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β7 integrins contribute to demyelinating disease of the central nervous system
Journal of Neuroimmunology 103 Ž2000. 146–152 www.elsevier.comrlocaterjneuroim
b7 integrins contribute to demyelinating disease of the central nervous system Jagat R. Kanwar a , Jane E.B. Harrison a , Dongmao Wang a , Euphemia Leung a , Werner Mueller b, Norbert Wagner b, Geoffrey W. Krissansen a,) a
Department of Molecular Medicine, School of Medicine and Health Science, UniÕersity of Auckland, Auckland, New Zealand b Institute for Genetics, UniÕersity of Cologne, 50937 Cologne, Germany Received 24 May 1999; received in revised form 18 August 1999; accepted 29 October 1999
Abstract A role for a4 integrins in different forms of the multiple sclerosis-like disease experimental autoimmune encephalomyelitis ŽEAE. has been demonstrated, but the individual contributions of a4b1, a4b7, and the related a Eb7 integrin have not been determined. The b7 integrins a4b7 and a Eb7 play a central role in chronic inflammation, mediating the trafficking, entry, andror adhesion of lymphocytes in the inflamed pancreas and gut, and their ligands MAdCAM-1, VCAM-1 and E-cadherin are expressed on brain endothelial cells andror on microvessels in the inflamed central nervous system. Here, we show that an antibody directed against the b7 subunit greatly attenuates a non-remitting form of EAE, induced by adoptive transfer of myelin oligodendrocyte peptide ŽMOG35 – 55 .-stimulated T cells. Combinational treatment with both anti-b7 and a4 integrin subunit antibodies led to more rapid and complete remission than that obtained with anti-a4 antibody alone, potentially implicating a role for a Eb7 in disease progression. Remission correlated with the down-regulation of the vascular addressins VCAM-1, MAdCAM-1, and ICAM-1 on cerebral blood vessels. Attenuated forms of disease were induced by adoptive transfer of either wild-type encephalitogenic T cells to b7-deficient gene knockout mice, or of b7yry encephalitogenic T cells to wild-type recipients. The former finding indicates that b7qve recruited cells contribute to disease progression. Thus a4b1, a4b7, and a Eb7 integrins may all play a contributory role in the progression of chronic forms of demyelinating disease, and together with their ligands could represent potential targets for improved treatment of some forms of multiple sclerosis. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Experimental autoimmune encephalomyelitis; b7 integrins; Cell adhesion molecules; Lymphocyte traffic; Central nervous system; Multiple sclerosis
1. Introduction Experimental autoimmune encephalomyelitis ŽEAE. is a disease of the central nervous system ŽCNS. similar to multiple sclerosis, in which encephalitogenic leukocytes penetrate the blood–brain barrier ŽBBB. and damage the myelin sheath of nerve fibres ŽRaine, 1991, 1994; Cannella and Raine, 1995; Martin and McFarland, 1995; Brosnan and Raine, 1996.. Both diseases lead to impaired nerve conduction and paralysis, which varies from relapsingrremitting to chronic progressive. The pathways by which encephalitogenic T cells access the CNS have not been fully delineated, but clearly involve integrin cell adhesion
) Corresponding author. Tel.: q64-9-373-7599 ext. 6280; fax: q64-9373-7674; e-mail: [email protected]
molecules that mediate lymphocyte trafficking and recirculation. The b7 integrin subfamily of cell adhesion molecules consists of two members, a4b7 ŽHolzmann and Weissman, 1989; Yuan et al., 1992. and a Eb7 ŽKrissansen et al., 1992; Parker et al., 1992. whose expression is restricted to leukocytes. a4b7 mediates the adherence of lymphocytes to high endothelial venules ŽHEV. via its preferred ligand MAdCAM-1 ŽBerlin et al., 1993; Briskin et al., 1993; Yang et al., 1995., whereas a Eb7 mediates the adhesion of intraepithelial lymphocytes of the intestine to the intestinal epithelium via an interaction with Ecadherin ŽCepek et al., 1994; Karecla et al., 1995.. In gene knockout mice deficient in the b7 integrin subfamily, the gut-associated lymphoid tissue ŽGALT. is severely impaired due to a failure of b7yry lymphocytes to adhere to blood vessel walls at the site of transmigration into the
0165-5728r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 5 7 2 8 Ž 9 9 . 0 0 2 4 5 - 3
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GALT ŽWagner et al., 1996.. Further, in non-obese diabetic ŽNOD. mice, treatment with anti-b7 or antiMAdCAM-1 mAbs provides protection against the spontaneous development of diabetes and insulitis, presumably by blocking lymphocyte migration into the inflamed pancreas ŽYang et al., 1997.. Hence a4b7-MAdCAM-1 interactions play critical roles in allowing the entry of leukocytes into chronically inflamed tissues. A role for a4 integrins in the establishment of EAE was demonstrated by in vivo administration of anti-a4 integrin antibodies which diminished the paralysis associated with EAE ŽYednock et al., 1992; Baron et al., 1993.. Further, the suppressive effects of TNFbp on the development of EAE appears to correlate with down-regulation of VCAM1rVLA-4 ŽSelmaj et al., 1998.. Hence, a4 integrins play a crucial role in allowing encephalitogenic T cells to enter the brain, but the individual contributions of the two related a4 integrins a4b1 and a4b7 to the various stages of the different forms of EAE has not been determined. A recent study of a relapsing–remitting form of EAE induced by proteolipid protein in the SJL mice, revealed that b7 integrins play no role in the early stages of EAE ŽEngelhardt et al., 1998.. However, this finding does not rule out a role for b7 integrins in the chronic phase of non-remitting forms of the disease, as it is established that MAdCAM-1, the preferential ligand for a4b7, is predominantly expressed on specialized HEV formed at chronically inflamed sites ŽStreeter et al., 1988.. Further, Ecadherin has also been found expressed on brain microvessel endothelial cells ŽPal et al., 1997.. Since MAdCAM-1 expression is particularly noticeable on HEV-like endothelia in relapsing brain lesions of mice with EAE ŽCannella et al., 1991., we sought to show a role for b7 integrins in the chronic phase of a non-remitting form EAE.
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Sigma, USA, served as a control for the latter antibodies in in vivo blocking and immunohistological studies. 2.2. Encephalitogenic lymphocytes
2. Materials and methods
Encephalitogenic lymphocytes were obtained by subcutaneous immunization of 8-10 week old wild-type and b7yry C57BLr6 mice with 300 mg of MOG35 – 55 peptide ŽMEVGWYRSPFSRVVHLYRNGK. ŽMendel et al., 1995.; synthesized by Chiron Technologies, Clayton, Australia and emulsified in CFA containing 500 mg of Mycobacterium tuberculosis H37RA ŽDIFCO Laboratories, Detroit, USA.. Administration of pertussis toxin was not necessary, and was omitted. Two injections were given 1 week apart in opposite flanks, and 14 days later spleen and draining lymph nodes were removed, and a single cell suspension obtained by teasing the tissue through a stainless steel wire mesh. Cells were then washed three times in Hanks’ balanced salt solution ŽHBSS, GIBCO, Grand Island, NY, USA. and resuspended to a concentration of 6 = 10 6 cellsrml in complete medium consisting of RPMI 1640 ŽGIBCO BRL, Gaithersburg, MD, USA. supplemented with 1 mM L-glutamine, pencillin–streptomycin, 10% FCS, and 2-mercaptoethanol. Cells were fed with 50 Urml of IL-2 ŽR & D Systems, Minneapolis, MN, USA. and 30 mgrml MOG peptide antigen, and incubated in a humidified 378C, 10% CO 2 incubator. After 4 d of activation lymphoblasts were isolated by centrifugation on Ficoll-paque ŽPharmacia LKB Biotechnology AB, Uppsala, Sweden. gradients. Blasts from the interface were washed three times, and resuspended in complete medium. Cell viability as determined by trypan blue exclusion was ) 97%. EAE was transferred to both wild-type and b7yry C57BLr6 mice by injecting 6 = 10 8 MOG peptide-stimulated lymphocytes into the tail vein Ž70%. and intraperitoneally Ž30%..
2.1. Mice and antibodies
2.3. ProliferatiÕe response of lymphocytes to MOG peptide
C57BLr6 wild-type and b7yry gene knockout mice were bred and housed by the Animal Resource Unit, School of Medicine and Health Science, University of Auckland, Auckland, New Zealand. They were randomly bred on a C57BLr6 background. Rat hybridomas secreting mAbs against mouse integrin a4 subunit ŽPSr2 rat IgG2b mAb. ŽMiyake et al., 1991., b7 subunit ŽFIB 504.64 rat IgG2a mAb. ŽAndrew et al., 1994., VCAM-1 ŽMK1.9 rat IgG1 mAb. ŽMiyake et al., 1991., and ICAM-1 ŽBE29G1 rat IgG2a mAb. ŽKuhlman et al., 1991. were purchased from the American Type Culture Collection, Rockville, MD. The rat hybridoma MECA-367 Žrat IgG2a. ŽBerlin et al., 1993., which secretes a mAb against mouse MAdCAM-1, was kindly provided by Dr. Eugene Butcher, Stanford University, Stanford, CA. Rat IgG obtained from
The cell proliferation enzyme-linked immunosorbent assay ŽELISA. was done with a non-radioactive and colorimetric immunoassay kit ŽBoehringer Mannheim, Mannheim, Germany. to measure the incorporation of 5-bromo-2-deoxyuridine ŽBrdU.. In brief, lymphocytes from the spleens of wild-type and b7yry C57BLr6 mice were purified on Ficoll-paque ŽPharmacia LKB Biotechnology AB, Uppsala, Sweden.. Equal numbers of wild-type and b7yry cells Ž1 = 10 4 . were incubated in the presence or absence of 30 mg of MOG35 – 55 peptide in 96-well flat-bottomed plates ŽBecton Dickinson Labware, Frankin Lakes, NJ, USA. in triplicate for 4 days. The cells were labelled with BrdU for 25 h, and fixed for 30 min at room temperature. The plates were washed and incubated with peroxidase-conjugated anti-BrdU Fab fragment. After
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washing, substrate was added, the reaction stopped, and absorbance measured in an ELISA plate reader.
3. Results
2.4. EAE induction, and inhibition with antibodies
3.1. In ÕiÕo administration of anti-b 7 integrin antibodies diminishes the paralysis associated with EAE
MOG peptide-stimulated lymphoblasts were injected into the tail vein Ž70% of cells. and intraperitoneally Ž30% of cells. into mice, and animals were observed daily for clinical signs of EAE. The mice were scored according to the following scale: 0, no clinical signs of EAE; 1, limp tail; 2, partial hind limb paralysis; 3, complete hind limb paralysis; 4, complete hind limb and partial fore limb paralysis; 5, paralysis extending to diaphragm; 6, hind and fore limb paralysis; 7, death due to EAE. Animals were cared for according to the guidelines of the University of Auckland Animal Ethics Committee. They had to be maintained in a state of hind-limb paralysis, hence care was shown in the provision of drinking water, food, and comfort. The daily mean clinical score for each group is the mean disease score of at least five mice. For antibody blocking, anti-b7 ŽFib 504. and anti-a4 ŽPSr2. subunit mAbs were administered into the tail vein Ž70% of mAb. and intraperitoneally Ž30% of mAb. at 10 mgrkg separately or in combination three times on alternate days when the clinical score reached 3 or 4.5. Control groups of mice received either PBS or rat IgG ŽSigma, USA.. Experiments were repeated to confirm reproducibility.
EAE was induced in C57BLr6 mice by adoptive transfer of myelin oligodendrocyte glycoprotein peptide ŽMOG35 – 55 .-stimulated lymphoblasts ŽMendel et al., 1995., resulting in chronic sustained paralysis. The first signs of clinical disease ensued within 4 to 5 days leading to paralysis of the hind limbs by day 10 ŽFig. 1.. Marked perivascular accumulation of leukocytes was observed at the peak of the disease; whereas inflamed blood vessels were not detectable in the brains of control animals Žrefer to Fig. 2a,b.. Sixteen days after adoptive transfer of MOG35 – 55-stimulated encephalitogenic lymphoblasts when the clinical score reached 4.5 Žhind limb paralysis., animals were given intravenous Ži.v.. and intraperitoneal Ži.p.. injections of mAbs against the integrin b7 ŽFib504. and a4 ŽPSr2. subunits, or of a rat IgG control antibody. Anti-a4 integrin mAb caused complete remission of the disease in all animals, such that no clinical symptoms could be observed 50 days after antibody treatment was suspended ŽFig. 1.. Anti-b7 integrin mAb caused a more gradual remission of disease symptoms such that the clinical scores of animals were reduced from 4.5 to 2 after 50 days, compared to undiminished disease scores in untreated controls.
2.5. Immunohistology Brains were frozen in isopentane at y708C, and transverse 10 mm sections were made through the cerebrum, cerebellum, and mid-brain. Sections were mounted on poly L-lysine-coated slides, and endogenous peroxidase blocking was done by incubating slides for 30 min in 0.3% H 2 O 2 in methanol. The sections were stained with antiCAM mAbs using VECTASTAIN ABC Elite Peroxidase ŽRat IgG. Kit ŽVector Laboratories, Burlingame, CA, USA.. In brief, sections were washed with PBS and blocked with rabbit serum, and incubated with different anti-CAM mAbs. After three washings with PBS, slides were incubated with biotinylated anti-rat secondary antibody for 10 min. They were then washed, incubated with ABC peroxidase reagent, and developed with SIGMA FAST DAB Ž3,3X-diaminobenzidine tetrahydrochloride. with metal enhancer CoCl 2 tablets. Slides were counterstained with haematoxylin and eosin. Controls for the staining protocol consisted of omitting the primary anti-CAM mAb, or substituting it with rat IgG ŽSigma.. Representative sections are reported in the case of analysis of leukocyte infiltration, whereas for visualisation of CAMs we deliberately chose blood vessels lacking extensive perivascular accumulation of leukocytes for clarity.
Fig. 1. In vivo administration of an anti-b7 integrin subunit mAb causes gradual remission of EAE. EAE was induced in C57BLr6 mice by injecting 6=10 8 MOG35 – 55-stimulated encephalitogenic lymphoblasts. On days 16, 18, and 20 animals received i.v. and i.p. injections of PBS Ždotted square., rat IgG control ŽI., anti-b7 Ž'. and a4 ŽB. subunit mAbs, or a combination of anti-b7 and anti-a4 mAbs Žv .. UU Indicates a significant difference at p- 0.001 from control groups of mice that received rat IgG.
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3.2. Combination of anti-b 7 and anti-a 4 antibodies proÕides enhanced therapeutic benefit Simultaneous administration of both mAbs caused a dramatic reversal in disease progression, such that complete remission was observed in only 5 days following suspension of antibody treatment. Rat IgG control antibody had no affect on the onset or severity of disease. Histochemistry of brain sections from animals receiving the control antibody revealed extensive leukocyte infiltration ŽFig. 2c., whereas infiltration into the brains of EAE animals was lessened by treatment with anti-b7 mAb ŽFig.
Fig. 3. Integrin b7-deficient mice are partially resistant to EAE, and generate lymphocytes that respond less well to MOG-peptide and are poorly encephalitogenic. ŽA. Proliferative response of wild-type and b7yry lymphocytes to MOG35 – 55-peptide. Ficoll-paque-purified lymphocytes from the spleens of wild-type and b7yry C57BLr6 mice were incubated in the presence or absence of 30 mg MOG peptide in triplicate for 4 days. The cells were labelled with BrdU, incubated with a peroxidase-conjugated anti-BrdU Fab fragment, and absorbance measured in an ELISA reader. ŽB. Induction of EAE in response to wild-type vs. b7yry lymphoblasts. EAE was induced in wild-type mice with 6=10 8 MOG35 – 55-stimulated wild-type Ždotted square. and b7yry lymphoblasts Žv .. EAE was induced in b7-deficient mice by injecting 6=10 8 MOG35 – 55-stimulated wild-type lymphoblasts ŽB.. UU Indicates a significant difference at p- 0.001 from wild-type groups of mice.
2e.. Leukocyte infiltration in mice receiving anti-a4 ŽFig. 2d., or anti-a4 plus anti-b7 ŽFig. 2f. mAbs was similar to Fig. 2. In vivo administration of anti-b7 and anti-a4 integrin subunit mAbs diminishes the accumulation of leukocytes in the CNS, and the expression of vascular addressins on cerebral blood vessels. Representative brain sections prepared from normal control mice Ža., EAE mice exhibiting tail, and fore and hind limb paralysis Žscore 6, 30 days after adoptive transfer. Žb., diseased mice treated with control rat IgG Žc., and from EAE animals treated with anti-a4 Žd., anti-b7 Že., or anti-a4 plus b7 Žf. subunit mAbs, were stained with haematoxylin and eosin. Sections c–f were obtained 77 days following adoptive transfer. Sections from the brains of EAE mice either untreated Žg, i, k; 30 days after adoptive transfer., or treated Žh, j, l; 77 days after adoptive transfer. with a combination of anti-a4 and anti-b7 mAbs were stained with an antiMAdCAM-1 Žg, h., anti-VCAM-1 Ži, j., and anti-ICAM-1 Žk, l. mAb. For sections g, i, k vessels not significantly occluded by infiltrated leukocytes were chosen for clarity; and hence the perivascular accumulation in these sections should not be regarded as a measure of the degree of inflammation. Sections b and c are shown at 60= magnification, while all others Ža, d–l. are at 100= magnification. All sections are either from the cerebellum or cortex. For staining of control sections, the first antibody was omitted Žm., or substituted with rat IgG Žn.; 60= magnification.
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that in healthy control mice. MAdCAM-1, VCAM-1, and ICAM-1 were all highly expressed at the peak of disease 30 days after adoptive ŽFig. 2g,i,k., whereas the expression of all three vascular addressins was dramatically downregulated in anti-a4 and anti-b7 mAb-treated mice by day 77 ŽFig. 2 h,j,l.. 3.3. AdoptiÕe transfer of wild-type MOG35 - 55-stimulated lymphoblasts induces an attenuated form of disease in b 7-deficient mice An attenuated form of EAE was induced in b7yry gene knockout mice following adoptive transfer of wild-type MOG35 – 55-stimulated lymphoblasts, where disease onset was delayed by 8 days compared to wild-type animals, and the mean clinical score at the peak of disease was reduced from 4.5 to 3 ŽFig. 3.. Administration of anti-a4 integrin antibodies alone to b7-deficient EAE mice led to rapid remission similar to that seen in wild-type animals receiving a combination of both anti-a4 and b7 mAbs ŽFig. 4.. 3.4. b 7 yry lymphocytes stimulated in Õitro with MOG35 - 55-peptide exhibit diminished encephalitogenicity Splenic lymphocytes recovered from b7yry gene knockout mice could be stimulated in vitro with MOG35 – 55-peptide, albeit to a lesser degree than their wild-type counterparts Ž45% reduction in proliferative capacity. ŽFig. 3a.. Their adoptive transfer into wild-type mice induced a severely attenuated form of EAE in which disease onset was delayed by 20 days, and disease severity reached a mean clinical score of only 2.25 ŽFig. 3b.. Histochemistry of brain sections of b7-deficient and wild-
Fig. 4. In vivo administration of an anti-a4 integrin subunit mAb causes rapid remission of EAE in b7-deficient mice. EAE was induced in b7-deficient mice by injecting 6=10 8 MOG35 – 55-stimulated wild-type encephalitogenic lymphoblasts. On days 21 Žclinical score of 3., 23, and 25 animals received i.v. and i.p. injections of PBS Ždotted square., rat IgG control ŽI., and anti-a4 subunit mAb ŽB.. UU Indicates a significant difference at p- 0.001 from the control groups of mice that received rat IgG.
type mice revealed as above that disease severity correlated with the degree of leukocyte infiltration Ždata not shown..
4. Discussion The data described herein indicate that b7 integrins play a contributory role in the establishment of a chronic non-remitting form of EAE induced in response to MOG35 – 55 , and suggest that both b7 and a4 integrins have a role to play in the disease. In this study, we sought to establish a relatively severe form of the disease in order to provide convincing data that anti-a4 and b7 antibody therapy was effective at causing the complete remission of crippling forms of EAE. Disease severity correlated with the numbers of encephalitogenic lymphoblasts administered Ždata not shown.. Many researchers administer 2.5 = 10 7 to 1 = 10 8 freshly stimulated lymph node cells, or long-term cultures of autoantigen-stimulated T cell lines ŽSelmaj et al., 1991; Baron et al., 1993; Mendel et al., 1995; Suen et al., 1997.. Often, the only consistent clinical symptoms of such regimes are a flaccid tail. This is particularly true for C57BLr6 mice where adoptive transfer of as many as 4 = 10 7 cells has been reported to cause only very mild clinical signs ŽSuen et al., 1997.. Several researchers administer pertussis toxin, or irradiate animals to exacerbate clinical symptoms. To avoid the employment of the latter strategies, particularly the former that affects cell migration pathways, we simply administered larger numbers of MOG-stimulated lymphoblasts. Smaller numbers of encephalitogenic T cell clones could have been prepared and used, but such clones are not representative of the diverse repertoire of T cell adhesive phenotypes. Antibody blocking experiments helped to define the relative contributions of a4 and b7 integrins, since anti-b7 mAbs target both a4b7 and a Eb7-expressing cells, whereas anti-a4 mAbs are directed against a4b7 and a4b1-expressing cells. Expression of a4b7 on lymphocytes is heterogeneous, and both b7 hi as well as b7yv e subsets exist ŽAndrew et al., 1996.. Encephalitogenic a4b1-expressing cells lacking b7-integrins would be expected to escape the inhibitory effects of anti-b7 mAbs, perhaps explaining the fact that anti-b7 mAbs attenuate the disease, but fail to cause complete remission. In contrast anti-a4 mAbs presumably cause complete remission by blocking both a4b1 and a4b7 receptors. Since coadministration of both anti-b7 and anti-a4 mAbs considerably shortened the time to remission over and above that achieved with anti-a4 mAb alone, it is possible that a Eb7 might also play a role. This notion is presently under investigation, in the light of the recent finding that Ecadherin has been found expressed on brain microvessel endothelial cells ŽPal et al., 1997.. Of interest, adoptive transfer of wild-type encephalitogenic lymphoblasts into
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b7-deficient mice induced an attenuated form of the disease, suggesting that b7 integrins may be important for the encephalitogenic functions of non-T leukocyte subsets such as granulocytes, and myeloid ŽCua et al., 1995. cells that facilitate the disease, andror for the recruitment of additional lymphocytes. We have previously shown that b7-integrins are expressed de novo on differentiating monocytesrmacrophages ŽYang et al., 1996.. All three leukocyte integrin ligands MAdCAM-1, VCAM-1, and ICAM-1 were highly expressed on brain microvessels at the height of disease severity, and were either absent or expressed at low levels in antibody-treated mice in remission. It was noticeable that MAdCAM-1 expression was not restricted to endothelia, but was also found on neighbouring cells. Whilst these MAdCAM-1 positive cells have not be identified, similar results have been reported elsewhere ŽCannella et al., 1991; O’Neill et al., 1991.. Cannella et al. Ž1991. noted that some astrocytes surrounding blood vessels stained with the antiMAdCAM-1 mAb MECA-89. MAdCAM-1 has also been detected on dendritic cells ŽSzabo et al., 1997., and choroid plexus epithelium ŽSteffen et al., 1996.. The b7 integrins could potentially act at various stages of the immune response including antigen presentation during epitope spreading, which occurs in MOG-induced disease ŽBernard et al., 1997.. It should be remembered that a4b7 ŽLehnert et al., 1998. and a Eb7 ŽBerg et al., 1999. mediate T cell costimulation, and their ligands VCAM-1 ŽKoopman et al., 1991., MAdCAM-1 ŽSzabo et al., 1997. and E-cadherin ŽEbner et al., 1998. are all expressed on dendritic cells. This notion seems unlikely given the demonstration that administration of anti-b7 integrin antibodies prior to the development of clinical signs of disease has no inhibitory effect on disease progression ŽEngelhardt et al., 1998.. However, it was noted that b7yry lymphocytes did not proliferate to the same extent as wild-type lymphocytes in response to MOG peptide, indicating that b7 integrins may be involved in T cell costimulation during antigen presentation. Alternatively, both a4 and b7 integrins may be required to allow transmigration of different encephalitogenic leukocyte subpopulations ŽT and non-T. across the BBB, as previously proposed for a4b1. Whatever the mechanism, effective therapies designed to treat chronic non-remitting forms of inflammatory diseases of the CNS may need to target multiple adhesion pathways, including those involving the b7 integrins.
Acknowledgements This work was supported in part by grants from the Multiple Sclerosis Society of New Zealand, the Royal Society of New Zealand, the Health Research Council of New Zealand, and The Lottery Grants Board of New Zealand. G.W.K. was a recipient of a Wellcome ŽUK.
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Koopman, G., Parmentier, H.K., Schuurman, H.J., Newman, W., Meijer, C.J., Pals, S.T., 1991. Adhesion of human B cells to follicular dendritic cells involves both the lymphocyte function-associated antigen 1rintercellular adhesion molecule 1 and very late antigen 4rvascular cell adhesion molecule 1 pathways. J. Exp. Med. 173, 1297–1304. Krissansen, G.W., Print, C.G., Prestidge, R.L., Hollander, D., Yuan, Q., Jiang, W.-M., Jenkins, D.R., Leung, E., Mead, P., Yong, R., Ameratunga, R.V., Cerf-Bensussan, N., Watson, J.D., 1992. Immunologic and structural relatedness of the integrin b7 complex and the human intraepithelial lymphocyte antigen HML-1. FEBS Lett. 296, 25–28. Kuhlman, P., Moy, V.T., Lollo, B.A., Brian, A.A., 1991. The accessory function of murine intercellular adhesion molecule-1 in T lymphocyte activation. J. Immunol. 146, 1773–1782. Lehnert, K., Print, C.G., Yang, Y., Krissansen, G.W., 1998. Mucosal addressin cell adhesion molecule-1 ŽMAdCAM-1. costimulates T cell proliferation exclusively through integrin a4b7 ŽLPAM-1., whereas VCAM-1 and CS-1 peptide use a4b1 ŽVLA-4.: Evidence for ‘‘remote’’ costimulation and induction of hyperresponsiveness to B7 molecules. Eur. J. Immunol. 28, 3605–3615. Martin, R., McFarland, H.F., 1995. Immunological aspects of experimental allergic encephalomyelitis and multiple sclerosis. Crit. Rev. Clin. Lab. Sci. 32, 121–182. Mendel, I., Kerlero de Rosbo, N., Ben-Nun, A., 1995. A myelin oligodendrocyte glycoprotein peptide induces typical chronic experimental autoimmune encephalomyelitis in H-2b mice: fine specificity and T cell receptor V beta expression of encephalitogenic T cells. Eur. J. Immunol. 25, 1951–1959. Miyake, K., Weissman, L.I., Greenberger, J.S., Kincade, P.W., 1991. Evidence for a role of the integrin VLA-4 in lympho-hemopoiesis. J. Exp. Med. 173, 599–607. O’Neill, J.K., Butter, C., Baker, D., Gschmeissner, S.E., Kraal, G., Butcher, E.C., Turk, J.L., 1991. Expression of vascular addressins and ICAM-1 by endothelial cells in the spinal cord during chronic relapsing experimental allergic encephalomyelitis in the Biozzi ABrH mouse. Immunology 72, 520–525. Pal, D., Audus, K.L., Siahaan, T.J., 1997. Modulation of cellular adhesion in bovine brain microvessel endothelial cells by a decapeptide. Brain Res. 747, 103–113. Parker, C.M., Cepek, K.L., Russell, G.J., Shaw, S.K., Posnett, D.N., Schwarting, R., Brenner, M.B., 1992. A family of b7 integrins on human mucosal lymphocytes. Proc. Natl. Acad. Sci. U.S.A. 89, 1924–1928. Raine, C.S., 1991. Multiple sclerosis: a pivotal role for the T cell in lesion development. Neuropathol. Appl. Neurobiol. 17, 265–274.
Raine, C.S., 1994. The Dale E. McFarlin Memorial Lecture: the immunology of the multiple sclerosis lesion. Ann. Neurol. 36, S61–72. Selmaj, K., Raine, C.S., Cross, A.H., 1991. Anti-tumor necrosis factor therapy abrogates autoimmune demyelination. Ann. Neurol. 30, 694– 700. Selmaj, K., Walczak, A., Mycko, M., Berkowicz, T., Kohno, T., Raine, C.S., 1998. Suppression of experimental autoimmune encephalomyelitis with a TNF binding protein ŽTNFbp. correlates with down-regulation of VCAM-1rVLA-4. Eur. J. Immunol. 28, 2035–2044. Steffen, B.J., Breier, G., Butcher, E.C., Schulz, M., Engelhardt, B., 1996. ICAM-1, VCAM-1, and MAdCAM-1 are expressed on choroid plexus epithelium but not endothelium and mediate binding of lymphocytes in vitro. Am. J. Pathol. 148, 1819–1838. Streeter, P.R., Berg, E.L., Rouse, B.T., Bargatze, R.F., Butcher, E.C., 1988. A tissue-specific endothelial cell molecule involved in lymphocyte homing. Nature 331, 41–46. Suen, W.E., Bergman, C.M., Hjelmstrom, P., Ruddle, N.H., 1997. A critical role for lymphotoxin in experimental allergic encephalomyelitis. J. Exp. Med. 186, 1233–1240. Szabo, M.C., Butcher, E.C., McEvoy, L.M., 1997. Specialization of mucosal follicular dendritic cells revealed by mucosal addressin-cell adhesion molecule-1 display. J. Immunol. 158, 5584–5588. Wagner, N., Lohler, J., Kunkel, E.J., Ley, K., Leung, E., Krissansen, G., Rajewsky, K., Muller, W., 1996. Critical role for b7 integrins in the formation of the gut associated lymphoid tissue. Nature 382, 366–370. Yang, X., Sytwn, H.-K., McDevitt, H.O., Michie, S.A., 1997. Involvement of b7 integrin and mucosal addressin cell adhesion molecule-1 ŽMAdCAM-1. in the development of diabetes in nonobese diabetic mice. Diabetes 46, 1542–1547. Yang, Y., Sammar, M., Harrison, J.E.B., Lehnert, K., Print, C.G., Leung, E., Prestidge, R., Krissansen, G.W., 1995. Construction and adhesive properties of a soluble MAdCAM-1-Fc chimera expressed in a baculovirus system: phylogenetic conservation of receptor-ligand interaction. Scand. J. Immunol. 42, 235–247. Yang, Y., Harrison, J.E.B., Print, C.G., Lehnert, K., Sammar, M., Lazarovits, A., Krissansen, G.W., 1996. Interaction of monocytoid cells with the mucosal addressin MAdCAM-1 via the integrins VLA-4 and LPAM-1. Immunol. Cell Biol. 74, 383–393. Yednock, T.A., Cannon, C., Fritz, L.C., Sanchez-Madrid, F., Steinman, L., Karin, N., 1992. Prevention of experimental autoimmune encephalomyelitis by antibodies against a4b1 integrin. Nature 356, 63–66. Yuan, Q., Jiang, W.-M., Leung, E., Hollander, D., Watson, J.D., Krissansen, G.W., 1992. Molecular cloning of the mouse integrin b7 subunit. J. Biol. Chem. 267, 7352–7358.
b7 integrins contribute to demyelinating disease of the central nervous system Jagat R. Kanwar a , Jane E.B. Harrison a , Dongmao Wang a , Euphemia Leung a , Werner Mueller b, Norbert Wagner b, Geoffrey W. Krissansen a,) a
Department of Molecular Medicine, School of Medicine and Health Science, UniÕersity of Auckland, Auckland, New Zealand b Institute for Genetics, UniÕersity of Cologne, 50937 Cologne, Germany Received 24 May 1999; received in revised form 18 August 1999; accepted 29 October 1999
Abstract A role for a4 integrins in different forms of the multiple sclerosis-like disease experimental autoimmune encephalomyelitis ŽEAE. has been demonstrated, but the individual contributions of a4b1, a4b7, and the related a Eb7 integrin have not been determined. The b7 integrins a4b7 and a Eb7 play a central role in chronic inflammation, mediating the trafficking, entry, andror adhesion of lymphocytes in the inflamed pancreas and gut, and their ligands MAdCAM-1, VCAM-1 and E-cadherin are expressed on brain endothelial cells andror on microvessels in the inflamed central nervous system. Here, we show that an antibody directed against the b7 subunit greatly attenuates a non-remitting form of EAE, induced by adoptive transfer of myelin oligodendrocyte peptide ŽMOG35 – 55 .-stimulated T cells. Combinational treatment with both anti-b7 and a4 integrin subunit antibodies led to more rapid and complete remission than that obtained with anti-a4 antibody alone, potentially implicating a role for a Eb7 in disease progression. Remission correlated with the down-regulation of the vascular addressins VCAM-1, MAdCAM-1, and ICAM-1 on cerebral blood vessels. Attenuated forms of disease were induced by adoptive transfer of either wild-type encephalitogenic T cells to b7-deficient gene knockout mice, or of b7yry encephalitogenic T cells to wild-type recipients. The former finding indicates that b7qve recruited cells contribute to disease progression. Thus a4b1, a4b7, and a Eb7 integrins may all play a contributory role in the progression of chronic forms of demyelinating disease, and together with their ligands could represent potential targets for improved treatment of some forms of multiple sclerosis. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Experimental autoimmune encephalomyelitis; b7 integrins; Cell adhesion molecules; Lymphocyte traffic; Central nervous system; Multiple sclerosis
1. Introduction Experimental autoimmune encephalomyelitis ŽEAE. is a disease of the central nervous system ŽCNS. similar to multiple sclerosis, in which encephalitogenic leukocytes penetrate the blood–brain barrier ŽBBB. and damage the myelin sheath of nerve fibres ŽRaine, 1991, 1994; Cannella and Raine, 1995; Martin and McFarland, 1995; Brosnan and Raine, 1996.. Both diseases lead to impaired nerve conduction and paralysis, which varies from relapsingrremitting to chronic progressive. The pathways by which encephalitogenic T cells access the CNS have not been fully delineated, but clearly involve integrin cell adhesion
) Corresponding author. Tel.: q64-9-373-7599 ext. 6280; fax: q64-9373-7674; e-mail: [email protected]
molecules that mediate lymphocyte trafficking and recirculation. The b7 integrin subfamily of cell adhesion molecules consists of two members, a4b7 ŽHolzmann and Weissman, 1989; Yuan et al., 1992. and a Eb7 ŽKrissansen et al., 1992; Parker et al., 1992. whose expression is restricted to leukocytes. a4b7 mediates the adherence of lymphocytes to high endothelial venules ŽHEV. via its preferred ligand MAdCAM-1 ŽBerlin et al., 1993; Briskin et al., 1993; Yang et al., 1995., whereas a Eb7 mediates the adhesion of intraepithelial lymphocytes of the intestine to the intestinal epithelium via an interaction with Ecadherin ŽCepek et al., 1994; Karecla et al., 1995.. In gene knockout mice deficient in the b7 integrin subfamily, the gut-associated lymphoid tissue ŽGALT. is severely impaired due to a failure of b7yry lymphocytes to adhere to blood vessel walls at the site of transmigration into the
0165-5728r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 5 7 2 8 Ž 9 9 . 0 0 2 4 5 - 3
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GALT ŽWagner et al., 1996.. Further, in non-obese diabetic ŽNOD. mice, treatment with anti-b7 or antiMAdCAM-1 mAbs provides protection against the spontaneous development of diabetes and insulitis, presumably by blocking lymphocyte migration into the inflamed pancreas ŽYang et al., 1997.. Hence a4b7-MAdCAM-1 interactions play critical roles in allowing the entry of leukocytes into chronically inflamed tissues. A role for a4 integrins in the establishment of EAE was demonstrated by in vivo administration of anti-a4 integrin antibodies which diminished the paralysis associated with EAE ŽYednock et al., 1992; Baron et al., 1993.. Further, the suppressive effects of TNFbp on the development of EAE appears to correlate with down-regulation of VCAM1rVLA-4 ŽSelmaj et al., 1998.. Hence, a4 integrins play a crucial role in allowing encephalitogenic T cells to enter the brain, but the individual contributions of the two related a4 integrins a4b1 and a4b7 to the various stages of the different forms of EAE has not been determined. A recent study of a relapsing–remitting form of EAE induced by proteolipid protein in the SJL mice, revealed that b7 integrins play no role in the early stages of EAE ŽEngelhardt et al., 1998.. However, this finding does not rule out a role for b7 integrins in the chronic phase of non-remitting forms of the disease, as it is established that MAdCAM-1, the preferential ligand for a4b7, is predominantly expressed on specialized HEV formed at chronically inflamed sites ŽStreeter et al., 1988.. Further, Ecadherin has also been found expressed on brain microvessel endothelial cells ŽPal et al., 1997.. Since MAdCAM-1 expression is particularly noticeable on HEV-like endothelia in relapsing brain lesions of mice with EAE ŽCannella et al., 1991., we sought to show a role for b7 integrins in the chronic phase of a non-remitting form EAE.
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Sigma, USA, served as a control for the latter antibodies in in vivo blocking and immunohistological studies. 2.2. Encephalitogenic lymphocytes
2. Materials and methods
Encephalitogenic lymphocytes were obtained by subcutaneous immunization of 8-10 week old wild-type and b7yry C57BLr6 mice with 300 mg of MOG35 – 55 peptide ŽMEVGWYRSPFSRVVHLYRNGK. ŽMendel et al., 1995.; synthesized by Chiron Technologies, Clayton, Australia and emulsified in CFA containing 500 mg of Mycobacterium tuberculosis H37RA ŽDIFCO Laboratories, Detroit, USA.. Administration of pertussis toxin was not necessary, and was omitted. Two injections were given 1 week apart in opposite flanks, and 14 days later spleen and draining lymph nodes were removed, and a single cell suspension obtained by teasing the tissue through a stainless steel wire mesh. Cells were then washed three times in Hanks’ balanced salt solution ŽHBSS, GIBCO, Grand Island, NY, USA. and resuspended to a concentration of 6 = 10 6 cellsrml in complete medium consisting of RPMI 1640 ŽGIBCO BRL, Gaithersburg, MD, USA. supplemented with 1 mM L-glutamine, pencillin–streptomycin, 10% FCS, and 2-mercaptoethanol. Cells were fed with 50 Urml of IL-2 ŽR & D Systems, Minneapolis, MN, USA. and 30 mgrml MOG peptide antigen, and incubated in a humidified 378C, 10% CO 2 incubator. After 4 d of activation lymphoblasts were isolated by centrifugation on Ficoll-paque ŽPharmacia LKB Biotechnology AB, Uppsala, Sweden. gradients. Blasts from the interface were washed three times, and resuspended in complete medium. Cell viability as determined by trypan blue exclusion was ) 97%. EAE was transferred to both wild-type and b7yry C57BLr6 mice by injecting 6 = 10 8 MOG peptide-stimulated lymphocytes into the tail vein Ž70%. and intraperitoneally Ž30%..
2.1. Mice and antibodies
2.3. ProliferatiÕe response of lymphocytes to MOG peptide
C57BLr6 wild-type and b7yry gene knockout mice were bred and housed by the Animal Resource Unit, School of Medicine and Health Science, University of Auckland, Auckland, New Zealand. They were randomly bred on a C57BLr6 background. Rat hybridomas secreting mAbs against mouse integrin a4 subunit ŽPSr2 rat IgG2b mAb. ŽMiyake et al., 1991., b7 subunit ŽFIB 504.64 rat IgG2a mAb. ŽAndrew et al., 1994., VCAM-1 ŽMK1.9 rat IgG1 mAb. ŽMiyake et al., 1991., and ICAM-1 ŽBE29G1 rat IgG2a mAb. ŽKuhlman et al., 1991. were purchased from the American Type Culture Collection, Rockville, MD. The rat hybridoma MECA-367 Žrat IgG2a. ŽBerlin et al., 1993., which secretes a mAb against mouse MAdCAM-1, was kindly provided by Dr. Eugene Butcher, Stanford University, Stanford, CA. Rat IgG obtained from
The cell proliferation enzyme-linked immunosorbent assay ŽELISA. was done with a non-radioactive and colorimetric immunoassay kit ŽBoehringer Mannheim, Mannheim, Germany. to measure the incorporation of 5-bromo-2-deoxyuridine ŽBrdU.. In brief, lymphocytes from the spleens of wild-type and b7yry C57BLr6 mice were purified on Ficoll-paque ŽPharmacia LKB Biotechnology AB, Uppsala, Sweden.. Equal numbers of wild-type and b7yry cells Ž1 = 10 4 . were incubated in the presence or absence of 30 mg of MOG35 – 55 peptide in 96-well flat-bottomed plates ŽBecton Dickinson Labware, Frankin Lakes, NJ, USA. in triplicate for 4 days. The cells were labelled with BrdU for 25 h, and fixed for 30 min at room temperature. The plates were washed and incubated with peroxidase-conjugated anti-BrdU Fab fragment. After
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washing, substrate was added, the reaction stopped, and absorbance measured in an ELISA plate reader.
3. Results
2.4. EAE induction, and inhibition with antibodies
3.1. In ÕiÕo administration of anti-b 7 integrin antibodies diminishes the paralysis associated with EAE
MOG peptide-stimulated lymphoblasts were injected into the tail vein Ž70% of cells. and intraperitoneally Ž30% of cells. into mice, and animals were observed daily for clinical signs of EAE. The mice were scored according to the following scale: 0, no clinical signs of EAE; 1, limp tail; 2, partial hind limb paralysis; 3, complete hind limb paralysis; 4, complete hind limb and partial fore limb paralysis; 5, paralysis extending to diaphragm; 6, hind and fore limb paralysis; 7, death due to EAE. Animals were cared for according to the guidelines of the University of Auckland Animal Ethics Committee. They had to be maintained in a state of hind-limb paralysis, hence care was shown in the provision of drinking water, food, and comfort. The daily mean clinical score for each group is the mean disease score of at least five mice. For antibody blocking, anti-b7 ŽFib 504. and anti-a4 ŽPSr2. subunit mAbs were administered into the tail vein Ž70% of mAb. and intraperitoneally Ž30% of mAb. at 10 mgrkg separately or in combination three times on alternate days when the clinical score reached 3 or 4.5. Control groups of mice received either PBS or rat IgG ŽSigma, USA.. Experiments were repeated to confirm reproducibility.
EAE was induced in C57BLr6 mice by adoptive transfer of myelin oligodendrocyte glycoprotein peptide ŽMOG35 – 55 .-stimulated lymphoblasts ŽMendel et al., 1995., resulting in chronic sustained paralysis. The first signs of clinical disease ensued within 4 to 5 days leading to paralysis of the hind limbs by day 10 ŽFig. 1.. Marked perivascular accumulation of leukocytes was observed at the peak of the disease; whereas inflamed blood vessels were not detectable in the brains of control animals Žrefer to Fig. 2a,b.. Sixteen days after adoptive transfer of MOG35 – 55-stimulated encephalitogenic lymphoblasts when the clinical score reached 4.5 Žhind limb paralysis., animals were given intravenous Ži.v.. and intraperitoneal Ži.p.. injections of mAbs against the integrin b7 ŽFib504. and a4 ŽPSr2. subunits, or of a rat IgG control antibody. Anti-a4 integrin mAb caused complete remission of the disease in all animals, such that no clinical symptoms could be observed 50 days after antibody treatment was suspended ŽFig. 1.. Anti-b7 integrin mAb caused a more gradual remission of disease symptoms such that the clinical scores of animals were reduced from 4.5 to 2 after 50 days, compared to undiminished disease scores in untreated controls.
2.5. Immunohistology Brains were frozen in isopentane at y708C, and transverse 10 mm sections were made through the cerebrum, cerebellum, and mid-brain. Sections were mounted on poly L-lysine-coated slides, and endogenous peroxidase blocking was done by incubating slides for 30 min in 0.3% H 2 O 2 in methanol. The sections were stained with antiCAM mAbs using VECTASTAIN ABC Elite Peroxidase ŽRat IgG. Kit ŽVector Laboratories, Burlingame, CA, USA.. In brief, sections were washed with PBS and blocked with rabbit serum, and incubated with different anti-CAM mAbs. After three washings with PBS, slides were incubated with biotinylated anti-rat secondary antibody for 10 min. They were then washed, incubated with ABC peroxidase reagent, and developed with SIGMA FAST DAB Ž3,3X-diaminobenzidine tetrahydrochloride. with metal enhancer CoCl 2 tablets. Slides were counterstained with haematoxylin and eosin. Controls for the staining protocol consisted of omitting the primary anti-CAM mAb, or substituting it with rat IgG ŽSigma.. Representative sections are reported in the case of analysis of leukocyte infiltration, whereas for visualisation of CAMs we deliberately chose blood vessels lacking extensive perivascular accumulation of leukocytes for clarity.
Fig. 1. In vivo administration of an anti-b7 integrin subunit mAb causes gradual remission of EAE. EAE was induced in C57BLr6 mice by injecting 6=10 8 MOG35 – 55-stimulated encephalitogenic lymphoblasts. On days 16, 18, and 20 animals received i.v. and i.p. injections of PBS Ždotted square., rat IgG control ŽI., anti-b7 Ž'. and a4 ŽB. subunit mAbs, or a combination of anti-b7 and anti-a4 mAbs Žv .. UU Indicates a significant difference at p- 0.001 from control groups of mice that received rat IgG.
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3.2. Combination of anti-b 7 and anti-a 4 antibodies proÕides enhanced therapeutic benefit Simultaneous administration of both mAbs caused a dramatic reversal in disease progression, such that complete remission was observed in only 5 days following suspension of antibody treatment. Rat IgG control antibody had no affect on the onset or severity of disease. Histochemistry of brain sections from animals receiving the control antibody revealed extensive leukocyte infiltration ŽFig. 2c., whereas infiltration into the brains of EAE animals was lessened by treatment with anti-b7 mAb ŽFig.
Fig. 3. Integrin b7-deficient mice are partially resistant to EAE, and generate lymphocytes that respond less well to MOG-peptide and are poorly encephalitogenic. ŽA. Proliferative response of wild-type and b7yry lymphocytes to MOG35 – 55-peptide. Ficoll-paque-purified lymphocytes from the spleens of wild-type and b7yry C57BLr6 mice were incubated in the presence or absence of 30 mg MOG peptide in triplicate for 4 days. The cells were labelled with BrdU, incubated with a peroxidase-conjugated anti-BrdU Fab fragment, and absorbance measured in an ELISA reader. ŽB. Induction of EAE in response to wild-type vs. b7yry lymphoblasts. EAE was induced in wild-type mice with 6=10 8 MOG35 – 55-stimulated wild-type Ždotted square. and b7yry lymphoblasts Žv .. EAE was induced in b7-deficient mice by injecting 6=10 8 MOG35 – 55-stimulated wild-type lymphoblasts ŽB.. UU Indicates a significant difference at p- 0.001 from wild-type groups of mice.
2e.. Leukocyte infiltration in mice receiving anti-a4 ŽFig. 2d., or anti-a4 plus anti-b7 ŽFig. 2f. mAbs was similar to Fig. 2. In vivo administration of anti-b7 and anti-a4 integrin subunit mAbs diminishes the accumulation of leukocytes in the CNS, and the expression of vascular addressins on cerebral blood vessels. Representative brain sections prepared from normal control mice Ža., EAE mice exhibiting tail, and fore and hind limb paralysis Žscore 6, 30 days after adoptive transfer. Žb., diseased mice treated with control rat IgG Žc., and from EAE animals treated with anti-a4 Žd., anti-b7 Že., or anti-a4 plus b7 Žf. subunit mAbs, were stained with haematoxylin and eosin. Sections c–f were obtained 77 days following adoptive transfer. Sections from the brains of EAE mice either untreated Žg, i, k; 30 days after adoptive transfer., or treated Žh, j, l; 77 days after adoptive transfer. with a combination of anti-a4 and anti-b7 mAbs were stained with an antiMAdCAM-1 Žg, h., anti-VCAM-1 Ži, j., and anti-ICAM-1 Žk, l. mAb. For sections g, i, k vessels not significantly occluded by infiltrated leukocytes were chosen for clarity; and hence the perivascular accumulation in these sections should not be regarded as a measure of the degree of inflammation. Sections b and c are shown at 60= magnification, while all others Ža, d–l. are at 100= magnification. All sections are either from the cerebellum or cortex. For staining of control sections, the first antibody was omitted Žm., or substituted with rat IgG Žn.; 60= magnification.
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that in healthy control mice. MAdCAM-1, VCAM-1, and ICAM-1 were all highly expressed at the peak of disease 30 days after adoptive ŽFig. 2g,i,k., whereas the expression of all three vascular addressins was dramatically downregulated in anti-a4 and anti-b7 mAb-treated mice by day 77 ŽFig. 2 h,j,l.. 3.3. AdoptiÕe transfer of wild-type MOG35 - 55-stimulated lymphoblasts induces an attenuated form of disease in b 7-deficient mice An attenuated form of EAE was induced in b7yry gene knockout mice following adoptive transfer of wild-type MOG35 – 55-stimulated lymphoblasts, where disease onset was delayed by 8 days compared to wild-type animals, and the mean clinical score at the peak of disease was reduced from 4.5 to 3 ŽFig. 3.. Administration of anti-a4 integrin antibodies alone to b7-deficient EAE mice led to rapid remission similar to that seen in wild-type animals receiving a combination of both anti-a4 and b7 mAbs ŽFig. 4.. 3.4. b 7 yry lymphocytes stimulated in Õitro with MOG35 - 55-peptide exhibit diminished encephalitogenicity Splenic lymphocytes recovered from b7yry gene knockout mice could be stimulated in vitro with MOG35 – 55-peptide, albeit to a lesser degree than their wild-type counterparts Ž45% reduction in proliferative capacity. ŽFig. 3a.. Their adoptive transfer into wild-type mice induced a severely attenuated form of EAE in which disease onset was delayed by 20 days, and disease severity reached a mean clinical score of only 2.25 ŽFig. 3b.. Histochemistry of brain sections of b7-deficient and wild-
Fig. 4. In vivo administration of an anti-a4 integrin subunit mAb causes rapid remission of EAE in b7-deficient mice. EAE was induced in b7-deficient mice by injecting 6=10 8 MOG35 – 55-stimulated wild-type encephalitogenic lymphoblasts. On days 21 Žclinical score of 3., 23, and 25 animals received i.v. and i.p. injections of PBS Ždotted square., rat IgG control ŽI., and anti-a4 subunit mAb ŽB.. UU Indicates a significant difference at p- 0.001 from the control groups of mice that received rat IgG.
type mice revealed as above that disease severity correlated with the degree of leukocyte infiltration Ždata not shown..
4. Discussion The data described herein indicate that b7 integrins play a contributory role in the establishment of a chronic non-remitting form of EAE induced in response to MOG35 – 55 , and suggest that both b7 and a4 integrins have a role to play in the disease. In this study, we sought to establish a relatively severe form of the disease in order to provide convincing data that anti-a4 and b7 antibody therapy was effective at causing the complete remission of crippling forms of EAE. Disease severity correlated with the numbers of encephalitogenic lymphoblasts administered Ždata not shown.. Many researchers administer 2.5 = 10 7 to 1 = 10 8 freshly stimulated lymph node cells, or long-term cultures of autoantigen-stimulated T cell lines ŽSelmaj et al., 1991; Baron et al., 1993; Mendel et al., 1995; Suen et al., 1997.. Often, the only consistent clinical symptoms of such regimes are a flaccid tail. This is particularly true for C57BLr6 mice where adoptive transfer of as many as 4 = 10 7 cells has been reported to cause only very mild clinical signs ŽSuen et al., 1997.. Several researchers administer pertussis toxin, or irradiate animals to exacerbate clinical symptoms. To avoid the employment of the latter strategies, particularly the former that affects cell migration pathways, we simply administered larger numbers of MOG-stimulated lymphoblasts. Smaller numbers of encephalitogenic T cell clones could have been prepared and used, but such clones are not representative of the diverse repertoire of T cell adhesive phenotypes. Antibody blocking experiments helped to define the relative contributions of a4 and b7 integrins, since anti-b7 mAbs target both a4b7 and a Eb7-expressing cells, whereas anti-a4 mAbs are directed against a4b7 and a4b1-expressing cells. Expression of a4b7 on lymphocytes is heterogeneous, and both b7 hi as well as b7yv e subsets exist ŽAndrew et al., 1996.. Encephalitogenic a4b1-expressing cells lacking b7-integrins would be expected to escape the inhibitory effects of anti-b7 mAbs, perhaps explaining the fact that anti-b7 mAbs attenuate the disease, but fail to cause complete remission. In contrast anti-a4 mAbs presumably cause complete remission by blocking both a4b1 and a4b7 receptors. Since coadministration of both anti-b7 and anti-a4 mAbs considerably shortened the time to remission over and above that achieved with anti-a4 mAb alone, it is possible that a Eb7 might also play a role. This notion is presently under investigation, in the light of the recent finding that Ecadherin has been found expressed on brain microvessel endothelial cells ŽPal et al., 1997.. Of interest, adoptive transfer of wild-type encephalitogenic lymphoblasts into
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b7-deficient mice induced an attenuated form of the disease, suggesting that b7 integrins may be important for the encephalitogenic functions of non-T leukocyte subsets such as granulocytes, and myeloid ŽCua et al., 1995. cells that facilitate the disease, andror for the recruitment of additional lymphocytes. We have previously shown that b7-integrins are expressed de novo on differentiating monocytesrmacrophages ŽYang et al., 1996.. All three leukocyte integrin ligands MAdCAM-1, VCAM-1, and ICAM-1 were highly expressed on brain microvessels at the height of disease severity, and were either absent or expressed at low levels in antibody-treated mice in remission. It was noticeable that MAdCAM-1 expression was not restricted to endothelia, but was also found on neighbouring cells. Whilst these MAdCAM-1 positive cells have not be identified, similar results have been reported elsewhere ŽCannella et al., 1991; O’Neill et al., 1991.. Cannella et al. Ž1991. noted that some astrocytes surrounding blood vessels stained with the antiMAdCAM-1 mAb MECA-89. MAdCAM-1 has also been detected on dendritic cells ŽSzabo et al., 1997., and choroid plexus epithelium ŽSteffen et al., 1996.. The b7 integrins could potentially act at various stages of the immune response including antigen presentation during epitope spreading, which occurs in MOG-induced disease ŽBernard et al., 1997.. It should be remembered that a4b7 ŽLehnert et al., 1998. and a Eb7 ŽBerg et al., 1999. mediate T cell costimulation, and their ligands VCAM-1 ŽKoopman et al., 1991., MAdCAM-1 ŽSzabo et al., 1997. and E-cadherin ŽEbner et al., 1998. are all expressed on dendritic cells. This notion seems unlikely given the demonstration that administration of anti-b7 integrin antibodies prior to the development of clinical signs of disease has no inhibitory effect on disease progression ŽEngelhardt et al., 1998.. However, it was noted that b7yry lymphocytes did not proliferate to the same extent as wild-type lymphocytes in response to MOG peptide, indicating that b7 integrins may be involved in T cell costimulation during antigen presentation. Alternatively, both a4 and b7 integrins may be required to allow transmigration of different encephalitogenic leukocyte subpopulations ŽT and non-T. across the BBB, as previously proposed for a4b1. Whatever the mechanism, effective therapies designed to treat chronic non-remitting forms of inflammatory diseases of the CNS may need to target multiple adhesion pathways, including those involving the b7 integrins.
Acknowledgements This work was supported in part by grants from the Multiple Sclerosis Society of New Zealand, the Royal Society of New Zealand, the Health Research Council of New Zealand, and The Lottery Grants Board of New Zealand. G.W.K. was a recipient of a Wellcome ŽUK.
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