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ModelArthritis in a Novel CD64 Transgenic Rat CD64-Directed Immunotoxin Inhibits

McLaughlin, Jan G. J. van de Winkel and Theo ThepenWalraven, Ilonka Stuij, Martin C. Harmsen, Pamela M. J. Anneke J. van Vuuren, Joel A. G. van Roon, Vanessa

http://www.jimmunol.org/content/176/10/5833doi: 10.4049/jimmunol.176.10.5833

2006; 176:5833-5838; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/176/10/5833.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2006 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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CD64-Directed Immunotoxin Inhibits Arthritis in a NovelCD64 Transgenic Rat Model1

Anneke J. van Vuuren,2* Joel A. G. van Roon,*† Vanessa Walraven,* Ilonka Stuij,*Martin C. Harmsen,‡ Pamela M. J. McLaughlin,‡ Jan G. J. van de Winkel,*§

and Theo Thepen*¶�

Macrophages are known to play a key role during inflammation in rheumatoid arthritis (RA). Inflammatory macrophages haveincreased expression of CD64, the high-affinity receptor for IgG. Targeting this receptor through a CD64-directed immunotoxin,composed of an Ab against CD64 and Ricin A, results in effective killing of inflammatory macrophages. In this study, we showelevated levels of CD64 on synovial macrophages in both synovial lining and synovial fluid in RA patients. The CD64-directedimmunotoxin efficiently eliminates activated synovial macrophages in vitro, while leaving quiescent, low CD64-expressing mac-rophages unaffected. To examine whether killing of CD64 macrophages results in therapeutic effects in vivo, we established anadjuvant arthritis (AA) model in newly generated human CD64 (hCD64) transgenic rats. We demonstrate that hCD64 regulationin this transgenic rat model is similar as in humans. After AA induction, treatment with CD64-directed immunotoxin results insignificant inhibition of disease activity. There is a direct correlation between immunotoxin treatment and decreased macrophagenumbers, followed by diminished inflammation and bone erosion in paws of these hCD64 transgenic rats. These data supportsynovial macrophages to play a crucial role in joint inflammation in AA in rats and in human RA. Selective elimination ofinflammatory macrophages through a CD64-directed immunotoxin may provide a novel approach for treatment of RA. TheJournal of Immunology, 2006, 176: 5833–5838.

R heumatoid arthritis (RA)3 is characterized by an inflam-matory process in synovium resulting in progressive de-struction of cartilage and bone in affected joints. The

abundance and activation of synovial macrophages in inflamedsynovium correlates with severity and chronicity of RA (1, 2).Macrophages are very versatile cells and exert a multitude of bi-ological functions contributing to maintenance of inflammationand bone destruction. For instance, they can secrete a range ofproinflammatory cytokines, chemokines, and metalloproteinases,which can directly trigger tissue damage (3, 4). Macrophages alsoregulate T cell and dendritic cell functions, and serve as APCs, inwhich capacity they might be involved in epitope spreading. Incontrast, macrophage functioning is subjected to control from thelocal milieu, like IFN-� produced by inflammatory T cells. Theinteraction between macrophages and their environment may lead

to a “vicious” circle, which maintains inflammation without exter-nal stimuli, resulting in a chronic phase.

At present, blockade of proinflammatory cytokines representsone approach to treat RA. Especially TNF-�, produced by mac-rophages, proved to be a key cytokine in destructive arthritis. Anti-TNF-� therapy results in impressive protection against joint in-flammation and joint damage in RA patients (5, 6), althoughsignificant numbers of patients do not respond to this anti-TNF-�therapy. In these nonresponding patients, blockade of a singlemacrophage effector function is probably not sufficient enough tocontrol disease. Therefore, counteracting complete macrophageactivity, rather than inhibition of individual inflammatory media-tors, may prove more efficacious. This idea is confirmed by ex-periments in which macrophages were eliminated through intra-articular injections of chlodronate containing liposomes relyingupon strong phagocytic capacity of macrophages (7).

One hallmark of inflammatory macrophages is strongly en-hanced expression of CD64 (Fc�RI), the high-affinity receptor forIgG (8, 9). CD64 expression is limited to cells from the myeloidlineage and can be up-regulated by several cytokines like IL-10and IFN-�, both of which are enhanced in inflammatory RA joints.In addition, CD64 expression can be induced on neutrophils inhumans (10). Endocytosis and phagocytosis have proven to bevery efficient through the CD64 receptor which qualifies this re-ceptor as a potential avenue to target macrophages (11).

In this study, we targeted inflammatory macrophages throughCD64, aiming to selectively eliminate the activated inflammatorymacrophages from arthritic joints. Therefore, we constructed ananti-CD64 Ab, chemically linked to the plant toxin Ricin A (RiA).Ricin is a well-defined catalytic inhibitor of protein synthesis at thelevel of the 60S ribosome. The structure of Ricin consists of a verypotent ribosome-inactivating A chain linked by a disulfide bond toa galactose-specific lectin (B chain or binding chain). The A and Bchains were separated, and after purification, the A chain was

*Department of Immunology, Immunotherapy Laboratory, University Medical Cen-ter, Utrecht, The Netherlands; †Department of Rheumatology and Clinical Immunol-ogy, University Medical Center, Utrecht, The Netherlands; ‡Department of Pathologyand Laboratory Medicine, University of Groningen, Groningen, The Netherlands;§Genmab, Utrecht, The Netherlands; ¶Medarex, Annandale, NJ 08801; and �Depart-ment of Pharmaceutical Product Development, Fraunhofer Institut fur Molekular undangewandte Ockologie, Aachen, Germany

Received for publication August 4, 2004. Accepted for publication March 4, 2006.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by the Dutch Technology Foundation.2 Address correspondence and reprint requests to Dr. Anneke J. van Vuuren at thecurrent address: Department of Gastroenterology and Hepatology, Erasmus Univer-sity Medical Center, L 459, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands.E-mail address: a.vanvuuren@erasmusmc.nl3 Abbreviations used in this paper: RA, rheumatoid arthritis; RiA, Ricin A; hCD64,human CD64; RANKL, receptor activator of NF-�B ligand; AA, adjuvant arthritis;ADCC, Ab-dependent cellular cytotoxicity; BsAb, bispecific Ab; MC, mononuclearcell; MFI, mean fluorescence intensity.

The Journal of Immunology

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00

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coupled to CD64 Abs to generate a very specific cell-reactiveconjugate. Recently, we have shown that this CD64-directed im-munotoxin (CD64-RiA) is very efficient in a chronic inflammatoryskin model in human CD64 (hCD64) transgenic mice (12). Alsosuccessful antitumor activity using RiA-conjugated Abs, directedagainst several tumors, have been reported in cancer patients(13, 14).

In the present study, we determined levels of CD64 on activatedmacrophages in both synovial lining as in synovial fluid from RApatients, and examined the in vitro susceptibility of these cells forCD64-RiA-mediated killing.

To test whether killing of CD64 macrophages results in thera-peutic effects in vivo, we established an adjuvant arthritis (AA)model in rats which bears a very close pathological resemblance toRA in patients (15, 16). For this purpose, we generated hCD64transgenic rats. First, we characterize these hCD64 transgenic rats,by determining hCD64 expression, regulation of this hCD64 ex-pression using the cytokines IFN-� and G-CSF, as well as func-tionality of this hCD64 receptor in transgenic rats. After successfulinduction of AA in these hCD64 transgenic rats, we performedstudies in which activated macrophages were eliminated in vivothrough CD64-RiA. Furthermore, front paws of these transgenicrats were extensively analyzed for inflammation, and bone erosionusing macrophage markers ED1, ED2, and receptor activator ofNF-�B ligand (RANKL). RANKL is produced by activated T cells(17), and mediates differentiation and activation of osteoclasts in-volved in bone erosion, a key event in arthritis. In addition,RANKL regulates lymphocyte development, and augments T cell/dendritic cell cooperative interactions (17, 18). We document ef-fective elimination of activated macrophages accompanied by di-minished inflammation and bone erosion at the histological levelresulting in significant inhibition of disease activity. The data pre-sented here indicate the employability of CD64 targeted immuno-toxins for the treatment of RA.

Materials and MethodsImmunotoxins

Immunotoxin CD64-RiA was prepared by Medarex. Humanized CD64mAb (H22) was chemically conjugated to two molecules of low-glycosy-lated RiA using a cleavable cross-linker N-succinimidyl-3-(2-piryldyldi-thio) propionate. All procedures were performed under good laboratorypractice conditions according to the manufacturer’s instructions. Conju-gated CD64-RiA was purified using size exclusion chromatography andpurity was checked on SDS-PAGE (12).

Cell depletion experiments

Mononuclear cells (MC) were isolated from synovial fluid of RA patientsas previously described (19). Viable synovial fluid macrophages (5 � 106

cells/ml) were cultured for 24 h in the absence or presence of CD64-RiA(2 � 10�10 to 2� 10�8 M RiA). The cytotoxic effect was measured bydetermining apoptosis.

Apoptosis

To assess macrophage apoptosis, nuclear DNA fragmentation, which is ahallmark of the apoptosis process, was determined using propidium iodidestaining (20). Because apoptotic MC lost CD14 expression (21), CD68served to identify apoptotic macrophages. Under control conditions nearlyall CD14� cells were CD68�. MC were fixed by adding 500 �l of coldethanol (70%) for at least 2 h at �20°C. After washing with PBS, the MCwere incubated for 20 min at 37°C in buffer containing 50 mM Na2HPO4,2.5 mM citric acid, and 0.1% Triton X-100 to extract intracellular DNA.During this time, macrophages were stained with CD68Fitc (clone EBN11;DAKO). Afterward propidium iodide (10 �g/ml; Sigma-Aldrich) wasadded to stain nuclear DNA, and fluorescence was analyzed by flowcytometry.

Generation CD64 transgenic rats

An 18-kb linear genomic DNA fragment encoding the entire 9.4-kb codingregion of the human Fc�RIA gene as well as its own promoter and regu-latory elements was microinjected into fertilized oocytes of 4-wk-oldWistar rats (Harlan). The same construct was previously used in transgenicmice and extensively studied (11). Injected oocytes were transferred topseudopregnant Wistar foster mothers (22). One transgenic founder wasmated with Wistar rats, and hemizygous transgenic offspring was identifiedby Southern blot analysis and genomic PCR. Offspring was routinelychecked for CD64 expression, for which 25 �l of blood was incubated with10.1Fitc, followed by lysis of erythrocytes, fixation of white blood cells inFACS Lysing Solution (BD Biosciences), and flow cytometry. TransgenicWistar rats were crossed to Lewis (University of Maastricht, Maastricht,The Netherlands) background for induction of AA. The F4 and F5 gener-ations were used in the studies presented in this manuscript. Rats weremaintained at the Central Laboratory Animal Institute (Utrecht University)and all experiments were approved by the Utrecht University animal ethicscommittee.

Antibodies

To detect hCD64 expression, we used FITC-conjugated mAb 10.1 (Sero-tec) or PE-conjugated CD64 mAb 22 (BD Biosciences). To identify spe-cific rat cells ED9Biotin as a monocyte marker, His48 or RP-1Biotin (BDBiosciences) as neutrophil marker, Ox33PE as B cell marker, and Ox19PE

as T cell marker were used. ED2Fitc and ED1Fitc (Serotec) were used tostudy macrophages and monocytes in histochemistry analyses, andRANKL (Santa Cruz Biotechnology) measured activated T cells. Strepta-vidinPE was used to detect biotinylated mAb. In phagocytosis experiments,we used the bispecific Ab (m22 � rabbit anti-Candida albicans IgG) (23),and in Ab-dependent cellular cytotoxicity (ADCC) experiments the bispe-cific Ab MDX-H210 (18). ED9, His48, Ox33, and Ox19 were gifts from E.Dopp (Free University, Amsterdam, The Netherlands).

Cytokine regulation of CD64 in transgenic rats

To investigate regulation of hCD64 expression by cytokines, transgenicrats were injected i.v. with recombinant rat IFN-� (1 � 106 U/rat) (pro-vided by Dr P. van der Meide, U-Cytech, Utrecht, The Netherlands).hCD64 expression was measured 24 h later. Recombinant human pegylatedG-CSF (75 �g/200 �l saline) (provided by Dr. J. Andresen, Amgen, Thou-sand Oaks, CA) was injected i.p. in transgenic rats, and blood collected 3days later. Erythrocytes were lysed from blood samples, and remainingleukocytes were analyzed for hCD64 expression by flow cytometry usingmAb 10.1FITC.

Phagocytosis

C. albicans (American Type Culture Collection (ATCC), 448585) phago-cytosis was performed as previously described (23). C. albicans was cul-tured overnight at 37°C in Sabouraud maltose broth (Difco), then centri-fuged, washed three times with PBS, and counted. C. albicans was FITClabeled at concentration of 0.1 mg/ml FITC (Sigma-Aldrich) in 0.1 Msodium phosphate buffer (pH 9.6) for 30 min at room temperature, washedthree times with PBS, aliquoted, and stored at �20°C until use. For phago-cytosis 1 � 105 peg-G-CSF primed rat neutrophils were incubated with4 � 105 FITC-labeled cells C. albicans in RPMI 1640 medium (InvitrogenLife Technologies) in the absence or presence of bispecific Ab (m22 ��-Can) (10 �g/ml). Yeast binding to neutrophils was quantified by flowcytometry. In addition, phagocytosis was studied in cytospin preparationsby light microscopy.

ADCC assay

The killing capacity of rat neutrophils was investigated in 51Cr-releaseassays (26). 51Cr-labeled SKBR-3 cells (human breast carcinoma, HTB-30;ATCC) (5 � 103 cells/well) were plated in RPMI 1640 medium, peg-G-CSF primed rat neutrophils (4 � 105 cells/well) were then added in thepresence or absence of different concentrations of bispecific Ab (BsAb)MDX-H210 (0.1 �g/ml, 0.4 �g/ml, and 2.0 �g/ml). BsAb MDX-H210recognizes the proto-oncogene product HER-2/neu, and CD64 (24). Afterincubation at 37°C for 4 h, 51Cr release was measured in supernatants.These experiments were performed without serum complement.

Induction and clinical assessment of AA

Experimental arthritis was induced by an intradermal injection of Myco-bacterium tuberculosis (strain H37Ra) (1 mg/100 �l IFA; Difco) at the tailbase. Rats were examined daily for developing clinical signs of arthritis ina blinded fashion. Severity of arthritis was determined by scoring of each

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paw on a scale of 0–4 based on the degree of swelling, erythema, anddeformation of joints (maximum score � 16) (15, 16). When individualrats achieved a total score of 4, they were i.v. injected at days 0, 1, 2, 4, 6,and 8 with CD64-RiA (750 �l, 10�6 M RiA) or with saline (placebotreated). At day 9 after treatment, rats were killed and paws removed forimmunohistochemical analyses.

Immunohistochemistry

From all experimental animals we dissected the front paws, as well astissue samples from spleen, kidney, and liver. Tissues were frozen in liquidnitrogen, and stored at �80°C before use. Sections (6 �m) were cut on afreezing microtome and mounted on coated slides. Nondecalcified frontpaws were cut using a carbide-tipped knife and attached to adhesive plastictape (25). Human synovial tissue samples were preincubated with 10%normal human serum for 20 min before CD64 (10.1Fitc or isotype (IgG1Fitc)staining in PBS containing 1% normal human serum for 45 min. All rattissue samples were preincubated with 10% normal rat serum, before slideswere stained for CD64. Tissue macrophages were stained with ED2Fitc, andmonocytes/macrophages with ED1Fitc. Alkaline phosphatase-conjugatedsheep �-FITC (Boehringer Mannheim) was used for detection as described(12). Activated T cells were stained with RANKL. Goat �-rabbit Biotin,and streptavidin peroxidase were used for detection. Peroxidase activitywas assayed with H2O2 as substrate and diaminobenzidine (Sigma-Aldrich)as chromogen. All slides were counterstained with hematoxylin. Processedtissues were evaluated and scored by two independent observers in ablinded fashion. Amount of staining per evaluated section was expressedfrom no staining (�), through few cells staining (�, refer to see Fig. 3b),to very abundant staining (����, refer to Fig. 3d). An independent,pathologist validated randomly selected evaluated slides.

Statistical analyses

The Student t test was used to compare arthritis scores over time of theCD64-RiA-treated group, and nontreated group. Data were considered sig-nificant at p � 0.05.

ResultsTargeting CD64 expressing macrophages in RA

To investigate whether CD64 expression levels were enhanced inchronic inflammation, we first studied CD64 expression in syno-vial tissues obtained from RA patients by immunohistochemistry.Synovial biopsies of RA patients, obtained from knee joint re-placement surgery, consistently showed abundant staining forCD64 in lining, sublining, perivasculair area, and stroma (Fig. 1, aand b). The staining pattern was congruent to CD68 staining,which is a human macrophage marker (data not shown). In con-trast, control synovial biopsies from individuals, which do not suf-fer from RA, showed occasional CD64 expression on lining cellsonly (data not shown). Macrophages isolated from synovial fluidof RA patients also showed high levels of CD64 expression com-pared with monocytes isolated from peripheral blood by FACSanalyses (21).

Apoptosis is a controlled form of cell death, associated withnormal physiology, as opposed to necrosis, which is associatedwith acute injury to cells. We investigated whether synovial fluidmacrophages, identified by CD68 expression, from RA patientscould be killed by CD64-RiA via apoptosis. Addition of CD64-RiA (2 � 10�8 M RiA) for 24 h to synovial fluid macrophagecultures resulted in strongly increased numbers of apoptoticCD68� cells with reduced DNA content (on average 55.4 �13.5%, p � 0.01, n � 5) (Fig. 1c), indicating apoptosis induction.

To test whether killing of CD64 macrophages would have ther-apeutic effects in vivo, we established an AA model in hCD64transgenic rats.

Characterization of hCD64 transgenic rats

To generate hCD64 transgenic rats, we used a genomic DNA frag-ment encompassing the entire human Fc�RI gene (11). Eight cop-ies of the transgene were incorporated, as determined by quanti-

tative southern analyses (data not shown), in a stable transgenicWistar line.

Peripheral blood samples from nontransgenic littermatesshowed no hCD64 expression (Fig. 2a) in contrast to blood sam-ples from transgenic rats, in which hCD64 expression (mean flu-orescence intensity (MFI) � 370 � 45, n � 12) was determined(Fig. 2b). No hCD64 expression was observed on transgenic Bcells (data not shown), nor on transgenic T cells (Fig. 2c). How-ever, neutrophils (data not shown), and monocytes (Fig. 2d) ex-pressed hCD64.

To investigate regulation of hCD64 expression in transgenicrats, we used the cytokines pegylated G-CSF and IFN-�. Growthfactor G-CSF increased hCD64 expression (MFI � 1350 � 47,n � 6) and in addition, the number of neutrophils (44 vs 8% innon-G-CSF-treated transgenic littermates) (Fig. 2e). IFN-� alsoincreased hCD64 expression levels (MFI � 1049 � 53, n � 4)(Fig. 2f) in blood of transgenic rats, compared with blood samplesof non-IFN-�-treated transgenic littermates (MFI � 370 � 45).

Next, functionality of hCD64 on rat cells in mediating phago-cytosis and ADCC was studied. Hereto, a BsAb (m22 � �Can),recognizing both hCD64 and C. albicans, was used. In the pres-ence of this hCD64-BsAb, 69.9 � 4.9% of transgenic neutrophilsshowed binding to C. albicans, vs 15.3 � 4.7% of nontransgenicneutrophils (n � 6, p � 0.005). Phagocytosis of C. albicans wasdemonstrated by light microscopy in cytospin preparations (Fig. 2,g and h). Furthermore, ADCC experiments were performed inwhich human breast carcinoma cells (SKBR-3 cells) were lysed byG-CSF-primed transgenic rat neutrophils via a BsAb (MDXH210)in 51Cr-release assays (26). Transgenic neutrophils efficientlykilled SKBR-3 cells (73.5 � 5%), in contrast to nontransgenic

FIGURE 1. CD64 expression in human synovial RA tissue. Sectionswere stained for CD64 (10.1Fitc) (a), and isotype control (IgG1Fitc) (b).Data are representative for biopsies of three RA patients. Magnification is�40. Apoptosis due to CD64-RiA treatment of synovial fluid macrophagesisolated from RA patients. These macrophages were cultured in vitro withseveral concentrations of CD64-RiA, using 2 � 10�9 M, and 2 � 10�8 MRiA significant killing was induced compared with macrophages culturesin medium alone (con), n � 5 (c).

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neutrophils (6.4 � 2.6%, n � 3). These data demonstrated expres-sion of hCD64 to be regulated similarly as in man, and confirmedfunctionality of hCD64 in this novel transgenic rat model.

Effect of CD64-RiA on AA

To study the in vivo effect of CD64-RiA, AA was induced inhCD64 transgenic rats by s.c. injection of M. tuberculosis in IFA.AA represents an experimental model that closely resembles ar-thritis pathology in humans (15, 16). Animals were monitoreddaily for development of clinical signs. Each paw was scored forjoint swelling and erythema. In transgenic as well as in nontrans-genic animals, arthritis developed with similar kinetics and to thesame extent, excluding an influence of the hCD64 gene in thetransgenic animals (data not shown). When individual animalsreached a total score of four, they were injected with CD64-RiAi.v., or saline as control (total of six injections). CD64-RiA treat-ment showed significant inhibition of arthritis progression (n � 7),compared with control animals (n � 8), in which arthritis scores

reached maximum levels ( p � 0.005) (Fig. 3a). A total of fourexperiments were performed and showed reproducible data.

In addition to the clinical scoring, extensive immunohistochem-ical analyses of front paws of all experimental animals wereperformed. These analyses confirmed inflammation in all non-treated animals, manifested by severe infiltration with lymphocytesand mononuclear cells, including CD64-expressing monocytes andmacrophages (Table I). Infiltrates were observed both intra- andperiarticularly, reaching out into the upper dermal layers. Thesewere accompanied by cartilage destruction and mild to severe ero-sion of tarsal and metatarsal bones (Fig. 3b). In CD64-RiA-treatedanimals, however, no or only mild inflammation was observed,primarily detectable as small, well delineated periarticular infil-trates, while only very minor cartilage or bone destruction wasobserved (Table I, Fig. 3c). The nontreated animals also showedincreased new bone formation, resulting in deformation of affectedjoints (Fig. 3, a and b).

FIGURE 2. Cell distribution and function of hCD64 in CD64-trans-genic rats. Peripheral blood samples were analyzed with 107�1Fitc for CD64expression. Blood samples of nontransgenic litters did not show any CD64expression (a), compared with samples of CD64-transgenic rats (mean flu-orescence intensity (MFI) was 370 � 45, n � 12) (b). No double stainingfor CD64 (x-axis; Fl-1) and T cells (Ox19Pe on y-axis; Fl-2) was observed(c). However, the same population of cells was positive for CD64 (x-axis;Fl-1) as well as for the monocyte marker ED9Pe (y-axis; Fl-2) (d). G-CSFincreased CD64 expression in transgenic rats, filled curve (MFI: 1350 �47, n � 6) (e). CD64 expression was increased by IFN-� (filled curve)(MFI: 1049 � 53, n � 4), compared with nontreated transgenic rat (opencurve) (f). The function of CD64 in transgenic rats was studied by phago-cytosis of C. albicans particles via BsAb (22 � �Can). Using G-CSFprimed neutrophils of nontransgenic animals, no phagocytosis was found(g) whereas G-CSF-primed neutrophils of transgenic rats showed extensivephagocytosis (n � 4) (h).

FIGURE 3. CD64-RiA treatment inhibits arthritis progression in CD64-transgenic rats. The median arthritis score of one representative experiment offour is shown. Seven transgenic rats were injected with CD64-RiA, and eightcontrols were injected with saline on days 0, 1, 2, 4, 6, and 8 (p � 0.005) (a).Immunohistochemical analyses of front paws (�10 objective). Ratswere killed at day 9 after treatment started, paws were isolated, andsections were stained for CD64. Positive CD64 staining and bone ero-sion observed in a placebo-treated rat (b). Less CD64 staining and nobone erosion was present in CD64-RiA-treated animals (c). CD64-pos-itive cells were present in upper dermis layer of placebo-treated animals(d), but not in CD64-RiA-treated rats (e).

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In synovial lining, ED2 staining, representing tissue macro-phages, showed expression patterns comparable to CD64. In infil-trates, however, far less ED2 expression was observed comparedwith CD64. Staining for ED1, a broader marker than ED2, thatstains both monocytes, macrophages, and dendritic cells (25),showed a staining pattern comparable to CD64 (Table I). Oste-oclasts are involved in bone erosion, therefore we determinedRANKL, which differentiates and activates these osteoclasts.RANKL staining of front paws was decreased in CD64-RiA-treated animals, compared with nontreated animals (Table I). Thiscorresponded with the observed mild bone erosion in CD64-RiA-treated rats compared with nontreated animals.

DiscussionAddition of hCD64-RiA to macrophage cultures isolated from sy-novial fluid of RA patients showed apoptosis of CD68� cells. Wepreviously showed that only IFN-� stimulated monocytic U937cells were effectively killed by CD64-RiA, whereas nonstimulatedU937 cells, which do have baseline CD64 expression, remainedunaffected (12). This indicates that apart from CD64 expression,activation of target cells may be a prerequisite for the cytotoxiceffect of CD64-RiA. Recent work indicates that insufficient apo-ptosis of inflammatory cells in RA joints might contribute topathogenesis (27, 28). Besides selective elimination, a strong in-hibition of proinflammatory cytokines like TNF-� and IL-1� wasobserved (21). Due to the versatile role of macrophages in bothinflammation and bone destruction, we hypothesize that reductionof the number of activated macrophages in RA may be more ben-eficial than blockade of single cytokines like TNF-�, which isfrequently used in the clinic at the moment. In addition, the newstrategy to eliminate activated macrophages only via targetingCD64 may further clarify the role of these macrophages in devel-opment and progression of RA.

Analysis of the hCD64 transgenic rats documented CD64 ex-pression on monocytes and neutrophils, and showed expression tobe increased by IFN-� and G-CSF. Human neutrophils do not con-tinuously express CD64, although they can express CD64 underinflammatory conditions (8). The higher hCD64 expression levelson both monocytes and neutrophils in transgenic rats, comparedwith humans, may be attributable to the number of gene copies intothe genome, which has also been observed in a murine hCD64transgenic line (11). However, phagocytosis and ADCC experi-ments established functionality of hCD64 in transgenic rats. Inconclusion, this hCD64 transgenic rat represents a novel animalmodel, in which CD64 expression, regulation of expression, andreceptor functioning is similar to that in humans.

AA has been demonstrated to be a rat model, that closely re-sembles RA in humans. Therefore, the in vivo effect of CD64-RiAwas studied in these CD64 transgenic rats, after induction of AA.

Injection of CD64-RiA in arthritic animals induced a significantinhibition of disease progression compared with control animals,injected with saline. In all experiments however, a small number ofrats did not respond to CD64-RiA treatment, which might be at-tributable to the mixed genetic Wistar/Lewis background of thetransgenic animals.

Immunohistochemical staining of front paws showed extensivestaining for CD64 and ED1 in inflammatory infiltrates in non-treated animals, while ED2, a tissue macrophage marker, showedfar less staining, suggesting specific elimination of activated mac-rophages in CD64-RiA-treated animals. These data indicate thatincreased CD64 expression is already induced in the very earlystages of differentiation from monocyte to inflammatory macro-phage. RANKL staining was also decreased in CD64-RiA-treatedanimals, and corresponded to an observed mild bone erosion inthese rats. This indicates that elimination of activated macrophagesmay result in decreased numbers of activated T cells, subsequentlyleading to diminished bone erosion and joint damage. This estab-lishes a direct correlation between CD64-RiA treatment, and de-creased macrophages numbers observed in vivo, followed by di-minished inflammation and bone erosion. It can, however, not beexcluded that CD64-expressing neutrophils, which may also beaffected by CD64-RiA treatment, play a role in the pathophysiol-ogy of arthritis. Part of the observed effects in our experimentscould, therefore, be attributed to depletion of neutrophils. Previousin vitro studies using the same CD64-RiA immunotoxin on humansynovial fluid cells (17), however, clearly showed anti-inflammatoryeffects due to elimination of macrophages, rather than neutrophils.

Overall, a direct correlation between microscopic observationsand macroscopic arthritis scores was found in the in vivo experi-ments. Histological examination of liver, spleen, and kidney sec-tions from all animals showed no abnormalities. Adjuvant arthritisrepresents an animal model in which it is notorious difficult tointervene as the inflammatory processes involving cytokine pro-duction, cell recruitment, and activation are already ongoing,thereby closely resembling the situation in RA patients. Despitethis, CD64-RiA treatment was found to significantly inhibit arthri-tis progression in the transgenic animals. In addition, we observedthe presence of activated CD64-positive macrophages, in both sy-novial lining and fluid of RA patients, as well as efficient killing ofthese cells with CD64-RiA. Taken together, these data indicatethat elimination of CD64-positive activated macrophages througha CD64-immunotoxin may provide a novel approach for treatmentof RA.

AcknowledgmentsWe thank Prof. Bijlsma for providing human RA material, Prof. Slootwegfor evaluation of the immunohistochemical analyses, and employees of theCentral Animal Laboratory for excellent animal care.

DisclosuresThe authors have no financial conflict of interest.

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Marker

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IA EA IA EA IA EA

ED1 ���� ���� �� � � �ED2 �� �� � � � �CD64 ���� ���� � � � �RANKL ��� ��� � � NT NT

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