Patent Application: US-37178406-A

Abstract:
the present invention relates to novel markers for alternatively activated macrophages . more specifically , the present invention relates to the use of galactose - type c - type lectins as surface markers that allows rapid identification and sorting of the alternative macrophages . such identifications can be useful in diseases where there is an imbalance between proinflammatory and anti - inflammatory immune reactions .

Description:
the invention is further explained with the aid of the following illustrative examples . f1 ( c57bi / 6 × balb / c ) mice were infected with plc −/− t . b . brucei ( namangala et al ., 2001 ) and balb / c with toi strain taenia crassiceps metacestodes as described ( rodriguez - sosa et al ., 2002 ). isolation of plastic - adherent peritoneal macrophages from infected animals and generation and in vitro cytokine stimulation of thioglycollate - elicited macrophages were as described ( raes et al ., 2002 ). after induction of airway inflammation in c57bi / 6 mice ( pynaert et al ., 2003 ) and collection of bronchoalveolar lavage fluid ( pynaert et al ., 2003 ), alveolar macrophages were first enriched via a dynal ( oslo , norway ) magnetic particle concentrator , using cellection dynabeads and cd1 c antibodies ( pharmingen , san diego , calif . ), followed by fluorescence - activated sorting of high - autofluorescent cells . in vitro cytokine treatment of murine macrophages and human monocytes [ 00181 the plastic - adherent population of pec from balb / c mice , injected intraperitoneally with 3 ml thioglycollate broth ( biomerieux , marcy i ′ etoile , france ) four days prior to collection , was cultured in the presence of 100 iu / ml mouse recombinant il - 4 ( pharmingen ) or 100 iu / ml mouse recombinant ifn - γ ( pharmingen ) for 48 hours . human peripheral blood monocytes were prepared as previously described ( vanham et al ., 2000 ). briefly , pbmc isolated from donor buffy coats were separated into lymphocyte - and monocyte - enriched fractions by counter - flow elutriation . the pooled monocyte - enriched fractions were treated with sheep erythrocytes , after which the e - rosette - negative fraction was obtained using density gradient separation . 6 × 10 6 cells were dispensed in six - well tissue culture dishes ( falcon ) in 3 ml rpmi 1640 medium supplemented with 10 % heat - inactivated fetal calf serum , 2 mm l - glutamine , 100 iu / ml penicillin and 100 μg / ml streptomycin ( gibco - invitrogen ) and incubated in vitro for three days with human recombinant ifn - γ ( 1000 iu / ml ) or human recombinant il - 4 ( 15 ng / ml ) at 37 ° c . in a humidified incubator containing 5 % co 2 in air . after preparation of total rna and cdna ( raes et al ., 2002 ), quantitative real - time pcr was performed in a bio - rad ( hercules , calif .) icycler , with bio - rad iq sybr green supermix . primers and pcr conditions were as described before for mouse fizz1 and ym ( raes et al . 2002 ) and for human amac - 1 ( kodelja et al . 1998 ). other primers used were : mouse ribosomal protein s12 sense ( 5 ′- cctcgatgacatccttggcctgag - 3 ′) ( seq id no : 18 ), mouse ribosomal protein s12 antisense ( 5 ′- ggaaggcatagctgctggaggtgt - 3 ′) ( seq id no : 8 ), mmgl1 sense ( 5 ′- atgatgtctgccagagaacc - 3 ′) ( seq id no : 1 ), mmgl1 antisense ( 5 ′- atcacagatttcagcaacctta - 3 ′) ( seq id no : 2 ), mmgl2 sense ( 5 ′- gataactggcatggacatatg - 3 ′) ( seq id no : 3 ), mmgl2 antisense ( 5 ′- tttctaatcaccataacacattc - 3 ′) ( seq id no : 4 ), mouse mmr ( mrc1 ) sense ( 5 ′- ctcgtggatctccgtgacac - 3 ′) ( seq id no : 9 ), mouse mmr ( mrc1 ) antisense ( 5 ′- gcaaatggagccgtctgtgc - 3 ′) ( seq id no : 10 ), mouse arginase i sense ( 5 ′- atggaagagaccttcagctac - 3 ′) ( seq id no : 5 ), mouse arginase i antisense ( 5 ′- gctgtcttcccaagagttggg - 3 ′) ( seq id no : 6 ), human ribosomal protein s12 sense ( 5 ′- gaattcgcgaagctgccaaa - 3 ′) ( seq id no : 11 ), human ribosomal protein s12 antisense ( 5 ′- gactccttgccatagtcctt - 3 ′) ( seq id no : 12 ), hmgl sense ( 5 ′- cctcagtgaccctgaagga - 3 ′) ( seq id no : 13 ), hmgl antisense ( 5 ′- aaaggcagctcagtgactct - 3 ′) ( seq id no : 14 ), human mmr ( mrc1 ) sense ( 5 ′- cctctggtgaacggaatgat - 3 ′) ( seq id no : 15 ), human mmr ( mrcl ) antisense ( 5 ′- aggccagcacccgttaaaat - 3 ′) ( seq id no : 16 ), human arginase i sense ( 5 ′- ggcaaggtgatggaagaaac - 3 ′) ( seq id no : 17 ) and human arginase 1 antisense ( 5 ′- agtccgaaacaagccaaggt - 3 ′) ( seq id no : 7 ). for all these primers , each pcr cycle consisted of 1 minute denaturation at 94 ° c ., 45 seconds annealing at 55 ° c . and 1 minute extension at 72 ° c . gene expression was normalized using ribosomal protein s12 as housekeeping gene . similar results were obtained using other housekeeping genes . all comparisons were tested for statistical significance ( p & lt ; 0 . 05 ) using the unpaired t test . one of the clones picked up from the ssh library contained a fragment of mgl2 , a recently identified macrophage c - type lectin with a high homology ( 91 . 5 % amino acid identity ) but a distinct carbohydrate specificity from the originally identified mgl , which has now been called mgl1 ( tsuiji et al ., 2002 ). mgl ( 1 ) was found to be mainly restricted to macrophages in connective tissues ( imai et al ., 1995 ) and to act as recognition molecule for glycosylated antigens on cancer cells ( ichii et al ., 2000 ). human mgl , for which so far one gene locus but several mrna species , apparently derived from alternative splicing , were identified ( higashi et al ., 2002 ), was shown to recognize tn antigen , a carcinoma - associated epitope , consisting of a cluster of serine or threonine - linked n - acetylgalactosamine ( suzuki et al ., 1996 ). recent studies demonstrated that both human and mouse mgl are expressed by immature dendritic cells and are involved in the uptake of glycosylated antigens ( higashi et al ., 2002 ; denda - nagai et al ., 2002 ). mgl1 and mgl2 expression in aamf elicited during plc −/− t . b . brucei infection to analyze the expression of mgl1 and mgl2 in camf and aamf , quantitative rt - pcr was first performed on rna from peritoneal macrophages elicited via the plc −/− t . b . brucei model used to generate the ssh library . in this model , correlating with a switch from a type i cytokine environment in the early stage of infection to a type ii cytokine environment in the late and chronic phases , macrophages from early stage infected mice are camf , while those from the late and chronic stages of infection are aamf ( namangala et al ., 2001 ). mgl1 and mgl2 , similar to arginase 1 , fizz1 and ym , were found to be significantly induced in plastic - adherent peritoneal exudate cells ( pecs ) from chronic stage plc −/− t . b . brucei - infected f1 mice ( aamf ) as compared to early stage infected ( camf ) or non - infected mice ( fig1 , column 1a ). similar results were obtained in peritoneal macrophages purified from pecs via facs sorting , indicating that the observed modulations of gene expression are not due to the plastic adherence procedure . distinct kinetics of mgl1 and mgl2 modulation during taenia crassiceps infections infections with helminths such as taenia crassiceps are characterized by a gradual progression to polarized type ii immune responses and the generation of aamf ( rodriguez - sosa et al ., 2002 ). as shown in fig1 , column 1b , the pattern of mgl2 induction in peritoneal macrophages during infection with t . crassiceps is similar to arginase 1 , fizz 1 and ym , with a gradual increase in expression until a plateau is reached a few weeks after inoculation . although mgl1 is also induced during t . crassiceps infection , its induction level as compared to non - infected animals never reaches the several 100 - fold observed for mgl2 and the pattern of induction is quite distinct : a gradual increase in the first week of infection , after which the induction level falls back to five - to ten - fold . these differences suggest mgl1 and mgl2 may exert distinct functions in vivo . to examine whether the enhanced expression of mgl1 and mgl2 in aamf occurs in macrophage populations besides peritoneal macrophages and in disease models besides parasite infections , allergic , type ii cytokine - dependent pulmonary inflammation was induced in sensitized mice using ovalbumin ( ova ) aerosols and alveolar macrophages were purified from bronchoalveolar lavage ( bal ) fluid . these alveolar aamf were characterized by a high induction of both mgl1 and mgl2 as compared to alveolar macrophages from control animals , wherein the induction level was more pronounced for mgl2 than mgl1 ( fig1 , column 1c ). on the other hand , mgl2 , but not mgl1 , expression was dramatically reduced in alveolar macrophages from mice with experimental type i inflammation , induced via intratracheal administration of lps . an lps - induced reduction of gene expression was also observed for fizz1 ( fig1 , column ic ). in vitro cytokine modulation and in vivo dependence on cytokine signaling of mmgl1 and mmgl2 expression to verify the association of mmgl1 and mmgl2 with type ii cytokine - induced aamf , thioglycollate - elicited peritoneal macrophages were incubated with the type ii cytokines il - 4 or il - 13 . both cytokines moderately induced mmgl1 expression and strongly induced mmgl2 expression ( fig2 , column 2a ). a similar behavior was observed for the previously identified aamf markers arginase 1 , mmr , fizz 1 and ym . to investigate the contribution of type ii cytokines to the in vivo induction of the expression of these markers , t . crassiceps infections were performed in wild - type ( wt ), il - 4 - deficient ( il - 4 ko ) and il - 4rα - deficient ( il - 4rα ko ) balb / c mice . in peritoneal macrophages from non - infected animals , no significant differences were detected in the expression levels of arginase 1 , mmr , fizz1 , ym , mmgl1 or mmgl2 between the three types of mice . also , upon t . crassiceps infection of the three types of mice , a similar parasite burden was recorded up to the time when peritoneal macrophages were isolated . yet , induction of mmgl1 and mmgl2 , similar to arginase 1 and fizz1 , was marginal or not significant in il - 4 ko as compared to wt mice during infection ( fig2 , column 2b ). although the in vivo induction of ym and mmr was also drastically reduced in the absence of il - 4 , a significant induction of these two genes was still recorded in infected il - 4 ko mice . this residual induction of mmr and ym may be due to il - 13 signaling , since their fold of induction was further reduced in il - 4rα ko mice , lacking both il - 4 and il - 13 signaling ( murata et al ., 1999 ). hence , in this infection model , expression of this set of aamf markers , including the novel mmgl1 and mmgl2 markers , requires il - 4 receptor - mediated signaling . this is in accordance with what has been documented before for arginase 1 ( hesse et al ., 2001 ) and mmr ( linehan et al ., 2003 ) during schistosoma mansoni infection and for fizz1 and ym during trypanosome infections ( raes et al ., 2002 ). after having established that mgl mrna is induced in alternatively activated macrophages in vivo and in vitro , we wanted to assess if alternative activation of macrophages was also associated with an enhanced surface expression of mgl and , hence , whether this lectin would represent a useful surface marker for alternatively activated macrophages . to this aim , expression of mgl was tested on peritoneal macrophages from mice infected with the helminth taenia crassiceps . mgl was detected by the mgl - specific antibody er - mp23 ( bma biomedicals ag ; leenen et al ., 1994 ) that is described as specific for macrophages of connective tissue in mice . as shown in fig3 , alternatively activated macrophages exhibited increased expression of mgl , as compared to a low basal expression in peritoneal macrophages from non - infected mice . as is the case in murine aamf , expression of mmr was reported to be induced in human aamf ( mcnally et al ., 1996 ). yet , for fizz1 and ym , so far , no human homologues associated with alternative activation of macrophages have been described and also human arginase 1 has not been documented as a marker for aamf . on the other hand , using comparative gene expression profiling of human peripheral blood monocytes incubated in vitro with il - 4 versus ifn - γ , expression of certain genes , with amac - 1 ( alternative macrophage activation - associated cc - chemokine - 1 ) as most promising example , has been identified to be associated with alternative activation of human macrophages ( kodelja et al ., 1998 ). yet no murine homologue of amac - 1 , acting as a marker for aamf , has currently been defined . to analyze if human mgl ( hmgl ), for which so far only one single gene locus has been identified ( higashi et al ., 2002 ), is acting as a marker for human aamf , human peripheral blood monocytes were treated in vitro with il - 4 or ifn - γ . similar to human mmr and amac - 1 , but unlike human arginase 1 , hmgl expression in monocytes was significantly induced by il - 4 , but not by ifn - γ ( fig4 ). 1 . denda - nagai k ., n . kubota , m . tsuiji , m . kamata , and t . irimura . macrophage c - type lectin on bone marrow - derived immature dendritic cells is involved in the internalization of glycosylated antigens . glycobiology 2002 ; 12 : 443 - 450 . 2 . goerdt s . and c . e . orfanos . other functions , other genes : alternative activation of antigen - presenting cells . immunity 1999 ; 10 : 137 - 142 . 3 . gordon s . alternative activation of macrophages . nat . rev . immunol . 2003 ; 3 : 23 - 35 . 4 . gratchev a ., p . guillot , n . hakiy , o . politz , c . e . orfanos , k . schledzewski , and s . goerdt . alternatively activated macrophages differentially express fibronectin and its splice variants and the extracellular matrix protein βig - 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