Leukemia Inhibitory Factor Deficiency Modulates the Immune Response and Limits Autoimmune Demyelination: A New Role for Neurotrophic in This information is current as Neuroinflammation of September 26, 2021. Ralf A. Linker, Niels Kruse, Stephanie Israel, Tao Wei, Silvia Seubert, Anja Hombach, Bettina Holtmann, Fred Luhder, Richard M. Ransohoff, Michael Sendtner and Ralf

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Leukemia Inhibitory Factor Deficiency Modulates the Immune Response and Limits Autoimmune Demyelination: A New Role for Neurotrophic Cytokines in Neuroinflammation1

Ralf A. Linker,2*† Niels Kruse,* Stephanie Israel,* Tao Wei,‡ Silvia Seubert,*† Anja Hombach,* Bettina Holtmann,§ Fred Luhder,* Richard M. Ransohoff,‡ Michael Sendtner,§ and Ralf Gold*

The neurotrophic cytokines ciliary neurotrophic factor and leukemia inhibitory factor (LIF) play a key role in neuronal and oligodendrocyte survival and as protective factors in neuroinflammation. To further elucidate the potential of endogenous LIF in modulating neuroinflammation, we studied oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis in LIF knockout mice (LIF؊/؊ mice). In the late phase of active myelin oligodendrocyte glycoprotein- Downloaded from -induced experimental autoimmune encephalomyelitis, LIF؊/؊ mice exhibited a markedly milder disease course. The inflam matory infiltrate in LIF؊/؊ mice was characterized by an increase in neutrophilic granulocytes early and fewer infiltrating associated with less demyelination later in the disease. In good correlation with an effect of endogenous LIF on the immune response, we found an Ag-specific T cell-priming defect with impaired IFN-␥ production in LIF؊/؊ mice. On the molecular level, the altered recruitment of inflammatory cells is associated with distinct patterns of chemokine production /in LIF؊/؊ mice with an increase of CXCL1 early and a decrease of CCL2, CCL3, and CXCL10 later in the disease. These http://www.jimmunol.org data reveal that endogenous LIF is an immunologically active molecule in neuroinflammation. This establishes a link between LIF and the immune system which was not observed in the ciliary neurotrophic factor knockout mouse. The Journal of Immunology, 2008, 180: 2204–2213.

eurotrophic cytokines play a key role in neuronal and cently, the role of CNTF and LIF was investigated in experi- oligodendrocyte survival, among them leukemia in- mental autoimmune encephalomyelitis (EAE), a model disease hibitory factor (LIF)3 and ciliary neurotrophic factor reflecting some of the typical features of the human disease N Ϫ/Ϫ

(CNTF). In vitro and in vivo, these factors support differenti- (7). EAE in CNTF knockout (CNTF ) by guest on September 26, 2021 ation and survival of oligodendrocyte precursor cells (1, 2) and mice takes a more severe course with enhanced oligodendrocyte prevent oligodendrocyte in response to serum with- apoptosis and axonal damage (8). On the other side, treatment drawal or challenge (3, 4). LIF and CNTF also support with LIF ameliorates EAE by preventing oligodendrocyte cell motoneuron survival in vitro and in vivo under different exper- death (9), whereas administration of CNTF can also interfere imental conditions (5). Thus, neurotrophic cytokines may be of with the immune system (10). great interest as protective factors not only for neurodegenera- Besides its effects on neuronal and glial cells, LIF possesses tive diseases, but also in autoimmune demyelination (6). Re- pleiotropic functions in many cell types and organs (see Ref. 11 for review) including the inhibition of embryonic stem cell differen- tiation, promotion of survival of hemopoietic precursor cells or * Institute for Multiple Sclerosis Research, University of Goettingen and Gemeinnu- † support of blastocyst implantation; the latter resulting in infertility etzige Hertie-Stiftung, Goettingen, Germany; Department of Neurology at St. Josef- Ϫ/Ϫ Hospital Ruhr-University Bochum, Bochum, Germany; ‡Neuroinflammation Re- in LIF-deficient (LIF ) mice (12). Furthermore, LIF seems to search Center, Department of Neurosciences, Lerner Research Institute, Cleveland interact with the immune system. LIFϪ/Ϫ mice display decreased Clinic Foundation, Cleveland, OH 44195; and §Institute for Clinical Neurobiology, Julius-Maximilians-Universita¨t, Wuerzburg, Germany numbers of hemopoietic stem cells in spleen and bone marrow Received for publication November 2, 2006. Accepted for publication December and an impaired Con A-mediated thymocyte stimulation (12). 1, 2007. Overexpression of LIF in T cells leads to altered immune organ The costs of publication of this article were defrayed in part by the payment of page morphology (13). Analyzing the immune response to peripheral charges. This article must therefore be hereby marked advertisement in accordance nerve injury in LIFϪ/Ϫ mice reveals a role for LIF in macro- with 18 U.S.C. Section 1734 solely to indicate this fact. phage recruitment (14, 15). Likewise, LIF deficiency modulates 1 This work was supported by the Deutsche Forschungsgemeinschaft, SFB 581, TPA1, the Institute for Multiple Sclerosis Research, University of Goettingen, the / response in a model of Bereich Humanmedizin and Gemeinnuetzige Hertie-Stiftung, and the U.S. Na- injury (16). In summary, these data suggest a proinflammatory tional Institutes of Health (RO1 NS 32151 to R.M.R.). function of LIF. Yet, other studies focusing on the immune 2 Address correspondence and reprint requests to Dr. Ralf A. Linker, Department reaction in LIFϪ/Ϫ mice after injection of CFA even point at a of Neurology, St. Josef-Hospital/Ruhr-University Bochum, Gudrunstrasse 56, D-44791 Bochum, Germany. E-mail-address: [email protected] prominent anti-inflammatory role for this cytokine (17, 18). So 3 Abbreviations used in this paper: LIF, leukemia inhibitory factor; CNTF, ciliary far, little is known on the interaction of LIF with the immune neurotrophic factor; EAE, experimental autoimmune encephalomyelitis; MOG, my- system during autoimmune inflammation of the CNS. Previous elin oligodendrocyte glycoprotein; WT, wild type; bmDC, bone marrow dendritic studies in EAE models mainly focused on the beneficial impact cell; IP10, IFN-␥-inducible protein 10. of exogenous or endogenous LIF on glial cells (9, 19). How- Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 ever, the consequences of LIF deficiency on initiation and www.jimmunol.org The Journal of Immunology 2205 course of neuroinflammatory diseases have not been investi- berglass filter paper with a 96-well harvester (Pharmacia), and radio- gated so far. activity was measured with a 96-well Betaplate liquid scintillation In this study, we have induced the model disease EAE with counter (Pharmacia). For proliferation assay of propagated MOG-specific T cells in cul- myelin oligodendrocyte glycoprotein peptide 35–55 (MOG 35–55) ture, 20,000 or 40,000 T cells were cultured with 125,000 or 250,000 Ϫ/Ϫ in LIF mice. We show that LIF deficiency results in attenuation spleen cells, respectively; otherwise, the protocol remained unchanged. of disease in the late phase of MOG-EAE with an altered compo- In some of these experiments, Ly6G-positive cells were isolated from sition and altered maintenance of the inflammatory infiltrate. These spinal cord and spleen of EAE diseased WT mice at the first maximum of disease and then titrated into the assay. All experiments were at least data suggest that endogenous LIF is an important regulator that repeated once. orchestrates T cell and macrophage responses in EAE. ELISA Materials and Methods Lymph node cells were prepared and cultured as above with medium alone Mice or in the presence of 10 ␮g/ml MOG 35–55 peptide. Supernatants were ␥ LIFϪ/Ϫ mice were backcrossed on a C57BL/6 background for Ͼ10 gen- harvested after 3 days of culture. Cytokines or chemokine (IL-2, IFN- , erations and bred at the in-house animal care facilities of the Institute of IL-6, IL-12 p35/p70, IL-5, and CCL2 (MCP-1)) were determined by sand- Ϫ/Ϫ wich ELISA, as described (21). mAb pairs and recombinant cytokine stan- Clinical Neurobiology (Wu¨rzburg, Germany). Because female LIF ␥ mice are infertile, they were maintained on a heterozygous background. dards were purchased from R&D Systems for IFN- or BD Pharmingen for Previous studies suggest a gene-dose effect in LIFϩ/Ϫ littermates (12). all other cytokines and CCL2 (MCP-1). All experiments were at least Therefore, age- and gender-matched control C57BL/6 animals were repeated once. purchased from Harlan Laboratories (Harlan Winkelmann) for all ex- periments. Mice were 8–12 wk old and body weight was in a range of Histology Downloaded from 20–25 g. Animals were housed in a room with controlled light cycle Different time points were chosen for histologic analysis (day 13 postin- and were given commercial food pellets and water ad libitum. All fection (p.i.) for the early phase of MOG-EAE, day 27 p.i. for the inter- experiments were approved by the Bavarian and Lower Saxony state mediate phase, and day 60 p.i. for the late phase of MOG-EAE). Animals authorities for animal experimentation. were deeply anesthetized with pentobarbital or ketamine and transcardially Induction and clinical evaluation of active MOG-EAE perfused with saline followed by 4% of paraformaldehyde. The complete spinal cord—and, in some mice, also spleen, thymus, and lymph nodes— For active induction of EAE, mice received a s.c. injection at flanks and were carefully removed. Thymus, lymph nodes, spleen, and six to eight http://www.jimmunol.org/ tail base of 200 ␮g of MOG 35–55 peptide (Bio-Rad and Charite) in axial spinal cord cross-sections per animal were further processed for rou- PBS emulsified in an equal volume of CFA containing Mycobacterium tine paraffin embedding. Histologic evaluation was done from at least two tuberculosis H37RA (Difco) at a final concentration of 1 mg/ml. Two independent experiments per time point. Paraffin sections were subjected to injections of pertussis toxin (400 ng/mouse i.p.; Sigma-Aldrich or List H&E staining to assess the structure of immune organs or parameters of Biochemicals) were given 24 and 72 h later. Animals were weighed and inflammation. Spinal cords were also stained with Luxol Fast Blue for scored for clinical signs of disease on a daily basis. Disease severity demyelination. was assessed using a scale ranging from 0 to 10; scores were as follows (8): 0, normal; 1, reduced tone of tail; 2, limp tail, impaired righting; 3, Immunohistochemistry absent righting; 4, gait ataxia; 5, mild paraparesis of hind limbs; 6, Immunohistochemistry was performed with 5-␮m paraffin sections as

moderate paraparesis; 7, severe paraparesis or paraplegia; 8, tetrapare- by guest on September 26, 2021 described (8). If necessary, Ag unmasking was achieved by heat pre- sis; 9, moribund; 10, death. treatment of sections for 30 min in 10 mM citric acid buffer (Mac-3, Generation of a MOG 35–55-specific T cell line and induction CD3, neutrophilic granulocytes) in a microwave oven (850 W). After of adoptive transfer MOG-EAE inhibition of unspecific binding with 10% BSA, sections were incubated overnight at 4°C with the appropriate primary Ab in 1% BSA. Second- MOG 35–55-specific T cells were generated as described earlier (20). ary Abs were used as indicated below. After blocking of endogenous Briefly, wild-type (WT) C57BL/6 mice were immunized with 200 ␮gof peroxidase with H2O2, the peroxidase-based ABC detection system MOG 35–55 in CFA. Nine to 12 days later, draining lymph nodes and (DakoCytomation) was used with diaminobenzidine as the chromogenic spleen were harvested and single-cell suspensions were prepared. substrate. Specificity of staining was confirmed by omitting the primary Lymph node cells were then cultured at a density of 3–6 ϫ 106 cells/ml Ab as a negative control. T cells were labeled by rat anti-CD3 (1: 300; in 35-mm plastic dishes (Nunc) in the presence of 20 ␮g/ml MOG Serotec) and macrophages by rat anti-mouse Mac-3 (1:200; BD Pharm- 35–55 in RPMI 1640 supplemented with 100 U/ml penicillin, 100 ingen), each with a rabbit anti-rat secondary Ab (1:100; Vector via ␮g/ml streptomycin (Biochrom), 1% L-glutamine, 1% sodium pyruvate, Linaris). Staining for neutrophilic granulocytes was done by immuno- and 1% nonessential amino acids (Invitrogen Life Technologies) and histochemistry for the 7/4 Ag (1:300; Serotec MCA 771GA (22)) with 10% FCS (heat-inactivated FCS; PAA Laboratories). Ag-specific T a rabbit anti-rat secondary Ab or by Naphtol AS-D chloroacetate reac- cells were selected by repeated propagation cycles in medium with tion (kit no. 91C; Sigma-Aldrich). 6–10% supernatant from Con A-treated Lewis rat spleen cells and 10% FCS followed by Ag-specific restimulation using irradiated (30 Gy) FACS analysis syngeneic spleen cells at a 6:1 ratio after 7–12 days of primary culture. Ϫ Ϫ Thymocyte and lymph node single-cell suspensions were stained in For induction of adoptive transfer-EAE, WT, or LIF / recipients re- PBS/1% BSA/0.03% NaN and analyzed by triple-color flow cytometry on ceived 4–6 ϫ 106 freshly activated MOG-specific T cell blasts from a 3 a FACSCalibur (BD Biosciences). Data were analyzed using FACScan stable T cell line (MOG.10) i.v. A total of 400 ng of pertussis toxin was software (BD Biosciences). The following Abs were used for analysis: administered i.p. immediately after cell transfer and 2 days later. Dis- FITC-labeled anti-CD8a (clone 53-6.7); PE-labeled anti-CD4 (clone GK ease severity was assessed as above. 1.5), FITC-labeled anti-CD25 (clone 7D4), and FITC-labeled anti-CD69 Proliferation assay (clone H1.2F3, all obtained from BD Biosciences). For lymph node and spleen cell proliferation assays, single-cell suspen- Preparation of T cells, neutrophilic granulocytes, macrophages, sions of spleen and inguinal lymph nodes from MOG 35–55-immunized and bone marrow dendritic cells (bmDC) LIFϪ/Ϫ and WT mice were prepared 12 days after immunization of mice with 200 ␮g of MOG 35–55 (8). A total of 2 ϫ 105 cells were seeded in T cells were isolated from mouse spleens using a MACS pan-T cell iso- 96-well microtiter plates (Nunc) in 100 ␮l of medium with addition of Ag. lation kit by negative selection (Miltenyi Biotec). Neutrophilic granulo- In some assays, T cells were isolated by MACS (see below) and cocultured cytes were isolated from mouse spinal cord and spleen by MACS using with freshly prepared APC from spleen of different donors. Ag concentra- Ly6G beads (Miltenyi Biotec). Resident peritoneal macrophages were ob- tions were 10–20 ␮g/ml, except for Con A (1.25–2.5 ␮g/ml). Triplicate tained by peritoneal lavage. Murine bmDC were prepared in adaptation of cultures were maintained at 37°C in a humidified atmosphere with 5% CO2 a protocol by Grauer et al. (23). The generation of mature bmDC was for 56 h and harvested following a 16-h pulse with 0.2 ␮Ci/well [3H]dT proven by FACS staining for MHC class II, B7-1, B7-2 CD40, and CD11c (tritiated thymidine; Amersham-Buchler). Cells were collected on fi- expression (all Abs via BD Biosciences). 2206 EAE IN LIF KNOCKOUT MICE

Table I. Clinical characteristics of MOG 35–55 EAE in LIFϪ/Ϫ micea

WT LIFϪ/Ϫ

Body weight before immunization (g) 22.7 Ϯ 3.6 20.5 Ϯ 3.6*** Incidence of MOG 35–55 EAE (%) 100% 96.9% Mortality (%) 7.1% 0% Onset of disease (days p.i.) 14.0 Ϯ 3.9 12.8 Ϯ 1.7 Disease severity day 60 p.i. 4.4 Ϯ 0.9 1.8 Ϯ 0.6*

a LIFϪ/Ϫ mice display reduced body weights and a reduced disease severity on day 60 p.i. of MOG-EAE. There was no difference in incidence, mortality, or onset .p Ͻ 0.001 ,ءءء ;p Ͻ 0.05 ,ء .of disease. Data are given as mean Ϯ SD

In vitro migration of murine peritoneal macrophages and bmDC Recombinant murine LIF was a gift from H. Butzkueven (University of Melbourne, Melbourne, Australia). Murine peritoneal macrophages and bmDC were prepared as described above. Murine bmDC were used on days 10–12 after preparation and maturation for 3 days in the presence of 500 U/ml TNF-␣. Chemotactic activity was assayed in multiwell micro- Downloaded from chambers (Costar/Corning via Omni Life Science) using a modified pro- tocol according to Ref. 24, with a polyvinylpyrrolidone-free polycarbonate filter, pore size 5 ␮m. After 100- to 180-min incubation at 37°C and 5%

CO2 in a humidified atmosphere, cells that had migrated through the filter into the lower chamber were counted by FACS. Measurements were per- formed in triplicates, outliers exceeding or dropping below 40% of the respective mean values were not considered for further analysis. Data are http://www.jimmunol.org/ pooled from two independent experiments and presented as chemotactic index which is the quotient of cells migrating in the presence of LIF and FIGURE 1. Clinical course of active and passive MOG-EAE in LIFϪ/Ϫ cells migrating in the presence of medium alone (14). Ϫ Ϫ mice. A, Clinical course of active MOG-EAE in LIF / (n ϭ 32, gray line) vs In vivo migration of murine bmDC WT control mice (n ϭ 28, black line). Data are summarized from a total of six experiments. Ten LIFϪ/Ϫ and 9 WT mice from a total of three experiments In vivo migration of murine bmDC was investigated following a protocol were followed until the late phase of the disease (day 60 p.i.). At that time by Del Prete et al. (25). Briefly, WT mature bmDC after 9 days of culture point, LIFϪ/Ϫ mice exhibited a significantly milder disease course (p ϭ 0.02). were labeled in vitro with (CFSE. A total of 2 ϫ 106 cells were injected s.c. Ϫ Ϫ B, Clinical course after adoptive transfer of 4–6 million MOG 35–55-specific in each hind footpad of a total of four WT or LIF / mice. In parallel, mice WT T cell blasts (MOG.10 T cell line) into LIFϪ/Ϫ mice (n ϭ 8, gray line) and were immunized s.c. with 25 ␮g of MOG 35–55 in CFA. Three days later, by guest on September 26, 2021 ϭ popliteal lymph nodes were recovered and disaggregated. The cell suspen- WT (n 7, black line) recipients. Both groups displayed a similar disease sion was evaluated separately for each leg by FACS. Inguinal lymph nodes course early and late during MOG-EAE with gait impairment. Data are pooled served as internal negative control. from a total of two experiments; error bars represent SEM. RT-PCR SPSS). Data are given as mean values Ϯ SEM or mean values Ϯ SD as p Ͻ 0.05 and highly ,ء indicated. Values of p were considered significant at .p Ͻ 0.001 ,ءءء p Ͻ 0.01 or ,ءء Total RNA from spinal cord, spleen, or freshly prepared T cells, macro- significant at phages, and DCs was purified over RNeasy columns (Qiagen). Reverse transcription was performed with 12 ␮l of purified RNA with 200 U of Superscript II reverse transcriptase. Quantification of ␤-actin was achieved with primers ␤-actin S2 (5Ј-ATTGCCGACAGGATGCAGAA-3Ј), ␤-actin AS2 (5Ј-GCTGATCCACATCTGCTGGAA-3Ј), and ␤-actin Son2 (5Ј- FAM-CAAGATCATTGCTCCTCCTGAGCGCA-TAMRA-3Ј) (26). For quantification of murine CCL2 (MCP-1), CXCL1 (KC), CXCL10 (IFN-␥- inducible protein 10 (IP10)), CCL3 (MIP-1␣), CCL5 (RANTES), GM- CSF, IFN-␥, and IL-17, we used predeveloped assays from Applied Bio- systems. Murine (m) LIFR␤ mRNA expression was measured with mLIFR S(5ЈGGATACCAACTGTTACGTTCCATAATT-3Ј), mLIFR AS (5Ј- TATCGAGTCTGCCGACGTATCTT-3Ј), and mLIFR Son (5Ј-FAM- AGAACTGGCTCCCATTGTTGCGCT-TAMRA-3Ј) as primers. All PCR were performed on a 7500 Real-Time PCR System (Applied Biosystems) in quadruplicate; relative quantification was performed according to Livak and Schmittgen (27). Statistical analysis Quantitative evaluation of histopathological changes was essentially per- formed as described (28). Coded sections were counted by blinded observ- ers by means of overlaying a stereological grid onto the sections and count- ing inflammatory infiltrates per mm2 white matter (29). The extent of demyelination was assessed according to Storch et al. (7). CD3, Mac-3- positive cells, and neutrophilic granulocytes were quantified on three rep- resentative sections, each one from cervical, thoracic, and lumbar spinal FIGURE 2. RT-PCR analysis for LIFR␤ expression in different resting cord by counting two defined areas with the most intense under ␤ a 400-fold magnification. For statistical evaluation of the clinical course, and activated immune cell types. LIFR mRNA was present in macrophages data were pooled from different experiments. Analysis was performed us- (Ⅺ) and bmDC (f) at baseline and after stimulation with 100 ng/ml LPS or ing the Mann-Whitney U test or for histology and clinical course and t test 500 U/ml TNF-␣ (TNF), respectively. Naive T cells did not display LIFR␤ for ELISA, RT-PCR, proliferation, and migration data (SPSS program; mRNA while it was clearly present in MOG-specific T cell blasts (u). The Journal of Immunology 2207

(Table I), but otherwise appeared grossly normal. MOG 35–55 EAE was induced in 32 LIFϪ/Ϫ and 28 WT mice in a total of six independent experiments. Disease incidence and mortality did not differ between both groups (Table I). Moreover, there was no dif- ference in onset of disease between LIFϪ/Ϫ mice and WT control mice. In the early phase of disease (day 17–20 p.i.), WT and LIFϪ/Ϫ mice suffered from mild paraparesis. Yet, in the late phase of MOG-EAE, LIFϪ/Ϫ mice displayed a significantly milder dis- ease course with only tail weakness while disability in the WT mice remained unchanged (Fig. 1A, p Ͻ 0.05).

LIF deficiency does not influence the disease course of adoptive transfer EAE To shed further light on the role of endogenous LIF in the initiation phase compared with the effector phase of MOG-EAE, we used a newly generated encephalitogenic MOG 35–55-specific T cell line from WT C57BL/6 mice in adoptive transfer experiments. MOG specificity of cultured blasts was shown in vitro by Ag-specific proliferation (data not shown). After adoptive transfer, MOG 35– Downloaded from 55-specific T cells led to a chronic course of disease with promi- nent gait ataxia as well as tail tremor and—in severe cases—also spasticity, but no tail weakness. The clinical course of adoptive transfer EAE in LIFϪ/Ϫ vs WT mice in a total of two experiments (n ϭ 8 vs 7; Fig. 1B) was without significant difference. Histologic

FIGURE 3. Composition of the inflammatory infiltrate in the early and analysis revealed predominantly meningeal and, in severe cases, http://www.jimmunol.org/ late phase of active MOG-EAE. A and B, Anti-neutrophil staining for 7/4 also parenchymal infiltrates (data not shown). These data speak for Ag 13 days after active immunization with MOG 35–55. Representative a role of endogenous LIF already in the initiation phase of the spinal cord cross-sections from the anterior columns of a WT control disease impacting on the further course of active MOG-EAE. mouse (A) and a LIFϪ/Ϫ mouse (B) are shown. Although WT mice dis- played some granulocytic infiltration, numbers of neutrophilic granulo- Ϫ Ϫ LIFR␤ expression on resting and activated immune cells cytes were clearly increased in the spinal cord of LIF / mice (infiltrate extension is marked by arrows; bar, 100 ␮m). 7/4-positive cells were also We next wanted to dissect whether endogenous LIF may be able to labeled by histochemistry for chloroacetate-esterase (see inset in B). C and directly act on T cells or APC. To that end, we investigated LIFR␤ D, Anti-Mac-3 staining for macrophage infiltration in LIFϪ/Ϫ mice in the

expression in a RT-PCR analysis of T cells, macrophages, and by guest on September 26, 2021 late phase of active MOG-EAE (day 60 p.i.). Representative anterior col- DCs (Fig. 2). LIFR␤ mRNA was easily detected in naive DCs as Ϫ/Ϫ umns from spinal cord cross-sections are shown. LIF mice (D) exhib- well as peritoneal macrophages without further up- or down-reg- ited a clearly reduced infiltration of Mac-3-positive cells in comparison to ␣ ␮ ulation after adherence or stimulation with TNF- or LPS. In con- WT control mice (C, infiltrate marked by arrows; bar, 100 m). E and F, ␤ Luxol Fast Blue staining of spinal cord cross-sections from WT (E) and trast, LIFR mRNA was neither found in naive nor mitogen-stim- LIFϪ/Ϫ mice (F) revealed a clearly reduced demyelination (marked by ulated T cell cultures. Yet, MOG-specific T cell blasts displayed a arrows) in LIFϪ/Ϫ mice on day 60 p.i. Representative sections are shown; clear LIFR␤ message. Thus, LIFR␤ is present on several relevant bar, 200 ␮m. immune cell subsets. In particular, Ag-specific T cell activation leads to LIFR␤ up-regulation thus rendering these cells responsive to LIF. Results Milder disease course of active MOG 35–55 EAE in Altered composition of the inflammatory infiltrate in LIFϪ/Ϫ LIFϪ/Ϫ mice mice early and late during active MOG-EAE In a first set of experiments, LIFϪ/Ϫ mice on a C57BL/6 back- The results from active in comparison to passive MOG-EAE in ground were compared with age- and gender-matched WT control LIFϪ/Ϫ mice speak for a role of endogenous LIF in the initiation mice for their susceptibility to EAE induction. As described earlier phase of the disease. We wondered why LIFϪ/Ϫ mice nevertheless (12), LIFϪ/Ϫ mice displayed an ϳ10% reduction in body weight develop a clinical disability similar to WT controls in the early

Table II. Blinded histological analysis of spinal cord cross-sections at different time points of MOG 35–55 EAEa

Day 13/14 p.i. Day 60 p.i.

WT LIFϪ/Ϫ WT LIFϪ/Ϫ

Mac-3-positive cells 881 Ϯ 92 813 Ϯ 171 46 Ϯ 22.5 25 Ϯ 22* CD-3-positive cells 592 Ϯ 247 644 Ϯ 343 234 Ϯ 171 144 Ϯ 121 7/4 Ag-positive cells 228 Ϯ 49 590 Ϯ 23* None None Demyelination score (LFB)b 0.8 Ϯ 0.3 0.9 Ϯ 0.4 2.8 Ϯ 0.7 1.6 Ϯ 0.6**

a LIFϪ/Ϫ mice display increased numbers of 7/4 Ag-positive cells on day 13 p.i. and less Mac-3-positive cells and demy- elination on day 60 p.i. p Ͻ 0.01. Data ,ءء ;p Ͻ 0.05 ,ء .b Demyelination was assessed according to Storch et al. (7); cell numbers are given per mm2 are pooled from at least two independent experiments per time point and displayed as mean Ϯ SD. 2208 EAE IN LIF KNOCKOUT MICE phase of MOG-EAE. Therefore, we performed immunohistochem- istry to investigate the composition of the inflammatory infiltrate in situ. Early in the course of disease (day 13 p.i.), 7/4 Ag-positive cells were abundant in the inflammatory infiltrate in LIFϪ/Ϫ mice (Fig. 3, A and B, Table II). The identity of 7/4-positive cells as neutrophilic granulocytes was confirmed by positive histochemis- try for chloroacetate-esterase (see inset Fig. 3B) as well as a poly- morphonuclear appearance in an H&E staining in situ and ex vivo after MACS isolation from spinal cord (data not shown). On con- secutive sections, 7/4 Ag-positive cells were not positive for Mac-3. On day 13 p.i., numbers of Mac-3-positive macrophages/ microglia and T cells were not different in comparison to WT controls. In the intermediate as well as late phase of MOG-EAE (days 27 and 60 p.i., respectively), 7/4-positive cells could not be found in the lesions in LIFϪ/Ϫ or WT mice. We also investigated the infiltrate composition in the later stages of the disease (intermediate phase, day 27 p.i. and late phase, day 60 p.i., respectively). Immunohistochemical analysis of spinal cord cross-sections revealed a significant reduction of Mac-3-positive Downloaded from macrophages and microglia in LIFϪ/Ϫ mice in the intermediate as well as late phase of MOG-EAE (Fig. 3, C and D; Table II) as well as a reduction in T cell infiltration. To investigate whether fewer macrophages in the lesions of LIFϪ/Ϫ mice also have a functional impact on the target tissue, Luxol Fast Blue staining was per-

formed to analyze a parameter of tissue destruction. The extent of http://www.jimmunol.org/ demyelination was not different in the early phase of disease on day 13 p.i., but clearly reduced in LIFϪ/Ϫ mice on day 27 as well as day 60 p.i. (Fig. 3, E and F, Table II). Ag-specific T cell proliferation is impaired after immunization of LIFϪ/Ϫ mice To gain more insight into the mechanisms governing T cell func- tion in LIFϪ/Ϫ mice, we investigated the proliferative capacity of by guest on September 26, 2021 LIF-deficient T lymphocytes in primary culture of lymph node FIGURE 4. Impaired T cell priming and T cell activation in LIFϪ/Ϫ Ϫ Ϫ cells. After immunizing LIF / and WT control mice with MOG mice. A, Investigation of T cell priming in LIFϪ/Ϫ mice after immunization 35–55 and CFA, draining lymph nodes were prepared 10 days later with MOG 35–55. T cell proliferation in lymph node primary cell culture and proliferation was assessed by [3H]thymidine incorporation. was assessed ex vivo by [3H]thymidine incorporation 72 h after re- Unspecific polyclonal activation with the mitogens phytohemag- stimulation. Representative data from a total of two experiments are Ϫ/Ϫ glutinin (data not shown) or Con A) revealed a small, but signif- shown; each marker represents a single animal. LIF mice displayed icant, increase in T cell proliferation in LIFϪ/Ϫ mice as compared a small increase to unspecific polyclonal activation with Con A, yet showed a significantly impaired proliferation in comparison to WT mice with WT controls (Fig. 4A, p Ͻ 0.05). Yet, in response to purified after recall with MOG 35–55 (p ϭ 0.002). B, Investigation of T cell protein derivative (a component of CFA), there was only few and proliferation in a MOG recall assay with coculture of WT T cells with 3 in response to MOG there was hardly any [ H]thymidine incorpo- LIF-deficient APC and vice versa. Although LIF deficiency of only T Ϫ/Ϫ ration detectable in LIF mice 72 h after restimulation while cells or APC did not alter T cell proliferation, deficiency of both T cells WT mice displayed a clear Ag-specific response (Fig. 4A, p Ͻ and APC together resulted in a significant impairment of T cell prolif- 0.01). Similar results were seen at an earlier time point, 24 h after eration (p Ͻ 0.05). C, Titration of WT neutrophilic granulocytes in recall with MOG 35–55 and a similar trend was observed after relation to several long-term MOG-specific T cell lines (MOG.6, stimulation with OVA as another protein Ag (data not shown). MOG.10, MOG.33). Representative data with neutrophilic granulocytes Yet, addition of exogenous LIF to MOG recall assays or MOG- from spleen are shown. Upon induction of Ag (MOG) specific and specific T cell lines did not influence WT T cell responses in vitro lectin-induced (Con A) unspecific T cell proliferation, the proliferative capacity (assessed by [3H]thymidine incorporation) increased with the (data not shown). number of added neutrophils. Data are pooled from three different ex- To investigate the cellular target for LIF, we next measured T periments with different cell lines at various restimulation cycles (R2, cell proliferation in MOG recall assays where WT T cells were R8, and R9). Error bars represent SDs; all experiments were performed cocultured with LIF-deficient APC and vice versa. Although LIF in triplicates; p Ͻ 0.05 in comparison to MOG or Con A stimulation of deficiency of only T cells or APC did not significantly alter T cell the respective line alone. D, IFN-␥ expression in lymph node primary proliferation, LIF deficiency of both T cells and APC resulted in a cultures after immunization with MOG 35–55 and recall with MOG significant impairment of T cell proliferation (Fig. 4B, p Ͻ 0.05). 35–55 ex vivo. Supernatants were analyzed by ELISA. Data are pooled In a next step, we investigated the role of neutrophilic granulo- from two independent experiments; each marker represents a single Ϫ/Ϫ ␥ cytes in the regulation of T cell responses. To that end, Ly6G- animal. In LIF mice, protein levels of IFN- were significantly re- positive cells were isolated from spleen and spinal cord by MACS. duced after recall with MOG 35–55 in comparison to WT control mice. E, RT-PCR analysis for mRNA expression of IFN-␥ and IL-17 in the In the immunocytological analysis, these cells were to 93% 7/4 Ag Ϫ Ϫ and spinal cord (SC) of LIF / and WT control mice on days 13 positive and in the morphological analysis 96% had a polymor- and 27 p.i. Levels of IFN-␥ are reduced in the spinal cord of LIFϪ/Ϫ phonuclear appearance, thus proving that the majority were neu- mice on day 27 p.i. (p Ͻ 0.05). trophilic granulocytes. When they were added to several stable, The Journal of Immunology 2209 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 4. (continued) long-term MOG-specific T cell lines in the presence of MOG Ag stimulation (Fig. 4C). Addition of neutrophils in MOG recall assay or Con A, neutrophils significantly increased T cell proliferation in cultures yielded similar results, while neutrophilic granulocytes a dose-dependent manner, both after Ag-specific and unspecific alone did not proliferate (data not shown). 2210 EAE IN LIF KNOCKOUT MICE

The proliferation defect in LIFϪ/Ϫ mice might be paralleled by a lack of cytokines which stimulate T cell proliferation or exert T cell effector functions Therefore, supernatants from primary lymph node tissue culture were assessed for IL-2 and IL-6, two cytokines implicated in T cell proliferation. Production of IL-2, and IL-6 was not different between LIFϪ/Ϫ mice and WT controls (data not shown). In a next step, we tried to restore the proliferative capacity in LIF-deficient primary lymph node cell culture by addition of cytokines in vitro. Addition of exogenous IL-2 at different con- centrations (0.1–10 ng/ml) did not lead to an increase in thymidine incorporation neither in LIFϪ/Ϫ nor in WT cultures. Moreover, neither the addition of LIF itself nor addition of the related cyto- kine IL-6 (0.1–10 ng/ml) was able to reconstitute proliferation in LIFϪ/Ϫ cultures (data not shown). Next, we investigated the ex- pression of IFN-␥, a critical T cell effector cytokine, in superna- tants from primary lymph node tissue culture by ELISA. Three days after recall with MOG 35–55, levels of IFN-␥ were signifi- cantly reduced in LIFϪ/Ϫ cultures after addition of MOG 35–55

(Fig. 4D). In a RT-PCR analysis, levels of IFN-␥ mRNA were Downloaded from reduced in the spinal cord of LIFϪ/Ϫ mice on day 27 p.i, but not on day 13 p.i. IFN-␥ expression in the brain as well as IL-17 expression in the CNS was not altered in comparison to WT con- trol mice (Fig. 4E). The production of the “Th2” cytokine IL-5 and production of IL-12p70 and IL-12/IL-23p40, the latter implicated

in IFN-␥ production, were not different in supernatants from pri- http://www.jimmunol.org/ mary lymph node tissue culture from LIFϪ/Ϫ mice and WT con- trols (data not shown). LIF deficiency does not alter DC migration or APC function FIGURE 5. Intact APC function in LIFϪ/Ϫ mice. A, Investigating the To analyze the impact of endogenous LIF on APC, the ability of impact of LIF-deficient APC for Ag presentation in vitro. MOG-specific LIF-deficient APC for Ag presentation was assessed in vitro. To WT T cells were restimulated in vitro with MOG 35–55 peptide or rMOG this end, newly generated MOG-specific T cell lines were restim- using irradiated syngeneic spleen cells from WT or LIFϪ/Ϫ donors. Each Ϫ/Ϫ ulated in culture using irradiated spleen cells from LIF or syn- dot represents a single animal. There was no difference in T cell prolifer- geneic WT mice. There was no difference in the amount of ation after restimulation with WT or LIFϪ/Ϫ APC and addition of MOG by guest on September 26, 2021 [3H]thymidine incorporation after Ag-specific stimulation with 35–55 or whole MOG protein (rec MOG). Error bars represent SDs. B,In Ϫ Ϫ MOG peptide 35–55 or whole MOG protein in the presence of vivo DC migration in LIF / and WT mice. bmDC were prepared from LIF-deficient or WT APC (Fig. 5A, data are shown for a repre- WT donors. Nine days after culture, immature bmDC were labeled with sentative MOG-specific T cell line, MOG.6). Experiments with CFSE and injected in each hindpad of a total of four mice. After 72 h, two further MOG-specific T cell lines (MOG.2 and MOG.10) popliteal lymph nodes and inguinal lymph nodes were harvested and in- vestigated by FACS. After gating for APC, there was a clear increase in yielded similar results. Therefore, LIF deficiency does not influ- CFSE-positive cells in popliteal lymph nodes from WT control mice (WT, ence Ag-processing and presentation and costimulatory function f) and LIFϪ/Ϫ mice (u) in comparison to inguinal lymph nodes (Ⅺ, p ϭ of APC. 0.021), but without any difference between both genotypes. Error bars rep- In a next step, we evaluated migration of DCs to exclude im- resent SDs. paired migration of APC causing insufficient T cell priming in LIFϪ/Ϫ mice. Mature bmDC displayed only a mild migratory re- Ϫ Ϫ sponse toward LIF which was neither dose dependent nor statis- LIF / mice for pooled mesenteric, inguinal, axillary, and cervical tically significant in vitro (data not shown). Therefore, we inves- lymph nodes). H&E staining was performed to investigate the tigated the migratory capacity of bmDC directly in vivo. To this morphology of immune organs. Lymph node architecture as well Ϫ Ϫ end, CFSE-labeled mature WT bmDC were injected s.c. in the as the structure of spleen and thymus was not altered in LIF / hind pads of LIFϪ/Ϫ and WT control mice. Three days after si- mice (data not shown). To investigate T cell development and multaneous immunization with a low dose of MOG 35–55, drain- commitment, FACS analysis of thymus and lymph nodes was per- ing lymph nodes were harvested and investigated for the presence formed and confirmed the normal distribution of single-positive, of labeled cells. FACS analysis revealed a similar percentage of double-positive, and double-negative cells in the thymus as well as ϩ ϩ Ϫ Ϫ CFSE-positive DC in popliteal lymph nodes of LIFϪ/Ϫ mice and normal CD4 and CD8 compartments in the periphery in LIF / WT control mice. Thus, in vivo migration of WT bmDC was not mice as well as a similar percentage of early activation marker different in LIFϪ/Ϫ and WT mice (Fig. 5B). (CD69 and CD25) positive T cells compared with WT mice (Table III, data are shown as percentages). LIF deficiency does not lead to altered T cell subsets In summary, the priming defect in LIFϪ/Ϫ mice can neither be Because no differences were observed in APC function of LIFϪ/Ϫ explained by obvious qualitative or quantitative differences in T mice, we next investigated the T cell compartment. First, immune cell subsets nor by an impaired APC function or DC migration. Ϫ/Ϫ organs of immunized LIF mice and WT mice were analyzed. Ϫ/Ϫ Cell counts of peripheral blood and lymph nodes revealed similar Altered pattern of chemokine production in LIF mice cell numbers in LIFϪ/Ϫ WT blood and lymph nodes, respectively We were interested in the molecular mechanisms governing the (47.5 million cells Ϯ 0.5 in WT vs 46.2 million cells Ϯ 1.47 in distinct pattern of inflammatory infiltration in LIFϪ/Ϫ mice. To The Journal of Immunology 2211

Table III. Distribution of CD4-, CD8-, CD25-, and CD69-positive cells in thymus and lymph nodes of WT and LIFϪ/Ϫ micea

Thymus Lymph Node

CD4ϩCD8ϩ CD4ϩ CD8ϩ CD4ϩ CD8ϩ CD4:8 ratio CD4ϩCD25ϩ CD4ϩCD69ϩ

WT 85.6 Ϯ 4.5 9.7 Ϯ 3.3 1.6 Ϯ 0.9 28.8 Ϯ 0.7 17.9 Ϯ 1.5 1.6 Ϯ 0.1 8.8 Ϯ 1.1 5.5 Ϯ 1.6 LIFϪ/Ϫ 88.4 Ϯ 0.7 6.4 Ϯ 0.7 1.8 Ϯ 0.3 33.8 Ϯ 4.6 20.6 Ϯ 4.0 1.7 Ϯ 0.5 9.7 Ϯ 3.4 3.9 Ϯ 1.8

a There were no differences in the distribution of different T cell subsets in the thymus and lymph nodes between LIFϪ/Ϫ and WT mice. Data are shown as percentages and are summarized from a FACS analysis of three mice per group as mean Ϯ SD. investigate expression patterns of chemokines, isolated mRNA (Fig. 6A). At that time point, there was also an increase of GM- from spinal cord of LIFϪ/Ϫ mice and WT controls was investi- CSF, CCL3 (MIP-1␣), and milder also of CCL5 (RANTES) gated by RT-PCR at different time points of MOG-EAE. At the expression in LIFϪ/Ϫ mice, although these effects were not sta- onset of disease (day 13 p.i.), the neutrophil attracting chemokine tistically significant. At day 13 p.i., there were no differences in CXCL1 (KC) was increased in the spinal cord of LIFϪ/Ϫ mice expression of CCL2 and CXCL10 (IP10) between both groups. In the later phase of MOG-EAE, again patterns of immune cell infiltration were correlated with chemokine production in a RT-

PCR analysis. In the early late stage of MOG 35–55-EAE (day Downloaded from 27 p.i.), CXCL1 (KC), CCL2 (MCP-1), CCL3 (MIP-1␣), CXCL10 (IP10), and CCL5 (RANTES) mRNA expression were significantly reduced in the spinal cord of LIFϪ/Ϫ mice (Fig. 6B). At that time point, there was also a trend toward a reduced expression of GM-CSF.

In summary, the expression of chemokines at the RNA level http://www.jimmunol.org/ correlates with the observed infiltrates of different immune cells in the spinal cords. Neutrophil invasion in LIFϪ/Ϫ mice early during MOG-EAE is associated with increased CXCL1 production. The decreased inflammatory infiltration in the later phase of the disease is paralleled by a decreased expression of several chemokines in- cluding CCL2, CCL3, and CXCL10.

Discussion In this study, we show that a deficiency of the neuroprotective by guest on September 26, 2021 cytokine LIF affects the immune response in autoimmune demy- elination. Although the early phase of active MOG 35–55 EAE in LIFϪ/Ϫ mice is similar to WT controls, the late phase is charac- terized by an alleviated disease course. In search of the explanation for this phenotype, we found an Ag-specific T cell priming defect. Importantly, the inflammatory infiltrate in the LIF-deficient mice is dominated by massive infiltration of neutrophilic granulocytes in the early phase of the disease. On the molecular level, this is par- alleled by a distinct pattern of chemokine expression with an in- crease of CXCL1 (KC). In the later phases, a reduced macrophage infiltration accompanied by decreased levels of CCL2, CCL3, and CXCL10 were observed. In extension of previous studies by Escary et al. (12), LIFϪ/Ϫ mice display an impaired T cell priming. We could exclude structural abnormalities in the immune system of LIFϪ/Ϫ mice as a possible explanation. This is in contrast to LIF-overexpressing mice which are characterized by interconver- sion of thymic and lymph node morphologies (13). LIFR␤ is FIGURE 6. Chemokine production in LIFϪ/Ϫ mice. A, RT-PCR analy- present in Ag-activated T cell blasts and LIF is produced by mono- sis of chemokine mRNA expression in the spinal cord of LIFϪ/Ϫ and WT cytes, macrophages, and also T cells (30, 31), thus opening the control mice at the beginning of disease (day 13 p.i.). Data are presented possibility of paracrine actions. Yet, similar to previous studies (9, as relative expression with the mean chemokine expression in WT mice set 32), exogenous addition of LIF to MOG-primed T cell cultures as1(f). Error bars represent SEM. At day 13 p.i., levels of CXCL1 (KC) does not have any additional effect on T cell proliferation and Ϫ/Ϫ were significantly increased in LIF mice (p ϭ 0.013). In addition, there adoptive transfer of WT T cells in LIFϪ/Ϫ mice does not change was a trend toward an increased production of GM-CSF and CCL3 (MIP-1 the course of EAE. In conclusion, we could not delineate a direct ␣) in the spinal cord of LIFϪ/Ϫ mice. B, RT-PCR analysis of chemokine Ϫ Ϫ impact of LIF on the control of T cell function although indirect mRNA expression in the spinal cord of LIF / and WT control mice (WT) effects are certainly conceivable. in the later phase of MOG 35–55 EAE (day 27 p.i.). Data are pooled from Ϫ/Ϫ two experiments and depicted as in A. There was a significant decrease of In the early phase of MOG-EAE, infiltrates in LIF mice are CXCL1 (KC), CCL2 (MCP-1), CCL3 (MIP-1␣), CXCL10 (IP10), and characterized by an abundance of 7/4 Ag-positive cells. Expression CCL5 (RANTES) expression in the spinal cord of LIFϪ/Ϫ mice (p Ͻ 0.05) of the 7/4 Ag is confined to neutrophilic granulocytes and mono- and a trend toward a decreased GM-CSF expression (p ϭ 0.086). cytes, but not macrophages (22, 33). Most of the 7/4 Ag-positive 2212 EAE IN LIF KNOCKOUT MICE cells in EAE lesions of LIFϪ/Ϫ mice are also reactive for chloro- Although LIF was shown to prevent oligodendrocyte loss (9), the acetate esterase and display a typical polymorphonuclear config- administration of CNTF also interfered with the immune system, uration. Thus, we characterize these 7/4 Ag-positive cells as neu- and inhibits inflammatory infiltration into the CNS (10). In good trophilic granulocytes which usually represent a minor cell type in correlation with the data on LIF treatment, cuprizone-induced de- C57BL/6 MOG-EAE. Well in line with the results on neutrophils myelination in LIFϪ/Ϫ mice resulted in a more pronounced oligo- enhancing T cell proliferation, neutrophilic granulocytes were dendrocyte loss (43). Moreover, Ab-mediated neutralization of shown earlier to orchestrate inflammation and tissue damage lead- LIF doubled the extent of oligodendrocyte loss in an EAE model ing to clinical symptoms in the early phase of EAE in CCR2 (19). In extension of these previous studies, we show here that knockout mice (34). Yet, these cells can also regulate T cell re- besides its protective role, LIF can also act as an immunomodu- sponses (35), probably depending on the activation status of cells, lator. Our data do not challenge the value of LIF for oligodendro- type of T cell, and the milieu. Interestingly, EAE in mice deficient cyte protection in neuroinflammation, but bring in another level of for the CCL2 receptor, CCR2, is also characterized by neutrophil complexity by revealing that endogenous LIF is also an immuno- invasion (34). Moreover, reports describe neutrophil infiltration in logically active molecule. The profound interaction of LIF with the EAE lesions of IFN-␥ knockout mice (35, 36). LIF-deficient mice immune system will make it difficult to predict results of possible are characterized by a decreased expression of both CCL2 and treatment trials. IFN-␥. In view of these data, it is tempting to speculate that a decrease in expression of some cytokines and chemokines (or their Acknowledgments receptors) may lead to counterregulatory up-regulation of other We thank H. Bru¨nner, V. T. Wo¨rtmann, and A. Bohl for expert tech- chemoattractants which result in a qualitatively different compo- nical assistance and Steffi Gaupp for helpful discussions. LIF was pro- Downloaded from sition of the inflammatory infiltrate. Indeed, an up-regulation of vided by Dr. H. Butzkueven (Melbourne, Australia). We thank Prof. CXCL1 in LIFϪ/Ϫ mice may lead to granulocyte attraction. This Toyka (Wuerzburg, Germany) for helpful discussions at the beginning concept is further sustained by recent studies investigating LIF or of experimentation and Dr. D. Merkler (Goettingen, Germany) for the LIF-related cytokine IL-6 in peritoneal inflammation and in providing rMOG. Ϫ/Ϫ endotoxic shock. Reminiscent to MOG-EAE in LIF mice, IL-6 Disclosures knockout mice display increased levels of CXCL1 expression and http://www.jimmunol.org/ Ϫ Ϫ The authors have no financial conflict of interest. higher numbers of infiltrating neutrophils while LIF / mice are characterized by an increased neutrophil sequestration (37, 38). References In the late phase of MOG-EAE, LIF deficiency leads to impaired 1. Kerr, B. J., and P. H. Patterson. 2005. Leukemia inhibitory factor promotes ol- macrophage recruitment in EAE lesions. In good agreement with igodendrocyte survival after . Glia 51: 73–79. previous studies using radioiodinated LIF (39), we confirm expres- 2. Mayer, M., K. Bhakoo, and M. Noble. 1994. Ciliary neurotrophic factor and ␤ leukemia inhibitory factor promote the generation, maturation and survival of sion of LIFR in these cells. Previous in vitro studies revealed that oligodendrocytes in vitro. Development 120: 143–153. mouse peritoneal macrophages respond to LIF in a microchamber 3. Barres, B. A., R. Schmid, M. Sendnter, and M. C. Raff. 1993. Multiple extra- assay, thus demonstrating a chemotactic action of this cytokine cellular signals are required for long-term oligodendrocyte survival. Development

Ϫ/Ϫ 118: 283–295. by guest on September 26, 2021 (14). Analysis of LIF mice in neurotrauma models has also 4. Louis, J. C., E. Magal, S. Takayama, and S. Varon. 1993. CNTF protection of provided evidence for a role of LIF in macrophage chemotaxis in oligodendrocytes against natural and -induced death. Sci- vivo (16, 40). In these paradigms, it might well be possible that ence 259: 689–692. 5. Sendtner, M. 1995. Molecular biology of neurotrophic factors. Baillieres Clin. LIF acts indirectly on macrophage migration via induction of other Neurol. 4: 575–591. chemokines. In our model, a role of endogenous LIF for macro- 6. Ransohoff, R. M., C. L. Howe, and M. Rodriguez. 2002. Growth factor treatment of : at last, a leap into the light. Trends Immunol. 23: phage recruitment was exclusively observed in the later phases of 512–516. active MOG-EAE. In contrast, macrophage recruitment in the 7. Storch, M. K., A. Stefferl, U. Brehm, R. Weissert, E. Wallstrom, early phase of active MOG-EAE and in adoptive transfer EAE is M. Kerschensteiner, T. Olsson, C. Linington, and H. Lassmann. 1998. Autoim- Ϫ/Ϫ munity to myelin oligodendrocyte glycoprotein in rats mimics the spectrum of not different between LIF and WT mice. These results argue multiple sclerosis pathology. Brain Pathol. 8: 681–694. against a direct effect of LIF on macrophage recruitment in neu- 8. Linker, R. A., M. Maurer, S. Gaupp, R. Martini, B. Holtmann, R. Giess, roinflammation. Rather, LIF deficiency may influence inflamma- P. Rieckmann, H. Lassmann, K. V. Toyka, M. Sendtner, and R. Gold. 2002. CNTF is a major protective factor in demyelinating CNS disease: a neurotrophic tory infiltration by indirect mechanisms like the regulation of che- cytokine as modulator in neuroinflammation. Nat. Med. 8: 620–624. mokine expression (15). Previous studies revealed that CCL2, 9. Butzkueven, H., J. G. Zhang, M. Soilu-Hanninen, H. Hochrein, F. Chionh, K. A. Shipham, B. Emery, A. M. Turnley, S. Petratos, M. Ernst, et al. 2002. LIF CCL3, and also GM-CSF play a pivotal role in local macrophage receptor signaling limits immune-mediated demyelination by enhancing oligo- recruitment and Ag-specific Th1 immune response in EAE (41). dendrocyte survival. Nat. Med. 8: 613–619. Indeed, production of CCL2, CCL3, CXCL10, and CCL5 is re- 10. Kuhlmann, T., L. Remington, I. Cognet, L. Bourbonniere, S. Zehntner, Ϫ/Ϫ F. Guilhot, A. Herman, A. Guay-Giroux, J. P. Antel, T. Owens, and J. F. Gauchat. duced in LIF mice. These chemokines may be produced by 2006. Continued administration of ciliary neurotrophic factor protects mice from endothelial cells (42), but also monocytes themselves resulting in inflammatory pathology in experimental autoimmune encephalomyelitis. negative feedback loops: the reduced expression of macrophage Am. J. Pathol. 169: 584–598. 11. Metcalf, D. 2003. The unsolved enigmas of leukemia inhibitory factor. Stem Cells Ϫ/Ϫ and T cell-attracting chemokines in the spinal cord of LIF mice 21: 5–14. specifically during the late phase of MOG 35–55-EAE result in a 12. Escary, J. L., J. Perreau, D. Dumenil, S. Ezine, and P. Brulet. 1993. Leukaemia inhibitory factor is necessary for maintenance of haematopoietic stem cells and decreased inflammatory infiltration, while the decrease of inflam- thymocyte stimulation. Nature 363: 361–364. matory cells themselves leads to further reduced chemokine levels 13. Shen, M. M., R. C. Skoda, R. D. Cardiff, J. Campos-Torres, P. Leder, and and thus finally a milder disease course. D. M. Ornitz. 1994. Expression of LIF in transgenic mice results in altered thymic epithelium and apparent interconversion of thymic and lymph node morpholo- Although some previous studies mainly point at proinflamma- gies. EMBO J. 13: 1375–1385. tory actions of LIF (16), others argue for an anti-inflammatory role 14. Sugiura, S., R. Lahav, J. Han, S. Y. Kou, L. R. Banner, F. de Pablo, and of this cytokine (17, 18). Our data present evidence for an inverse P. H. Patterson. 2000. Leukaemia inhibitory factor is required for normal inflam- matory responses to injury in the peripheral and central nervous systems in vivo impact of LIF on macrophage and neutrophil recruitment. These and is chemotactic for macrophages in vitro. Eur. J. Neurosci. 12: 457–466. results point to an additional immunomodulatory role rather than a 15. Tofaris, G. K., P. H. Patterson, K. R. Jessen, and R. Mirsky. 2002. Denervated Schwann cells attract macrophages by secretion of leukemia inhibitory factor purely pro- or anti-inflammatory function. The role of LIF and (LIF) and monocyte chemoattractant protein-1 in a process regulated by inter- CNTF in EAE were also investigated in therapeutic approaches. leukin-6 and LIF. J. Neurosci. 22: 6696–6703. The Journal of Immunology 2213

16. Kerr, B. J., and P. H. Patterson. 2004. Potent pro-inflammatory actions of leu- 31. Vanderlocht, J., N. Hellings, J. J. Hendriks, F. Vandenabeele, M. Moreels, kemia inhibitory factor in the spinal cord of the adult mouse. Exp. Neurol. 188: M. Buntinx, D. Hoekstra, J. P. Antel, and P. Stinissen. 2006. Leukemia inhibitory 391–407. factor is produced by myelin-reactive T cells from multiple sclerosis patients and 17. Banner, L. R., P. H. Patterson, A. Allchorne, S. Poole, and C. J. Woolf. 1998. protects against tumor necrosis factor-␣-induced oligodendrocyte apoptosis. Leukemia inhibitory factor is an anti-inflammatory and analgesic cytokine. J. Neurosci. Res. 83: 763–774. J. Neurosci. 18: 5456–5462. 32. Metcalfe, S. M., P. A. Muthukumarana, H. L. Thompson, M. A. Haendel, and 18. Zhu, M., K. Oishi, S. C. Lee, and P. H. Patterson. 2001. Studies using leukemia G. E. Lyons. 2005. Leukaemia inhibitory factor (LIF) is functionally linked to inhibitory factor (LIF) knockout mice and a LIF adenoviral vector demonstrate a axotrophin and both LIF and axotrophin are linked to regulatory immune toler- key anti-inflammatory role for this cytokine in cutaneous inflammation. J. Im- ance. FEBS Lett. 579: 609–614. munol. 166: 2049–2054. 33. Taylor, P. R., G. D. Brown, A. B. Geldhof, L. Martinez-Pomares, and S. Gordon. 19. Butzkueven, H., B. Emery, T. Cipriani, M. P. Marriott, and T. J. Kilpatrick. 2006. 2003. Pattern recognition receptors and differentiation antigens define murine Endogenous leukemia inhibitory factor production limits autoimmune demyeli- myeloid cell heterogeneity ex vivo. Eur. J. Immunol. 33: 2090–2097. nation and oligodendrocyte loss. Glia 53: 696–703. 34. Gaupp, S., D. Pitt, W. A. Kuziel, B. Cannella, and C. S. Raine. 2003. Experi- 20. Gold, R., G. Giegerich, H. P. Hartung, and K. V. Toyka. 1995. T-cell receptor mental autoimmune encephalomyelitis (EAE) in CCR2Ϫ/Ϫ mice: susceptibility in (TCR) usage in Lewis rat experimental autoimmune encephalomyelitis: TCR multiple strains. Am. J. Pathol. 162: 139–150. ␤ ␤ -chain-variable-region V 8.2-positive T cells are not essential for induction 35. Zehntner, S. P., C. Brickman, L. Bourbonniere, L. Remington, M. Caruso, and and course of disease. Proc. Natl. Acad. Sci. USA 92: 5850–5854. T. Owens. 2005. Neutrophils that infiltrate the regulate T 21. Crowther, J. R. 1995. ELISA. Theory and practice. Methods Mol. Biol. 42: cell responses. J. Immunol. 174: 5124–5131. 1–218. 36. Glabinski, A. R., M. Krakowski, Y. Han, T. Owens, and R. M. Ransohoff. 1999. 22. Hirsch, S., and S. Gordon. 1983. Polymorphic expression of a neutrophil differ- Chemokine expression in GKO mice (lacking interferon-␥) with experimental entiation antigen revealed by monoclonal 7/4. Immunogenetics 18: autoimmune encephalomyelitis. J. Neurovirol. 5: 95–101. 229–239. 37. McLoughlin, R. M., S. M. Hurst, M. A. Nowell, D. A. Harris, S. Horiuchi, 23. Grauer, O., G. Wohlleben, S. Seubert, A. Weishaupt, E. Kampgen, and R. Gold. L. W. Morgan, T. S. Wilkinson, N. Yamamoto, N. Topley, and S. A. Jones. 2004. 2002. Analysis of maturation states of rat bone marrow-derived dendritic cells Differential regulation of neutrophil-activating chemokines by IL-6 and its sol- using an improved culture technique. Histochem. Cell Biol. 117: 351–362.

uble receptor isoforms. J. Immunol. 172: 5676–5683. Downloaded from 24. Penna, G., S. Sozzani, and L. Adorini. 2001. Cutting edge: selective usage of chemokine receptors by plasmacytoid dendritic cells. J. Immunol. 167: 38. Weber, M. A., S. Schnyder-Candrian, B. Schnyder, V. Quesniaux, V. Poli, 1862–1866. C. L. Stewart, and B. Ryffel. 2005. Endogenous leukemia inhibitory factor at- 25. Del Prete, A., W. Vermi, E. Dander, K. Otero, L. Barberis, W. Luini, tenuates endotoxin response. Lab. Invest. 85: 276–284. S. Bernasconi, M. Sironi, A. Santoro, C. Garlanda, et al. 2004. Defective den- 39. Hilton, D. J., N. A. Nicola, and D. Metcalf. 1991. Distribution and comparison of dritic cell migration and activation of adaptive immunity in PI3K␥-deficient receptors for leukemia inhibitory factor on murine hemopoietic and hepatic cells. mice. EMBO J. 23: 3505–3515. J. Cell. Physiol. 146: 207–215. 26. Kruse, N., M. Pette, K. Toyka, and P. Rieckmann. 1997. Quantification of cy- 40. Toews, A. D., C. Barrett, and P. Morell. 1998. Monocyte chemoattractant protein 1 is responsible for macrophage recruitment following injury to sciatic nerve. tokine mRNA expression by RT PCR in samples of previously frozen blood. http://www.jimmunol.org/ J. Immunol. Methods 210: 195–203. J. Neurosci. Res. 53: 260–267. 27. Livak, K. J., and T. D. Schmittgen. 2001. Analysis of relative gene expression 41. Huang, D. R., J. Wang, P. Kivisakk, B. J. Rollins, and R. M. Ransohoff. 2001. data using real-time quantitative PCR and the 2(Ϫ⌬⌬CT) method. Methods 25: Absence of monocyte chemoattractant protein 1 in mice leads to decreased local 402–408. macrophage recruitment and antigen-specific T helper cell type 1 immune re- 28. Linker, R. A., E. Rott, H. H. Hofstetter, T. Hanke, K. V. Toyka, and R. Gold. sponse in experimental autoimmune encephalomyelitis. J. Exp. Med. 193: 2005. EAE in ␤-2 microglobulin-deficient mice: axonal damage is not dependent 713–726. on MHC-I restricted immune responses. Neurobiol. Dis. 19: 218–228. 42. Man, S., E. E. Ubogu, and R. M. Ransohoff. 2007. Inflammatory cell migration 29. Eugster, H. P., K. Frei, R. Bachmann, H. Bluethmann, H. Lassmann, and into the central nervous system: a few new twists on an old tale. Brain Pathol. 17: A. Fontana. 1999. Severity of symptoms and demyelination in MOG-induced 243–250. EAE depends on TNFR1. Eur. J. Immunol. 29: 626–632. 43. Emery, B., H. S. Cate, M. Marriott, T. Merson, M. D. Binder, C. Snell, P. Y. Soo, 30. Anegon, I., D. Grolleau, and J. P. Soulillou. 1991. Regulation of HILDA/LIF S. Murray, B. Croker, J. G. Zhang, et al. 2006. Suppressor of cytokine signaling

gene expression in activated human monocytic cells. J. Immunol. 147: 3 limits protection of leukemia inhibitory factor receptor signaling against central by guest on September 26, 2021 3973–3980. demyelination. Proc. Natl. Acad. Sci. USA 103: 7859–7864.