (Promeg, Mnnheim, Germny) or the peroxidsesed ApopTg peroxidse in situ poptosis detection kit (Chemicon, Millipore, Schwlch, Germny) ccording to the instructions of the mnufcturer. Sections were susequently incuted with mcrophge-specific rhodmine-coupled ntiody (1: dilution, Serotec). Nuclei were counterstined using 4,6-dimidino-2-phenylindole stining (Sigm, Deisenhofen, Germny). Flow cytometry Two hundred microliter whole lood of o/o mice ws mixed with 5 ml RBC lysis uffer (0.74% NH 4 Cl) nd incuted for 30 minutes to lyse red lood cells. The remining leukocytes were wshed, plted t 10 5 cells/well nd incuted with 50 ml phycoerythrin-conjugted nti- ntiody (BD Biosciences, Heidelerg, Germny) (1: diluted in phosphte-uffered sline contining 1% fetl clf serum) for 30 minutes t 41C. Cells were wshed nd nlyzed for surfce F4/ 80 expression using FACSCliur flow cytometer (BD Biosciences). ELISA Totl wound lyste (25 mg), from 13 dy wounds, ws nlyzed for the presence of immunorective IL-1, TNF, or CCL2 y ELISA using the respective Quntikine murine ELISA kits (R&D systems, Wiesden, Germny). Determintion of lood glucose nd lood insulin levels Blood glucose levels were determined using the Accutrend sensor (Roche Biochemicls, Mnnheim, Germny) nd serum insulin ws nlyzed y ELISA (Crystl Chemicls, Chicgo) s descried y the mnufcturer. Sttisticl nlysis Dt re shown s mens7sd. Dt nlysis ws crried out using the unpired Student s t-test with rw dt. CONFLICT OF INTEREST The uthors stte no conflict of interest. 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