Proc. Natl. Acad. Sci. USA Vol. 91, pp. 12604-12608, December 1994 Immunology A predominant role of integrin a!4 in the spontaneous development of autoimmune diabetes in nonobese diabetic mice (lymphocyte/L-selectin/) XIAO-DONG YANG*t, SARA A. MICHIEt§, ROLAND TISCH*, NATHAN KARIN1, LAWRENCE STEINMAN1, AND HUGH 0. McDEVITT*II Departments of *Microbiology and Immunology, tPathology, ¶Neurology, and IlMedicine, Stanford University School of Medicine, Palo Alto, CA 94305-5402; and §Department of Veterans Affairs, Center for Molecular Biology, Palo Alto, CA 94304 Contributed by Hugh 0. McDevitt, September 20, 1994

ABSTRACT To elucidate the role of mole- damage and destruction in organ-specific autoimmune dis- cules in the pathogenesis of insulin-dependent diabetes mellitus ease. For IDDM, lymphocytic infiltration of the islets (insu- and to determine the predominant lymphocytic homing path- litis) is a prerequisite for the development of diabetes. Thus, way(s) involved in the selective lymphocytic infiltration of a better understanding of lymphocyte trafficking to the islets pancreatic islets (insulitis), nonobese diabetic mice were treated may be helpful in gaining insight into the events linked to with monoclonal antibodies specific for the L-selectin and disease pathogenesis and, more importantly, to develop an integrin a4 lymphocyte adhesion molecules. Treatment of anti- (CAM)-based therapy for treat- neonatal mice with either anti-L-selectin or anti-integrin aN ment of IDDM in patients. monoclonal antibodies for the first 4 weeks of life led to a We have demonstrated (2) that blocking L-selectin or significant and long-term protection against spontaneous oc- integrin a4 lymphocyte adhesion molecules in nonobese currence of insulitis and diabetes. The same treatment failed to diabetic (NOD) mice leads to partial or complete protection, inhibit lymphocytic infiltration of the salivary glands (sialad- respectively, against the development of diabetes in an enitis). This tissue-specific inhibition of inflammation may be adoptive transfer model. In contrast, anti-integrin A32 treat- attributed to differences between the pancreas and salivary ment does not prevent the onset of disease (3). This suggests gland in their expression of endothelial ligands for L-selectin that L-selectin and integrin a4 may be involved in lymphocyte (peripheral vascular addressin) and for integrin at4 (mucosal homing to the islets. This notion has been recently confirmed addressin cell adhesion molecule 1 and vascular cell adhesion by other investigators (4, 5). Nevertheless, with regard to the molecule 1). Mucosal addressin cell adhesion molecule 1 is spontaneous development ofIDDM, it is critical to determine highly expressed by vessels within the inflamed islets but was (i) which adhesion pathway(s) is essential for spontaneous not detected in the salivary glands. In contrast, peripheral disease progression, (ii) whether blockade of L-selectin or vascular addressin- and vascular cell adhesion molecule 1-ex- integrin a4 is sufficient to stop the disease after its onset, and pressing vessels can be found in almost (iii) the duration and the time at which the treatment should every area of inflam- be applied to achieve a significant therapeutic effect. In the mation within the salivary glands but are seen in only 40-50% present study, we have addressed these issues by adminis- of inflamed islets. Anti-L-selectin and anti-integrin a4 treat- tering anti-L-selectin and anti-integrin a4 monoclonal anti- ment had no demonstrable effect on anti-f-cell autoimmunity bodies (mAbs) at early or late stages of the spontaneous or on the immune responses to foreign antigens. Therapeutic disease process and by determining the expression of the treatment with anti-L-selectin after the onset of insulitis from endothelial CAMs in the islets and other lymphoid and 10 to 14 weeks of age delayed the onset but failed to prevent extralymphoid organs. spontaneous insulin-dependent diabetes mellitus, whereas anti- integrin a4 treatment resulted in a significant and long-lasting suppression of the disease. These data strongly suggest that MATERIALS AND METHODS integrin a4 plays a prominent role in the spontaneous devel- Mice. NOD/McD mice were bred and maintained at the opment of insulitis and diabetes in nonobese diabetic mice. Stanford University Animal Facility under barrier isolation conditions. In our NOD colony, the frequency of overt Insulin-dependent diabetes mellitus (IDDM) is the result of diabetes reaches 70-80% in females and 10-20% in males by organ-specific autoimmune destruction of the insulin- the age of 30 weeks. secreting /8 cells in the pancreatic islets of Langerhans. It has Treatment Regimen with Anti-Lymphocyte Adhesion Mol- become evident that IDDM is a multifactorial disease medi- ecule mAbs. Hybridomas that produce mAbs utilized in this ated by T cells in which both CD4+ and CD8+ T cells are study were purchased from American Type Culture Collec- required for ,-cell destruction (1). A great deal of effort has tion. mAbs Mel-14 (anti-L-selectin), R1-2 (anti-integrin a4), focused on the determination of genetic and environmental and immunoglobulin-isotype-matched controls M1/9.3.17 factors that trigger the diabetogenic response and the precise and M1/89.18.7 (anti-leukocyte common antigen) were pre- role autoreactive T cells play in mediating the diabetogenic pared using G affinity chromatography (Pharmacia) process. An issue that has only very recently been addressed as described (2). NOD mice from separate litters were is the mechanism by which autoreactive and effector lym- randomized before treatment. Two regimens were applied: (i) phocytes migrate to and enter target organs. Adherence oflymphocytes and other inflammatory cells to Abbreviations: IDDM, insulin-dependent diabetes mellitus; NOD, vascular endothelium, followed by transendothelial migra- nonobese diabetic; mAb, monoclonal antibody; PNAd, peripheral tion into the target organ, is essential for eventual tissue vascular addressin; CAM, cell adhesion molecule; MAdCAM-1, mucosal addressin CAM 1; VCAM-1, vascular CAM 1; OVA, ovalbumin; CPH, carboxypeptidase H; GAD65, 65-kDa isoform of The publication costs of this article were defrayed in part by page charge glutamic acid decarboxylase; HSP60, 60-kDa heat shock protein; payment. This article must therefore be hereby marked "advertisement" LN, lymph node; ICAM-1, intercellular CAM 1. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed.

12604 Downloaded by guest on September 28, 2021 Immunology: Yang et al. Proc. Natl. Acad. Sci. USA 91 (1994) 12605 Newborn mice were treated i.p. with 30 ,l of PBS only or 100 with Mel-14, R1-2, and control mAbs (M1/9.3.17 and M1/ 90 0 0 - 89.18.7) [each at 80 ,ug/g (body weight)] every other day from the first day after birth for 4 weeks. (ii) Ten-week-old female 80 mice were injected i.p. with Mel-14, R1-2, or control mAbs 70 (each at 1 mg) every other day for 4 weeks. '60 Histopathology. Histopathology for examination and eval- uation ofinsulitis and sialadenitis was performed as described 0~50 (6). rA40 ELISA for Determination of Anti-fl-Cell Autoantibody. An . 30 antigen-specific ELISA for determining autoantibody re- sponse to a panel of murine /-islet-cell antigens including : 20 carboxypeptidase H (CPH), the 65-kDa isoform of glutamic 10 acid decarboxylase (GAD65), 60-kDa murine heat shock 0 protein (HSP60), and peripherin was carried out as described 10 15 20 25 30 35 40 45 (7). Age (weeks) Determination of the Immune Response to Ovalbumin (OVA). Immune response to OVA was examined as de- FIG. 1. Prevention of spontaneous development of IDDM in scribed (6). NOD mice by anti-L-selectin or anti-integrin a4 treatment. Newborn Immunohistology. Pancreata, salivary glands, spleens, pe- female NOD mice were treated i.p. with PBS (o, n = 10), Mel-14 (A, n = 12), R1-2 (n, n = 16), M1/9.3.4 (A, n = 5), or M1/89.18.7 (n, n ripheral and mesenteric lymph nodes (LNs), and Peyer's = 5) for 4 weeks. Diabetes was determined by measuring glycouria patches were frozen, cut, and stained with a panel of mAbs biweekly and presented as diabetes incidence: number of diabetic using a three-step immunoperoxidase technique as described mice/total number of mice within the same treatment group. (8). mAbs included Mel-14, R1-2, MECA-79 [anti-peripheral vascular addressin (PNAd)], MECA-367 [anti-mucosal ad- mice, but in only 60% of Mel-14-treated and 57% of R1-2- dressin CAM 1 (MAdCAM-1)], MK-2.7 [anti-vascular CAM treated mice. Furthermore, in animals with insulitis, the 1 (VCAM-1)], 53-2.1 (anti-Thyl.2), 53-6.7 (anti-CD8), GK1.5 extent of inflammation in the islets of Mel-14- and R1-2- (anti-CD4), and RA3-6B2 (anti-CD45R/B220). MECA-79 and treated mice was markedly less than that seen in PBS-treated MECA-367 were generously provided by E. C. Butcher mice. Interestingly, the incidence of sialadenitis did not (Stanford University). MK2.7 and RA3-6B2 were kindly appear to be significantly affected by Mel-14 or R1-2 treat- provided by P. Kincade (University of Oklahoma) and by R. ment in the same mice (Table 1), suggesting that these Coffman (DNAX), respectively. antibodies affect lymphocyte homing to sites ofinflammation in a tissue-selective manner. RESULTS To further study the tissue-specific effect ofanti-L-selectin and anti-integrin a4 treatment, we examined the expression of Treatment of Neonatal NOD Mice with Mel-14 or Rl-2 various endothelial adhesion molecules in pancreata, sub- Prevents Development of Spontaneous IDDM. Insulitis can be mandibular salivary glands, and other lymphoid tissues. seen histologically in the pancreata ofmost NOD mice by 4-5 weeks of age (9), suggesting that the initial few weeks of life These molecules include PNAd (a ligand for L-selectin) (10, are critical for the development oflymphocytic infiltration of 11), MAdCAM-1 (a ligand for integrin a437) (12, 13), and the islets. This initial inflammation in turn triggers a subse- VCAM-1 (a ligand for integrin a4,81) (14). As illustrated in Fig. quent cascade of immunopathologic events leading to /-cell 2, MAdCAM-1 expression was found on vascular endothe- destruction. To verify this notion, newborn NOD mice were lium within or adjacent to every infiltrated islet (100%). In treated i.p. with PBS, Mel-14, R1-2, M1/9.3.17, and Ml/ contrast, no MAdCAM-1 expression was seen in the inflamed 89.18.7 from the first day after birth every other day for 4 salivary glands (0%). PNAd and VCAM-1 were expressed by weeks. The mice were then followed for the spontaneous vascular endothelium in areas of inflammation in both pan- onset ofdiabetes. Fig. 1 illustrates that all female mice treated creas and salivary gland. In the salivary gland, almost every with R1-2 remained completely free of diabetes. In addition area of inflammation contained several vessels expressing anti-L-selectin treatment led to a significant and long-lasting these two adhesion molecules (=100%), whereas only 40- protection from IDDM (30 vs. 85% diabetes in control group 50% of inflamed islets contained PNAd- or VCAM-1- at 42 weeks of age). In contrast, mice treated with control expressing vessels. No observable difference in the expres- mAbs developed IDDM with an incidence similar to that seen sion of MAdCAM-1, PNAd, and VCAM-1 by vascular en- for PBS-treated animals, suggesting that the protective effect dothelium in LNs and Peyer's patches was detected in PBS-, observed with the Mel-14 and R1-2 mAbs is specific. To Mel-14-, or R1-2-treated mice (data not shown). Therefore, determine whether administering Mel-14 and R1-2 mAbs our data suggest that autoreactive lymphocytes may primar- results in the depletion of L-selectin- or integrin a4- ily utilize an a43--MAdCAM-1 adhesion pathway to enter the expressing cells, respectively, mononuclear cells were ana- islets whereas sialadenitis is mediated by specific interactions lyzed by immunofluorescence staining and flow cytometry of a4/31-VCAM-1 and/or L-selectin-PNAd and that the tis- and by immunohistological examination offrozen sections of sue specificity of the homing process may be influenced by spleen, LNs, and Peyer's patches. No significant deletion distinct patterns of expression of endothelial adhesion mol- was observed in animals treated with Mel-14, R1-2, or control ecules in different organs. mAbs (data not shown). Baron et al. (4) have shown that integrin a4 preferentially Anti-L-Selectin- or Anti-Integrin <4-Mediated Protection mediates homing ofCD4+ effector cells to the islets, whereas May Result from Selective Inhibition of Lymphocytic Infiltra- anti-integrin a4 treatment has no effect on the entry of CD8+ tion into the Pancreatic Islets. To determine whether anti-L- T cells. To further address this issue, we analyzed the selectin and anti-integrin a4 treatment is indeed inhibiting proportions ofinfiltrating CD4+ and CD8+ T cells and B cells lymphocyte accumulation in the islets, we examined histo- in the islets and salivary glands. In mice that were treated logic sections of pancreata from 42-week-old NOD mice that with Mel-14 or R1-2 and displayed insulitis and sialadenitis, had been treated with mAbs from birth to 4 weeks of age. As no significant skewing toward a particular subset of lympho- demonstrated in Table 1, insulitis was seen in all PBS-treated cytes was found in situ (data not shown). Downloaded by guest on September 28, 2021 12606 Immunology: Yang et al. Proc. Natl. Acad. Sci. USA 91 (1994) Table 1. Treatment with Mel-14 (anti-L-selectin) or R1-2 (anti-integrin c4) inhibits development of insulitis but not sialadenitis Insulitis incidence, Sialadenitis severity, no. of mice no. of mice afflicted/ Insulitis severity, no. of islets afflicted/total no. of islets (%) afflicted/total no. of mice Treatment total no. of mice(%) - + ++ +++ - + ++ +++ +++ PBS 3/3 (100) 26/57 (45.6) 6/57 (10) 7/57 (12.3) 18/57 (31.6) 0/3 0/3 3/3 0/3 0/3 Mel-14 3/5 (60) 112/139 (80.8) 7/139 (5.0) 10/139 (7.1) 10/139 (7.1) 0/5 4/5 1/5 0/5 0/5 R1-2 4/7 (57) 169/184 (92.0) 11/184 (5.9) 4/184 (2.1) 0/184 (0) 0/8 4/8 4/8 0/8 0/8 Newborn NOD mice were treated i.p. with PBS, Mel-14, or R1-2 for 4 weeks. The diabetes-protected animals were sacrificed at 42 weeks of age and the histopathology of the islets and salivery glands was analyzed. Insulitis was scored by incidence and severity. A minimum of 13 islets per individual was examined. Insulitis was graded as described (2): -, no lymphocytic infiltration; +, few lymphocytes surrounding the islets; + +, many surrounding lymphocytes, + + +, intraislet lymphocyte infiltration and accumulation. Severity of sialadenitis was scored as follows: -, no inflammation; +, small foci of perivascular inflammation; + +, small foci of periductular/periacinar inflammation; + + +, large foci periductular/periacinar inflammation. Treatment with Anti-L-Selectin or Anti-Integrin a4 mAbs were enumerated and assayed in vitro for proliferative re- Does Not Block the Immune Responses to Self or Foreign sponses to OVA. Relative to PBS-treated animals, the num- Antigens. One ofthe major concerns ofusing anti-CAM mAb bers of lymphocytes found in the draining LNs of Mel-14- or as a therapy is the depletion of immune competent cells and Rl-2-treated mice were markedly reduced by 87 and 72%, the induction of immune suppression leading to potential respectively (data not shown). This suggests that both L-se- infections and tumor development. To address this issue, we lectin and integrin a4 may be involved in the process of carried out two sets of experiments. In the first, 15-week-old lymphocyte accumulation in local peripheral LNs in vivo mice that had been treated with PBS, Mel-14, or R1-2 from during an immune response. To further study whether these birth for 4 weeks were examined for both in vivo autoanti- mice were immunocompetent, lymphocytes from draining body responses and in vitro T-cell proliferative responses to LNs and spleens were tested for their ability to respond to apanel of(-islet-cell antigens. We have demonstrated (7) that OVA. Fig. 4 illustrates that T cells from PBS-, Mel-14-, or NOD mice develop spontaneous and age-dependent autoim- Rl-2-treated mice responded equally well to OVA, indicating mune responses to the panel of 3-islet-cell antigens and that that both local and systemic immune responses to a foreign the response to GAD65 appears to be correlated with the antigen remain unaffected by anti-L-selectin or anti-integrin onset of insulitis. The data in Fig. 3 indicate that the auto- Ca4 treatment. The fact that priming of an immune response antibodies specific for CPH, GAD65, HSP60, and peripherin was not altered by the blockade oflymphocyte trafficking and were detected in all PBS-, Mel-14-, and R1-2-treated mice and that a proliferative response could be detected in the splenic the extent of the responses was comparable among the cell cultures suggests that naive T cells may become sensi- treatment groups. In parallel to the antibody response, the in tized in other lymphoid tissues rather than in the draining vitro T-cell proliferative responses to these antigens were LNs in Mel-14- or Rl-2-treated mice. also unaffected by Mel-14 or R1-2 treatment in the first 4 As an additional test to determine whether anti-L-selectin weeks of life (data not shown). or anti-integrin a4 treatment can induce a general immuno- Secondly, to assess the immune responses to foreign suppressive effect, the ability ofmice pretreated with Mel-14, antigens, adult NOD mice that had been treated i.p. with PBS R1-2, or PBS to mount an anti-viral response was assessed. or 1 mg ofMel-14 or R1-2 every other day from 10 to 14 weeks Mice were inoculated i.p. with a sublethal dose of murine of age were immunized with chicken OVA. Ten days later, cytomegalovirus (3 x 106 plaque-forming units) (15). All of draining LNs and spleens were removed and lymphocytes the PBS-, anti-L-selectin-, or anti-integrin a4-treated animals were resistant to cytomegalovirus-induced death, and the 130 viral titers in the pancreata, salivary glands, and other organs 120 * Pancreas were comparable (data not shown), suggesting that the ability ED Salivary gland ='a l1110 c uet 100 * PBS (3) = cR 90 0l Mel-14 (4) T 0.8 [ E R1-2 (4) .3 80 0 ;;r, = 707 s W 60 0.6 o '- u 50 0) E _ T E 40 0 n 0.4 i- X 30 0- x 'o 20 e- 10 0.2

0 ~~~~~~~~~~~~~~~~~~~~~~~~~~F MAdCAM-1 PNAd VCAM-1 0.0 FIG. 2. Expression ofendothelial adhesion molecules in inflamed CPH GAD65 HSP60 Peripherin pancreatic islets and salivary glands. Frozen sections of pancreata and submandibular salivary glands from PBS-treated mice at 42 FIG. 3. Autoantibody responses to a panel of pancreatic p-cell weeks of age were stained with mAb against PNAd (MECA-79), antigens. Sera from mice treated with PBS, Mel-14, or R1-2 were MAdCAM-1 (MECA-367), and VCAM-1 (MK2.7). Each focus of examined for autoantibody responses to a panel of (-cell antigens lymphocytic inflammation was evaluated microscopically for pres- including CPH, GAD6S, HSP60, and peripherin by using an antigen- ence ofvessels expressing these endothelial adhesion molecules (n = specific ELISA. Data are presented as the mean ± SD of the OD405 6 mice; data are the mean ± SD). values in the ELISA. Downloaded by guest on September 28, 2021 Immunology: Yang et aL Proc. Natl. Acad. Sci. USA 91 (1994) 12607

'U treat mice with mAbs specific for adhesion molecules ex- * Spleen M NC pressed on lymphocytes and to determine their effect on the 9 l Lymph node MNC disease process. Depending on the age of the animal or the 8 extent of disease progression, both anti-L-selectin and anti- integrin a4 can partially or completely prevent and treat 7 spontaneous disease. This is consistent with our previous 6 finding using an adoptive transfer model of diabetes (2). In both cases, anti-integrin a4 treatment consistently provided a 5 more efficient protective effect than did blocking the L-se- 4 T lectin receptor. Similar data has been shown in the model of experimental allergic encephalomyelitis (16). The fact that 3 neonatal treatment with Mel-14 was able to prevent diabetes 2 while treatment during 10-14 weeks of age only delayed the

1 onset could be due to decreased levels of expression of PBS MVel-14 1R1-2 L-selectin on memory and/or activated autoreactive T cells in adult animals. Unlike the selectin family, which initiates FIG. 4. Anti-L-selectin or anti-integrin a4 treatment failed to the adhesion cascade and is functionally overlapping and inhibit the immune response to OVA. Mice treated with PBS, redundant, integrins are generally thought to be involved in Mel-14, or R1-2 were immunized with OVA. Ten days after immu- nization, the mononuclear cells (MNC) from spleens or from the the later steps ofthe adhesion process and are responsible for draining LNs were tested for their ability to respond to OVA in vitro. mediating strong adhesion to vascular endothelium and ex- The proliferative response was assayed by measuring the [3H]thy- tracellular matrix components such as fibronectin (17, 18). midine uptake (cpm) and is presented as stimulation index (mean cpm Therefore, integrin a4 mediated adhesion may be essential for in the presence of antigen/mean cpm with medium only). both naive and memory/activated T cells to enter the islets. Alternatively, the differential effect between Mel-14 or R1-2 to clear the virus was preserved in the Mel-14- and R1-2- may also be due to the different affmities of the antibody treated mice. and/or the epitopes they recognize. In the previous study (3), Therapeutic Potential for Treatment of Ongoing Anti-13Cell we have shown that anti-integrin A2 treatment failed to Autoimmunity in NOD Mice. To assess the potential appli- prevent IDDM. This has been confirmed and extended by cation of anti-L-selectin or anti-integrin a4 treatment to an Baron et al. (4) who demonstrated that blocking the ligand of ongoing diabetogenic response, NOD mice at 10 weeks of integrin (2, intercellular cell adhesion molecule 1 (ICAM-1), age, which exhibit extensive insulitis, were treated i.p. with had no protective effect in an adoptive transfer model of Mel-14, R1-2, or control antibodies for 4 weeks. As illustrated diabetes. Thus, these data suggest that during the late effector in Fig. 5, Mel-14 treatment delayed the onset of disease stages of the diabetogenic process, the integrin g32 pathway marginally and failed to protect mice from spontaneous may not have as significant a role as does the integrin a4 diabetes. In contrast, anti-integrin a4 treatment significantly pathway. reduced the incidence ofovert diabetes (30 vs. 84% ofcontrol Although the signaling functions of selectins and integrins mice at 42 weeks of age), further suggesting that integrin a4 are not fully understood, it has been shown that integrin 2 mediated lymphocyte adhesion plays an important role dur- and ICAM-1 can function as accessory molecules to induce ing the spontaneous development of IDDM at early and late signals leading to T-cell activation in vitro (19) and that stages of the disease process. interruption with this interaction induces tolerance in vivo (20). However, it is still unknown whether Mel-14 or R1-2 transduces a signal resulting in a functional change in autore- DISCUSSION active lymphocytes. What is clear is that both Mel-14 and In the present study, we have attempted to determine R1-2 are able to block neutrophil and lymphocyte migration which lymphocyte homing pathway(s) is essential for autore- in vivo without depleting L-selectin- and integrin a4 express- active and diabetogenic lymphocytes to migrate to the pan- ing cells (21, 22) and that Mel-14 or R1-2 treatment fails to to to mediate creatic islets. Two approaches were taken. The first was to induce immune tolerance self islet antigens, immunosuppression to foreign antigens, and to interrupt T-cell responses in vitro (2, 22). Moreover, the fact that 100 sialadenitis continues to develop in Mel-14- or Rl-2-treated 90 NOD mice provides further evidence that administering these mAbs does not jeopardize immune or other inflammatory I responses. Thus, these data indicate that the effect of anti- L-selectin or anti-integrin a4 treatment is not achieved by interfering with the ability of immune competent cells to

c 50 respond to antigen. This in turn suggests that anti-L-selectin I ._ or particularly anti-integrin a4 treatment may be a preferen- tial choice for the development of an anti-CAM-based im- .8 munotherapy. 20 The second approach was to analyze the expression of the a endothelial ligands for the various lymphocyte adhesion 10 molecules in the islets, salivary glands, and other tissues to gain insight into the mechanism oftissue-specific lymphocyte -rO 15 20 25 30 35 40 45 homing in autoimmunity. It is ofparticular interest that many Age (weeks) MAdCAM-1-expressing vessels are seen in the pancreas; are with inflamed islets FIG. 5. Therapeutic treatment with Mel-14 or Rl-2. Female NOD these vessels prominently associated mice at age of 10 weeks were treated i.p. with PBS (o, n = 7), Mel-14 but are also scattered throughout the exocrine pancreas. In (A, n = 6), R1-2 (o, n = 6), M1/9.3.4 (A, n = 5), or M1/89.18.7 (o, marked contrast, there is no MAdCAM-1 expression in NOD n = 5) every other day for 4 weeks. Mice were monitored weekly for salivary glands even on the vessels surrounding infiltrated the onset of diabetes. areas. Furthermore, PNAd- and VCAM-1-expressing vessels Downloaded by guest on September 28, 2021 12608 Immunology: Yang et al. Proc. Natl. Acad. Sci. USA 91 (1994) are detected in only some (40-50%) inflamed islets but in inflammatory cells from the circulation. If this is indeed the essentially all inflamed areas in the NOD salivary glands (Fig. case, an anti-CAM approach may have great potential to treat 2). Consistent with our finding, Hanninen et al. (8) and the ongoing autoimmune or inflammatory diseases in patients Faveeuw et al. (23) have recently demonstrated that (i) (25). MAdCAM-1 is primarily expressed on endothelium in the islets while VCAM-1 is constitutively expressed on dendritic We thank Drs. R. Cardin and J. Boname for helpful assistance in cells found in inflamed islets and on vessels in the exocrine carrying out the cytomegalovirus infection experiments. X.-D.Y., pancreas of NOD and control nonautoimmune-prone mice. R.T., and N.K. were recipients of Juvenile Diabetes Foundation Insulitis is associated with a marked increase in islet- International, National Institutes of Health, and Multiple Sclerosis associated vascular expression of MAdCAM-1 but not Society fellowships, respectively. This work was supported by VCAM-1 (8). In addition, ICAM-1 expression was detected grants ofthe National Institutes of Health (CA-49734 and DK-33880) on vessels found in the exocrine pancreas and on the endo- and the Department of Veterans Affairs. within The thelial and dendritic cells inflamed islets (23). (ii) 1. Castano, L. & Eisenbarth, G. S. (1990) Annu. Rev. Immunol. islet-infiltrating lymphocytes predominantly expressed inte- 8, 647-679. grin a437 as early as 4 weeks of age when insulitis is first 2. Yang, X.-D., Karin, N., Tisch, R., Steinman, L. & McDevitt, detectable, whereas L-selectin was only expressed at very H. 0. (1993) Proc. Natl. Acad. Sci. USA 90, 10494-10498. low levels up until 12 weeks of age (8). The majority of the 3. Yang, X.-D., Michie, S. A., Tisch, R., Karin, N., Steinman, L. infiltrating T cells found in inflamed islets and salivary glands & McDevitt, H. 0. (1994) J. Autoimmun., in press. have the memory/activated phenotype: L-selectinlOw and 4. Baron, J. L., Reich, E. P., Visintin, I. & Janeway, C. A. J. CD44(Pgp-l)hi*h (23). (iii) In vitro blockade of L-selectin or (1994) J. Clin. Invest. 93, 1700-1708. integrin a437, or its ligand MAdCAM-1 inhibits lymphocyte 5. Burkly, L. C., Jakubowski, A. & Hattori, M. (1994) Diabetes attachment to the inflamed vessels in NOD islets (8). Fur- 43, 529-534. thermore, anti-ICAM-1 and anti-integrin A32 treatment were 6. Yang, X.-D., Tisch, R., Singer, S. M., Cao, Z. A., Liblau, unable to block the transfer of diabetes in recipient mice (3, R. S., Schreiber, R. D. & McDevitt, H. 0. (1994) J. Exp. Med. 4). Thus, these data suggest that although multiple families of 180, 995-1004. 7. Tisch, R., Yang, X.-D., Singer, S. M., Liblau, R. S., Fugger, adhesion molecules are involved in lymphocyte trafficking to L. & McDevitt, H. 0. (1993) Nature (London) 366, 72-75. extranodal inflammatory sites in the NOD mouse, the inte- 8. Hanninen, A., Taylor, C., Streeter, P. R., Stark, L. S., Sarte, grin a4,37-MAdCAM-1 interaction may play a predominant J. M., Shizuru, J. A., Simell, 0. & Michie, S. A. (1993) J. Clin. role in mediating tissue-specific migration of diabetogenic Invest. 92, 2509-2515. cells into pancreatic islets. In contrast, interactions between 9. Miyazaki, A., Hanafusa, T., Yamada, K., Miyagawa, J., Fu- L-selectin and PNAd and between a4P1 and VCAM-1 may be jino-Kurihara, H., Nakajima, H., Nonaka, K. & Tarui, S. primarily involved in lymphocyte trafficking to the salivary (1985) Clin. Exp. Immunol. 60, 622-630. glands that causes sialadenitis. If this is correct, in addition 10. Streeter, P. R., Rouse, B. T. & Butcher, E. C. (1988) J. Cell to the antigen-specific T-cell receptor, organ-specific T cells Biol. 107, 1853-1862. may also express defined homing receptors facilitating se- 11. Berg, E. L., Robinson, M. K., Warnock, R. A. & Butcher, lective entry into their target tissues. E. C. (1991) J. Cell Biol. 114, 343-349. The long-term protective effect mediated by neonatal treat- 12. Berlin, C., Berg, E. L., Briskin, M. J., Andrew, D. P., Kilshaw, P. J., Holzmann, B., Weissman, I. L., Hamann, A. & ment may be due to the fact that the first few weeks after birth Butcher, E. C. (1993) Cell 74, 185-195. is an essential period for lymphocyte trafficking and migra- 13. Berg, E. L., McEvoy, L. M., Berlin, C., Bargatze, R. F. & tion to the islets (6, 9). Therefore, inhibiting the adhesion Butcher, E. C. (1993) Nature (London) 366, 695-698. process during this time may prevent autoreactive lympho- 14. Shimizu, Y., Newman, W., Tanaka, Y. & Shaw, S. (1992) cytes entering the islets and thus prevent subsequent events Immunol. Today 13, 106-112. leading to eventual ,3-islet-cell destruction. This is supported 15. Manning, W. C., Stoddart, C. A., Lagenaur, L. A., Abenes, by the finding that treatment with Mel-14 and R1-2, in G. B. & Mocarski, E. S. (1992) J. Virol. 66, 3794-3802. addition to preventing overt diabetes, markedly reduced the 16. Yednock, T. A., Cannon, C., Fritz, L. C., Sanchez-Madrid, incidence and severity of insulitis (Table 1). An alternative F., Steinman, L. & Karin, N. 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