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Peripheral lymphoid tissue-like adhesion molecule expression in nodular infiltrates in inflammatory myopathies
Neuromusc. Disord., Vol. 6. No. 4, pp. 255-260, 1996 Copyright © 1996 Elsevier Science BN. All fights reserved Printed in Great Britain. 0960-8966/96 Sl 5.00 + .00
Pergamon
PII: S0960-8966(96)00015-6 PERIPHERAL LYMPHOID TISSUE-LIKE ADHESION MOLECULE EXPRESSION IN NODULAR INFLAMMATORY
INFILTRATES IN
MYOPATHIES
J. L. DE BLEECKER,** A. G. ENGEL,* and E. C. BUTCHER~ *University Hospital, Department of Neurology, De Pintelaan 185, B-9000 Gent, Belgium; *Mayo Clinic, Neuromuscular Laboratory, Department of Neurology, 200 lSt Street SW, Rochester, MN 55905, U.S.A.; §Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, U.S.A. (Received 31 August 1995; revised 15 January 1996; accepted 8 February 1996)
Abstract--Non-granulomatous nodular accumulations of inflammatory cells in inflammatory myopathies were studied to characterize adhesion mechanisms used for leukocyte recruitment. The nodules had a B-cell-rich center surrounded by a helper T-cell-rich peripheral zone, resembling lymph nodes, The T-cell-rich zones harbored high-walled venules resembling high endothelial venules (HEV), whose endothelia frequently expressed ICAM-1, VCAM-1, and less constantly E-selectin. This endothelial adhesion molecule profile differs from that found in polymyositis, inclusion body myositis, or dermatomyositis, but resembles that in lymphoid tissues. Also, the peripheral lymph node addressin, a vascular addressin specific for peripheral lymphoid tissue HEV, was present on many HEV. This adhesion system is probably responsible for the excessive lymphocyte recruitment. The similar cellular organization and lymphocyte recirculation mechanisms of the nodular infiltrates in muscle and of lymph nodes suggest that the former may also produce antibodies.
Key words:inflammatorymyopathy, adhesion molecules,immunology,immunohistochemistry
INTRODUCTION
During immune surveillance and inflammation lymphocytes are recruited to lymphoid and extralymphoid sites by the interaction of lymphocyte adhesion receptors with endothelial ligands. We previously described the expression of adhesion molecules in the more common inflammatory myopathies [1]. In derrnatomyositis (DM), we found strong upregulation of intercellular adhesion molecule-1 (ICAM-1) on perimysial and perifascicular endothelial cells, further underscoring an immune effector response against the intramuscular microvasculature in this disease [2]. Vascular cell adhesion molecule-1 (VCAM-1) was inconsistently expressed and E-selectin was absent. In polymyositis (PM) and inclusion body myositis (IBM), we found induction of ICAM-1 on the surfaces of non-necrotic muscle fibers invaded by T cells, implicating this adhesion molecule in *Author to whom correspondence should be addressed.
the initiation or perpetuation of the myocytotoxic immune effector response. Most vessels surrounded by an inflammatory exudate in PM and IBM were upregulated for ICAM-1, but expressed no VCAM-1 or E-selectin [3-5]. Few inflammatory myopathy specimens harbor large, non-granulomatous nodular collections of inflammatory cells. These nodules often contain blood vessels that resemble high endothelial venules (HEV) found in lymphoid tissues. The HEV are sites of lymphocyte recruitment to lymphoid tissues via specific lymphocyte receptor/endothelial ligand interactions. Specific lymphocyte surface molecules, called homing receptors, and tissue-selective endothelial ligands, called vascular addressins, govern lymphocyte adhesion to the HEV. Peripheral lymphoid tissues differ from mucosal lymphoid tissues in the vascular addressins used for lymphocyte recruitment [6--8]. We here compare the adhesion molecules involved in lymphocyte recruitment to nodular infiltrates in muscle with those in lymphoid
255
256
J.L. de Bleeckeret al.
tissues and those in PM, IBM, and DM by immunolocalizing endothelial cell-associated adhesion molecules involved in several stages of leukocyte diapedesis. We also localized the peripheral lymph node addressin (PNAd), a vascular addressin expressed on human HEV in peripheral but not in mucosal lymphoid tissues [8, 91. EXPERIMENTAL PROCEDURES
Patients Seven proximal limb muscle specimens harboring large nodular collections of inflammatory cells were identified from a bank of diagnostic muscle biopsies of the Mayo Clinic Neuromuscular Laboratory. The clinical data on each patient are summarized in Table 1. The combined clinical and laboratory features in each case were consistent with an overlap syndrome. All patients had an inflammatory myopathy. In all biopsy specimens, diffusely scattered inflammatory cells appeared at perivascular, perimysial as well as endomysial sites. In addition, all specimens harbored large nodular collections of inflammatory cells. The nodules were perimysial and endomysial in three, only perimysial in three, and only endomysial in one. All muscle specimens also contained blood vessels with hyalinized mural elements. These vessels were 30-90 pm and appeared within the nodular infiltrates or at other perimysial, endomysial or epimysial sites. Necrotic and regenerating fibers and significant perimysial fibrosis were noted in all specimens. Disease controls consisted of four patients each with PM, IBM, and DM as defined by clinical and histological criteria [4,10]. Muscle specimens from non-weak subjects deemed to be free of neuromuscular disease on clinical, electromyographic and histologic grounds served as normal controls. Positive controls for adhesion molecule expression consisted of surgical tonsil and appendix tissue. No patients received immunosuppressive treatment within three months prior to biopsy. Primary immunoreagents The primary antibodies are listed in Table 2. Most antibodies recognizing cell markers or adhesion molecules were commercially obtained. PNAd was detected with the rat
monoclonal antibody MECA-79 [6, 11]. Fluorescein isothiocyanate (FITC)-labeled Ulex europaeus agglutinin-I (UEA-I) (Dako) was used as an endothelial cell marker [2]. Immunocytochemical procedures Consecutive 4-pm cryostat sections were used. The immunocytochemical procedures and secondary antibodies were described previously [1, 12]. Briefly, all antibodies were first studied by immunofluorescence or by the immunoperoxidase method. Subsequently, combinations of antibodies raised in different species were colocalized by two-color immunofluorescence, using non-crossreacting secondary antibodies. Control studies consisted of omission of the primary antibody and substitution of nonimmune and, when appropriate, isotype-specific immunoglobulins for the primary antibody. RESULTS Tonsil and appendix The staining pattern for each adhesion molecule was similar to that in previous studies [11, 13]. Many HEV strongly expressed PNAd in tonsil, but the expression was much less frequent and fainter in appendix [8]. Normal muscle and disease controls PNAD was absent from endothelial cells in normal muscle [8] and in PM, IBM and DM specimens. The immunolocalization of all other antibodies was as indicated in the Introduction and as reported before [1]. Nodular inflammatory collections The center of the nodules was composed of B-cells, whereas the periphery contained mainly CD3+ T-cells (Fig. 1, 2A). Most T-cells were CD4+ T-cells (Fig. 2B). Scarce CD8+ T-cells occurred at the boundaries of the nodules (Fig. 2C). CD56-positive NK cells were very rare. Macrophages occurred mainly in the muscle tissue immediately adjacent to the nodules. Blood vessels with HEV morphology appeared mainly at the periphery of the nodules. Many of these high-walled vessels were strongly positive for PNAd over their entire circumference (Fig. 3). PNAd staining occurred in the cytoplasm as well as in the lumenal and ablumenal cell surfaces of the
258
J. L. de Bleecker et al. Table 2. Primary antibodies
Antigen CD3 CD4 CD8 CD22 Macrophages CD56 ICAM-I E-selectin VCAM- 1 PNAd
Antibody Rabbit polyclonal Mouse monoclonal IgG1 Mouse monoclonal IgG1 Mouse monoclonal IgGl Mouse monoclonal lgGl Rat monoclonal IgGl Mouse monoelonal IgG1 Mouse monoclonal IgG2a Mouse monoclonal IgG 1 Mouse monoclonal lgGl Rat monoclonal IgM
Concentration (/ag/ml) 10 2 8 8 1 2 0.5 100 15 4 10
Source Dako Coulter Coulter Dako Dako Sanbio AMAC Becton-Dickinson W. Newman AMAC E.C. Butcher
Fig. 1. Two-color immunolocalization of CD3 (red) and CD22 (green) in a large perimysal nodule. A central Bcell-rich zone is surrounded by a mantle of CD3+ T-cells. Scleroderma. x250.
endothelial cells. VCAM-1 (Fig. 4A) and ICAM-1 (Fig. 4B) were also strongly expressed on these vessels, but E-selectin staining was less constant and intense (Fig. 4D). ICAM-1 and VCAM-1 were also strongly expressed on neighboring PNAd-negative arterioles and venules. Muscle fibers did not immunoreact for any anti-adhesion molecule antibodies. DISCUSSION
Although nodular infiltrates in inflammatory myopathies are not disease-specific, the clinical profile in our patients was remarkably similar. They all showed an overlap syndrome combining a subacute or chronic inflammatory myopathy with an associated connective tissue disorder. Cervical muscle weakness was common and we noted a high incidence of
Fig. 2. Two-color immunolocalizations in serial sections o f (A) CD3 (red) and CD22 (green); (B) CD3 (red) and CD4 (green); (C) CD3 (red) and CD8 (green). CD3+ Tcells and CD22+ B-cells tend to group (A). The abundant yellow cells in (B) are CD4+ T-cells. Rare yellow cells in (C) are CD8+ T-cells. Chronic inflammatory myopathy. x400.
Nodular Infiltrates in Myopathies
259
Fig. 3. Endothelia of several blood vessels with high endothelial venule morphology express the peripheral lymph node addressin. Immunoperoxidase stain. Chronic inflammatory myopathy. ×400.
interstitial pneumonitis and anti-nuclear autoantibodies. The present study shows that the nodules occurred mainly at endomysial and perimysial sites. They share several immunological characteristics with secondary lymphoid tissues: (1) Like in lymphoid tissue, the cell subsets segregate with a central B-cell-rich zone surrounded by a peripheral CD4+ T-cell zone. The similar organization suggests that these intramuscular nodules and lymph nodes share functional properties, possibly including local antibody production. (2) The T-cell-rich periphery harbors highwalled blood vessels with a broad adhesion molecule profile closely resembling that of lymphoid tissue HEV [12, 13]. Endothelial VCAM-1 expression is especially prominent in comparison with that observed in PM, IBM and DM specimens [1]. Unlike in PM, IBM and DM, a number of blood vessels also express Eselectin. The different adhesion molecule profile may reflect the activity of different cytokines. Locally synthesized cytokines, such as interferon-Tand tumor necrosis factor-a, are known to modulate postcapillary venule structure and function [9,14]. (3) The PNAd vascular addressin is induced on many postcapillary venules. This peripheral lymph node-specific adhesion system probably subserves lymphocyte recirculation in intramus-
D
Fig. 4. Immunofluorescence immunolocalizations of (A) VCAM-1 (green) and CD3 (red); (B) ICAM-I (red); (C) Ulex europaeus agglutinin-I (green); (D) E-selectin (red). Many postcapiUary venules with high endothelial cells react strongly for VCAM-I and ICAM-1, and weakly for E-selectin. Asterisks indicate the same postcapillary venule in non-consecutive serial sections. Chronic inflammatory myopathy, x400.
260
J.L. de Bleecker et al.
cular nodules and may significantly augment cellular recruitment. The lymphocyte accumulation in these nodules is indeed conspicuously more prominent than at perimysial, endomysial or perivascular sites in the other inflammatory myopathies. PNAd induction at extralymphoid sites of chronic inflammation has been noted previously in thyroid and cutaneous inflammatory diseases associated with extensive cellular infiltrations [8]. The selective enrichment in CD4+ T-cells is consistent with these cells being more efficient in recirculation than CD8+ T-cells [15].
6.
7.
8.
9.
Acknowledgements----Supported by NIH Grant NS-6277
and a Research Grant from the Muscular Dystrophy Association (to A. G. E.). J. L. De Bleecker was supported by the Belgian National Fund for Scientific Research and a Grant from Ghent State University.
10. 11.
REFERENCES 1. De Bleecker J L, Engel A G. Expression of cell adhesion molecules in inflammatory myopathies and Duchenne dystrophy. J Neuropathol Exp Neurol 1994; 53: 369-376. 2. Emslie-Smith A M, Engel A G. Microvascular changes in early and advanced dermatomyositis: a quantitative study. Ann Neurol 1990; 27: 343-356. 3. Arahata K, Engel A G. Monoclonal antibody analysis of mononuclear cells in myopathies. I: quantitation of subsets according to diagnosis and sites of accumulation and demonstration and counts of muscle fibers invaded by T cells. Ann Neurol 1984; 16: 193-208. 4. Engel A G, Arahata K. Mononuclear cells in myopathies: quantitation of functionally distinct subsets, recognition of antigen-specific cell-mediated cytotoxicity in some diseases, and implications for the pathogenesis of the different inflammatory myopathies, Hum Pathol 1986; 17: 704-721. 5. Hohlfeld R, Engel A G. Lysis of myotubes by allo-
12. 13. 14.
15.
reactive cytotoxic T cells and natural killer cells. Relevance to myoblast transplantation. J Clin Invest 1990; 86: 370-374. Berg E L, Robinson M K, Warnock R A, Butcher E C. The human peripheral lymph node vascular addressin is a ligand for LECAM-I, the peripheral lymph node homing receptor. J Cell Biol 1991; 114: 343-349. Imai Y, Singer M S, Fennie C, Lasky L A, Rosen S D. Identification of a carbohydrate-based endothelial ligand for a lymphocyte homing receptor. J Cell Biol 1991; 113: 1213-1221. Michie S A, Streeter P R, Bolt P A, Butcher E C, Picker L J. The human peripheral lymph node vascular addressin. An inducible endothelial antigen involved in lymphocyte homing. Am J Pathol 1993; 143: 1688-1698. Hendriks H R, Korn R E, Mebius R E, Kraal G. Interferon gamma increased adherence of lymphocytes to high endothelial venules. Immunology 1989; 68: 221-226. Lotz B P, Engel A G, Nishino H, Stevens J C, Litchy W J . Inclusion body myositis. Brain 1989; 112: 727-742. Streeter P R, Rouse B T N, Butcher E C. Immunohistologic and functional characterization of a vascular addressin involved in lymphocyte homing into peripheral lymph nodes. J Cell Biol 1988; 107: 1853-1862. De Bleecker J L, Engel A G. Immunocytochemical study of CD45 T cell isoforms in inflammatory myopathies. Am J Pathol 1995; 146: 1178-1187. Rice G E, Munro J M, Corless C, Bevilacqua M P. Vascular and non-vascular expression of ICAM-1. Am J Pathol 1991; 138: 385-393. Manolios N, Geczy C, Schrieber L. High endothelial venule morphology and function are inducible in germ-free mice: a possible role for interferon-gamma. Cell Immunol 1988; 117: 136-151. Abernethy N J, Hay J B, Kimpton W G, Washington E, Cahill R N P. Lymphocyte subset-specific and tissue-specific lymphocyte-endothelial cell recognition mechanisms independently direct the recirculation of lymphocytes from blood to lymph in sheep. Immunology 1991; 72: 239-245.
Pergamon
PII: S0960-8966(96)00015-6 PERIPHERAL LYMPHOID TISSUE-LIKE ADHESION MOLECULE EXPRESSION IN NODULAR INFLAMMATORY
INFILTRATES IN
MYOPATHIES
J. L. DE BLEECKER,** A. G. ENGEL,* and E. C. BUTCHER~ *University Hospital, Department of Neurology, De Pintelaan 185, B-9000 Gent, Belgium; *Mayo Clinic, Neuromuscular Laboratory, Department of Neurology, 200 lSt Street SW, Rochester, MN 55905, U.S.A.; §Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, U.S.A. (Received 31 August 1995; revised 15 January 1996; accepted 8 February 1996)
Abstract--Non-granulomatous nodular accumulations of inflammatory cells in inflammatory myopathies were studied to characterize adhesion mechanisms used for leukocyte recruitment. The nodules had a B-cell-rich center surrounded by a helper T-cell-rich peripheral zone, resembling lymph nodes, The T-cell-rich zones harbored high-walled venules resembling high endothelial venules (HEV), whose endothelia frequently expressed ICAM-1, VCAM-1, and less constantly E-selectin. This endothelial adhesion molecule profile differs from that found in polymyositis, inclusion body myositis, or dermatomyositis, but resembles that in lymphoid tissues. Also, the peripheral lymph node addressin, a vascular addressin specific for peripheral lymphoid tissue HEV, was present on many HEV. This adhesion system is probably responsible for the excessive lymphocyte recruitment. The similar cellular organization and lymphocyte recirculation mechanisms of the nodular infiltrates in muscle and of lymph nodes suggest that the former may also produce antibodies.
Key words:inflammatorymyopathy, adhesion molecules,immunology,immunohistochemistry
INTRODUCTION
During immune surveillance and inflammation lymphocytes are recruited to lymphoid and extralymphoid sites by the interaction of lymphocyte adhesion receptors with endothelial ligands. We previously described the expression of adhesion molecules in the more common inflammatory myopathies [1]. In derrnatomyositis (DM), we found strong upregulation of intercellular adhesion molecule-1 (ICAM-1) on perimysial and perifascicular endothelial cells, further underscoring an immune effector response against the intramuscular microvasculature in this disease [2]. Vascular cell adhesion molecule-1 (VCAM-1) was inconsistently expressed and E-selectin was absent. In polymyositis (PM) and inclusion body myositis (IBM), we found induction of ICAM-1 on the surfaces of non-necrotic muscle fibers invaded by T cells, implicating this adhesion molecule in *Author to whom correspondence should be addressed.
the initiation or perpetuation of the myocytotoxic immune effector response. Most vessels surrounded by an inflammatory exudate in PM and IBM were upregulated for ICAM-1, but expressed no VCAM-1 or E-selectin [3-5]. Few inflammatory myopathy specimens harbor large, non-granulomatous nodular collections of inflammatory cells. These nodules often contain blood vessels that resemble high endothelial venules (HEV) found in lymphoid tissues. The HEV are sites of lymphocyte recruitment to lymphoid tissues via specific lymphocyte receptor/endothelial ligand interactions. Specific lymphocyte surface molecules, called homing receptors, and tissue-selective endothelial ligands, called vascular addressins, govern lymphocyte adhesion to the HEV. Peripheral lymphoid tissues differ from mucosal lymphoid tissues in the vascular addressins used for lymphocyte recruitment [6--8]. We here compare the adhesion molecules involved in lymphocyte recruitment to nodular infiltrates in muscle with those in lymphoid
255
256
J.L. de Bleeckeret al.
tissues and those in PM, IBM, and DM by immunolocalizing endothelial cell-associated adhesion molecules involved in several stages of leukocyte diapedesis. We also localized the peripheral lymph node addressin (PNAd), a vascular addressin expressed on human HEV in peripheral but not in mucosal lymphoid tissues [8, 91. EXPERIMENTAL PROCEDURES
Patients Seven proximal limb muscle specimens harboring large nodular collections of inflammatory cells were identified from a bank of diagnostic muscle biopsies of the Mayo Clinic Neuromuscular Laboratory. The clinical data on each patient are summarized in Table 1. The combined clinical and laboratory features in each case were consistent with an overlap syndrome. All patients had an inflammatory myopathy. In all biopsy specimens, diffusely scattered inflammatory cells appeared at perivascular, perimysial as well as endomysial sites. In addition, all specimens harbored large nodular collections of inflammatory cells. The nodules were perimysial and endomysial in three, only perimysial in three, and only endomysial in one. All muscle specimens also contained blood vessels with hyalinized mural elements. These vessels were 30-90 pm and appeared within the nodular infiltrates or at other perimysial, endomysial or epimysial sites. Necrotic and regenerating fibers and significant perimysial fibrosis were noted in all specimens. Disease controls consisted of four patients each with PM, IBM, and DM as defined by clinical and histological criteria [4,10]. Muscle specimens from non-weak subjects deemed to be free of neuromuscular disease on clinical, electromyographic and histologic grounds served as normal controls. Positive controls for adhesion molecule expression consisted of surgical tonsil and appendix tissue. No patients received immunosuppressive treatment within three months prior to biopsy. Primary immunoreagents The primary antibodies are listed in Table 2. Most antibodies recognizing cell markers or adhesion molecules were commercially obtained. PNAd was detected with the rat
monoclonal antibody MECA-79 [6, 11]. Fluorescein isothiocyanate (FITC)-labeled Ulex europaeus agglutinin-I (UEA-I) (Dako) was used as an endothelial cell marker [2]. Immunocytochemical procedures Consecutive 4-pm cryostat sections were used. The immunocytochemical procedures and secondary antibodies were described previously [1, 12]. Briefly, all antibodies were first studied by immunofluorescence or by the immunoperoxidase method. Subsequently, combinations of antibodies raised in different species were colocalized by two-color immunofluorescence, using non-crossreacting secondary antibodies. Control studies consisted of omission of the primary antibody and substitution of nonimmune and, when appropriate, isotype-specific immunoglobulins for the primary antibody. RESULTS Tonsil and appendix The staining pattern for each adhesion molecule was similar to that in previous studies [11, 13]. Many HEV strongly expressed PNAd in tonsil, but the expression was much less frequent and fainter in appendix [8]. Normal muscle and disease controls PNAD was absent from endothelial cells in normal muscle [8] and in PM, IBM and DM specimens. The immunolocalization of all other antibodies was as indicated in the Introduction and as reported before [1]. Nodular inflammatory collections The center of the nodules was composed of B-cells, whereas the periphery contained mainly CD3+ T-cells (Fig. 1, 2A). Most T-cells were CD4+ T-cells (Fig. 2B). Scarce CD8+ T-cells occurred at the boundaries of the nodules (Fig. 2C). CD56-positive NK cells were very rare. Macrophages occurred mainly in the muscle tissue immediately adjacent to the nodules. Blood vessels with HEV morphology appeared mainly at the periphery of the nodules. Many of these high-walled vessels were strongly positive for PNAd over their entire circumference (Fig. 3). PNAd staining occurred in the cytoplasm as well as in the lumenal and ablumenal cell surfaces of the
258
J. L. de Bleecker et al. Table 2. Primary antibodies
Antigen CD3 CD4 CD8 CD22 Macrophages CD56 ICAM-I E-selectin VCAM- 1 PNAd
Antibody Rabbit polyclonal Mouse monoclonal IgG1 Mouse monoclonal IgG1 Mouse monoclonal IgGl Mouse monoclonal lgGl Rat monoclonal IgGl Mouse monoelonal IgG1 Mouse monoclonal IgG2a Mouse monoclonal IgG 1 Mouse monoclonal lgGl Rat monoclonal IgM
Concentration (/ag/ml) 10 2 8 8 1 2 0.5 100 15 4 10
Source Dako Coulter Coulter Dako Dako Sanbio AMAC Becton-Dickinson W. Newman AMAC E.C. Butcher
Fig. 1. Two-color immunolocalization of CD3 (red) and CD22 (green) in a large perimysal nodule. A central Bcell-rich zone is surrounded by a mantle of CD3+ T-cells. Scleroderma. x250.
endothelial cells. VCAM-1 (Fig. 4A) and ICAM-1 (Fig. 4B) were also strongly expressed on these vessels, but E-selectin staining was less constant and intense (Fig. 4D). ICAM-1 and VCAM-1 were also strongly expressed on neighboring PNAd-negative arterioles and venules. Muscle fibers did not immunoreact for any anti-adhesion molecule antibodies. DISCUSSION
Although nodular infiltrates in inflammatory myopathies are not disease-specific, the clinical profile in our patients was remarkably similar. They all showed an overlap syndrome combining a subacute or chronic inflammatory myopathy with an associated connective tissue disorder. Cervical muscle weakness was common and we noted a high incidence of
Fig. 2. Two-color immunolocalizations in serial sections o f (A) CD3 (red) and CD22 (green); (B) CD3 (red) and CD4 (green); (C) CD3 (red) and CD8 (green). CD3+ Tcells and CD22+ B-cells tend to group (A). The abundant yellow cells in (B) are CD4+ T-cells. Rare yellow cells in (C) are CD8+ T-cells. Chronic inflammatory myopathy. x400.
Nodular Infiltrates in Myopathies
259
Fig. 3. Endothelia of several blood vessels with high endothelial venule morphology express the peripheral lymph node addressin. Immunoperoxidase stain. Chronic inflammatory myopathy. ×400.
interstitial pneumonitis and anti-nuclear autoantibodies. The present study shows that the nodules occurred mainly at endomysial and perimysial sites. They share several immunological characteristics with secondary lymphoid tissues: (1) Like in lymphoid tissue, the cell subsets segregate with a central B-cell-rich zone surrounded by a peripheral CD4+ T-cell zone. The similar organization suggests that these intramuscular nodules and lymph nodes share functional properties, possibly including local antibody production. (2) The T-cell-rich periphery harbors highwalled blood vessels with a broad adhesion molecule profile closely resembling that of lymphoid tissue HEV [12, 13]. Endothelial VCAM-1 expression is especially prominent in comparison with that observed in PM, IBM and DM specimens [1]. Unlike in PM, IBM and DM, a number of blood vessels also express Eselectin. The different adhesion molecule profile may reflect the activity of different cytokines. Locally synthesized cytokines, such as interferon-Tand tumor necrosis factor-a, are known to modulate postcapillary venule structure and function [9,14]. (3) The PNAd vascular addressin is induced on many postcapillary venules. This peripheral lymph node-specific adhesion system probably subserves lymphocyte recirculation in intramus-
D
Fig. 4. Immunofluorescence immunolocalizations of (A) VCAM-1 (green) and CD3 (red); (B) ICAM-I (red); (C) Ulex europaeus agglutinin-I (green); (D) E-selectin (red). Many postcapiUary venules with high endothelial cells react strongly for VCAM-I and ICAM-1, and weakly for E-selectin. Asterisks indicate the same postcapillary venule in non-consecutive serial sections. Chronic inflammatory myopathy, x400.
260
J.L. de Bleecker et al.
cular nodules and may significantly augment cellular recruitment. The lymphocyte accumulation in these nodules is indeed conspicuously more prominent than at perimysial, endomysial or perivascular sites in the other inflammatory myopathies. PNAd induction at extralymphoid sites of chronic inflammation has been noted previously in thyroid and cutaneous inflammatory diseases associated with extensive cellular infiltrations [8]. The selective enrichment in CD4+ T-cells is consistent with these cells being more efficient in recirculation than CD8+ T-cells [15].
6.
7.
8.
9.
Acknowledgements----Supported by NIH Grant NS-6277
and a Research Grant from the Muscular Dystrophy Association (to A. G. E.). J. L. De Bleecker was supported by the Belgian National Fund for Scientific Research and a Grant from Ghent State University.
10. 11.
REFERENCES 1. De Bleecker J L, Engel A G. Expression of cell adhesion molecules in inflammatory myopathies and Duchenne dystrophy. J Neuropathol Exp Neurol 1994; 53: 369-376. 2. Emslie-Smith A M, Engel A G. Microvascular changes in early and advanced dermatomyositis: a quantitative study. Ann Neurol 1990; 27: 343-356. 3. Arahata K, Engel A G. Monoclonal antibody analysis of mononuclear cells in myopathies. I: quantitation of subsets according to diagnosis and sites of accumulation and demonstration and counts of muscle fibers invaded by T cells. Ann Neurol 1984; 16: 193-208. 4. Engel A G, Arahata K. Mononuclear cells in myopathies: quantitation of functionally distinct subsets, recognition of antigen-specific cell-mediated cytotoxicity in some diseases, and implications for the pathogenesis of the different inflammatory myopathies, Hum Pathol 1986; 17: 704-721. 5. Hohlfeld R, Engel A G. Lysis of myotubes by allo-
12. 13. 14.
15.
reactive cytotoxic T cells and natural killer cells. Relevance to myoblast transplantation. J Clin Invest 1990; 86: 370-374. Berg E L, Robinson M K, Warnock R A, Butcher E C. The human peripheral lymph node vascular addressin is a ligand for LECAM-I, the peripheral lymph node homing receptor. J Cell Biol 1991; 114: 343-349. Imai Y, Singer M S, Fennie C, Lasky L A, Rosen S D. Identification of a carbohydrate-based endothelial ligand for a lymphocyte homing receptor. J Cell Biol 1991; 113: 1213-1221. Michie S A, Streeter P R, Bolt P A, Butcher E C, Picker L J. The human peripheral lymph node vascular addressin. An inducible endothelial antigen involved in lymphocyte homing. Am J Pathol 1993; 143: 1688-1698. Hendriks H R, Korn R E, Mebius R E, Kraal G. Interferon gamma increased adherence of lymphocytes to high endothelial venules. Immunology 1989; 68: 221-226. Lotz B P, Engel A G, Nishino H, Stevens J C, Litchy W J . Inclusion body myositis. Brain 1989; 112: 727-742. Streeter P R, Rouse B T N, Butcher E C. Immunohistologic and functional characterization of a vascular addressin involved in lymphocyte homing into peripheral lymph nodes. J Cell Biol 1988; 107: 1853-1862. De Bleecker J L, Engel A G. Immunocytochemical study of CD45 T cell isoforms in inflammatory myopathies. Am J Pathol 1995; 146: 1178-1187. Rice G E, Munro J M, Corless C, Bevilacqua M P. Vascular and non-vascular expression of ICAM-1. Am J Pathol 1991; 138: 385-393. Manolios N, Geczy C, Schrieber L. High endothelial venule morphology and function are inducible in germ-free mice: a possible role for interferon-gamma. Cell Immunol 1988; 117: 136-151. Abernethy N J, Hay J B, Kimpton W G, Washington E, Cahill R N P. Lymphocyte subset-specific and tissue-specific lymphocyte-endothelial cell recognition mechanisms independently direct the recirculation of lymphocytes from blood to lymph in sheep. Immunology 1991; 72: 239-245.