American Journal of Pathology, Vol. 140, No. 6, June 1992 Copyight © American Association of Pathologists

Expression and Cell Distribution of the Intercellular Adhesion Molecule, Vascular Cell Adhesion Molecule, Endothelial Leukocyte Adhesion Molecule, and Endothelial Cell Adhesion Molecule (CD31) in Reactive Human Lymph Nodes and in Hodgkin's Disease Luigi P. Ruco,* Donatella Pomponi,* Rod Pigott,t Andy J. H. Gearing,t Andrea Baiocchini,* and Carlo D. Baroni* From the Second Division of Pathologic Anatomy, Department of Human Biopathology,* Sapienza University, Rome, Italy; and British Biotechnology,t Cowley, Oxford, United Kingdom

The immunocytochemical expression of intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1), endothelial leukocyte adhesion molecule (ELAM-1), endothelial cell adhesion molecule (EndoCAM CD31), and HLA-DR antigens was investigated in sections of 24 reactive lymph nodes and in 15 cases of Hodgkin's disease. ICAM-1 was detected in sinus macrophages, follicular dendritic reticulum cells (FDRCs), interdigitating reticulum cells (IDRCs), epithelioid macrophages, Hodgkin's cells (HCs), and vascular endothelium. ICAM-1 expression was often associated with that of HLA-DR antigens. VCAM-1 was detected in FDRCs, in fibroblast reticulum cells (FRCs), in macrophages, and in rare blood vessels. EndoCAM (CD31) was constitutively expressed in all types of endothelial cells, sinus macrophages, and in epithelioid granulomas. ELAM4- was selectively expressed by activated endothelial cells of high endothelium venules (HEVs). When expression of the inducible adhesion molecules ICAM-1, VCAM-1 and ELAM-4 was comparatively evaluated in HEVs, it wasfound that ICAM-I + HEVs were present in all reactive and HD nodes, whereas ELAM- I and/or VCAM- I were expressed only in those pathologic conditions characterized by high levels of interleukin-litumor necrosis factor (IL-1I TNF) production, such as granulomatosis and

Hodgkin's disease. In Hodgkin's disease, the expression of ELAM-I/VCAM-1 was more pronounced in cases of nodular sclerosis and was associated with a significantly higher content of perivascular neutrophils. (Am J Pathol 1992, 140:133 7-1344)

Intercellular adhesion molecule (ICAM-1), endothelial leukocyte adhesion molecule (ELAM-1), and vascular cell adhesion molecule (VCAM-1) are inducible proteins involved in cell-to-cell adhesion. ICAM-1 is either absent or is present at low levels in most cells. Many cell types including hematopoietic cells, epithelial cells, and endothelial cells can be stimulated to produce high levels of ICAM-1 after exposure to cytokines, endotoxin, or phorbol esters.1'2 ELAM-1 expression is restricted to cytokine (e.g., IL-1/TNF) activated endothelial cells and is involved in monocyte, neutrophil, and eosinophil adhesion.3 5 VCAM-1 expression is upregulated by IL-1, IL-4, TNF, and perhaps others; the molecule is present on activated endothelial cells, macrophages, interdigitating reticulum cells (IDRCs), and follicular dendritic reticulum cells (FDRCs), and is involved in lymphocyte and monocyte adhesion.6 8 Each of these molecules interacts with specific ligands present on leukocytes. ICAM-1 binds lymphocyte function antigen-1 (LFA-1), a ,2 integrin expressed on most types of blood cells.2 ELAM-1 recognizes sialylated 1,3-fucosylated carbohydrate structures such as Lewis X antigen9 present on neutrophils and monocytes. VCAM-1 binds very late antigen-4 (VLA-4), a ,i integrin expressed by activated lymphocytes and Supported by a grant from Istituto Pasteur-Fondazione Cenci Bolognetti and by MPI 40% Immunologia. Accepted for publication December 31, 1991. Address reprint requests to Dr. Luigi Ruco, II Cattedra di Anatomia

Patologica, Viale Regina Elena 324, 00161 Rome, Italy.

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monocytes.10 CD31, also termed endoCAM or PECAM1, is an adhesion molecule present in endothelial cells, monocytes, and platelets.11-14 CD31 is constitutively expressed at the cell-cell borders of contiguous endothelial cells and is probably involved in endothelium organization. 13 Adhesion molecules have many important functions in the lymphoid tissue. They regulate lymphocyte traffic, promote cell-to-cell adhesion during antigen presentation, and allow germinal center organization. This study investigated the expression and distribution of adhesion molecules in human lymph nodes. The study was done on hyperplastic nodes, and in pathologic conditions, including granulomatosis and Hodgkin's disease, where high levels of cytokines known to induce adhesion molecules are produced.1918

lingame, CA). Each incubation step lasted 30 minutes with 5-minute TRIS-buffered saline washes between each step. The sections were finally incubated with 0.03% H202 and 0.06% 3,3'diaminobenzidine (Sigma, St. Louis, MO) for 3 to 5 minutes. Slides were then washed for 5 minutes in running tap water, counterstained with hematoxylin for 5 minutes, and mounted in Canadian balsam. Endogenous peroxidase was visualized in only eosinophils. In some experiments endogenous peroxidase was inhibited by pretreatment with 0.01% v/v H202. Additional controls included omission of primary antibody to visualize nonspecific binding through Fc receptors. The alkaline phosphatase reaction was revealed using a New Fuchsin substrate (Sigma, St. Louis, MO) (30 minutes with continuous stirring).

Materials and Methods

Results

Enlarged lymph nodes were removed at surgery for diagnostic purpose. Normal, nonactivated lymph nodes were obtained from surgical specimens of gastric and breast resections; they were less than 0.5 cm in diameter, and did not show activated germinal centers and paracortical hyperplasia. Lymph-node fragments were embedded in optimal cryopreserved tissue (OCT) compound (Ames Division, Miles Laboratories, Elkhart, IN), snapfrozen in liquid nitrogen, and stored at -80°C until sectioning. Other fragments were formalin-fixed and paraffin-embedded for conventional histology procedures.

Reactive Lymphadenitis

Immunostaining Procedures Cryostat sections were fixed in acetone for 10 minutes at room temperature and were immunostained with monoclonal antibodies to ICAM-1 (P3.58b), VCAM-1 (BBIGV1), ELAM-1 (29F2; 5D11; iFll), CD31 (9G11), and HLA-DR (OKla-1)(Ortho Pharmaceutical, Raritan, NJ). Macrophages were recognized with Dako-mac (CD68), and follicular dendritic reticulum cells with DRC-1 (Dakopatts, Geostrup, Denmark). Specificity of 29F2, 5D1 1 and 1 Fl 1 for ELAM-1, and of BBIG-V1 for VCAM-1 was confirmed by binding to COS cells transfected with cDNA encoding respectively human ELAM-1 and human VCAM-1. Cryostat sections were preincubated with normal horse serum to prevent nonspecific binding, and then incubated with an optimal dilution (1:10/1 :100) of the primary antibody for 30 minutes. The slides were sequentially incubated with biotin-conjugated horse anti-mouse immunoglobulin antibodies followed by avidin-biotinperoxidase complex (PK 4002; Vector Laboratories, Bur-

Cryostat sections of 24 reactive lymph nodes were immunostained for CD31 (9G11), ICAM-1 (P3.58b), VCAM-1 (BBIG-V1), ELAM-1 (29F2,5D11,1F11), and HLA-DR (OKla-1 )(Table 1). Reactive nodes showed follicular, sinus, and/or diffuse lymphoid hyperplasia, or were sites of granulomatosis. Sinus macrophages of subcapsular, intermediary, and medullary sinuses were strongly immunoreactive for CD31. The staining was present in all sinus cells with equal intensity and was similar to that of vascular endothelium (Figure 1 a). Macrophages of hyperplastic sinuses were characterized by marked expression of ICAM-1 and HLA-DR antigens. Rare sinus macrophages were VCAM-1 +. In the B-cell follicles, follicular dendritic reticulum cells (FDRCs) of germinal centers were ICAM-1 + and markedly VCAM-1 + (Figure ic, d). Immunoreactivity for VCAM-1 was detected in some fibroblast reticulum cells (FRCs) of the mantle zone; furthermore, numerous VCAM-1 + FRCs were interposed between subcapsular sinus and cortical B-cell follicles (Figure 1 b). In the interfollicular areas and paracortex, immunoreactivity for VCAM-1 was mainly expressed by FRCs; they were located beneath subcapsular sinus, around B-cell follicles and around HEVs (Figure 1 e). FRCs were distinguished by endogenous alkaline phosphatase activity (Figure if), and by their typical morphology and tissue distribution. Some macrophages and/or interdigitating reticulum cells (IDRCs) were also VCAM-1 +. ICAM-1 was markedly expressed by scattered macrophages and by IDRCs with typical folded nucleus (Figure 2e). Macrophages and/or IDRCs were strongly HLA-DR + and were

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Table 1. Expression of Inducible Adhesion Molecules ICAM-1, VCAM-1, and ELAM-1 in Reactive Lymph Nodes (n = 24) Lymph node CD31 VCAM-1 ELAM-1 site ICAM-1 Weak reactivity in FDRCs Negative Strong reactivity in FDRCs Negative B-cell

follicles

and in FRCs of the mantle

Paracortex

zone Perivascular and perifollicular FRCs, some

Sinuses

Strong reactivity in HLA-DR+ IDRCs, in macrophages and in epithelioid macrophages Strong reactivity in HLA-DR+ epithelioid granulomas and in giant multinucleated cells HEVs are frequently positive Sinus macrophages show zonal reactivity In sinus hyperplasia, macrophages are markedly ICAM-1 +/DR+

Negative

Macrophages and IDRCs

Some perigranulomatous cells

Negative

Epithelioid

HEVs are rarely positive

HEVs may be positive

Plasma cells

macrophage-like cells macrophages

Negative Rare macrophages in hyperplastic sinuses

CD31 + with variable intensity. In tuberculous and sarcoid granulomas, epithelioid macrophages were HLADR+/ICAM-1 +/CD31 + and were negative for VCAM1/ELAM-1 (Figure 2c, d). ICAM-1 staining of epithelioid macrophages was often confined to cell membrane (Figure 2f). Clustered plasma cells were often characterized by membrane reactivity for CD31. In Table 2, we have compared the expression of ICAM-1, ELAM-1, VCAM-1, and HLA-DR in HEVs. A variable number of ICAM-1 + HEVs was detected in all cases (Figure 2b). ICAM-1 + HEVs were numerous, and could be seen throughout the lymph-node paracortex. ICAM-1 expression was often associated with marked endothelial reactivity for HLA-DR in cases of follicular hyperplasia. As previously reported, a few ELAM-1 + HEVs were detected in some cases of mixed and diffuse hyperplasia (Figure 2a). HEVs were consistently VCAM-1 in all hyperplastic nodes. In granulomatous lymphadenitis, some blood vessels located around granulomas were ICAM-1 +/ELAM-1 +NCAM-1 + and HLA-DR+. ELAM1 + and/or VCAM-1 + vessels were few and showed a focal distribution. HEVs of six normal nodes obtained from resected organs were negative for ICAM-1, ELAM1, VCAM-1, and HLA-DR.

Hodgkin's Disease In 15 cases of Hodgkin's disease, Hodgkin's and ReedSternberg cells (H/RS) were often positive for HLA-DR and ICAM-1 as previously reported19 and were negative for VCAM-1 and CD31. Immunoreactivity for ICAM-1 and/ or VCAM-1 was also present in numerous tumorassociated reactive macrophages and dendritic cells. In Hodgkin's disease, two distinctive patterns of vascular

Negative

All blood vessels All sinus macrophages All sinus macrophages

reactivity were noted (Table 3). In nodular sclerosis, blood vessels were ICAM-1 +/ELAM-1 +NCAM-1 + and HLA-DR-. In cases of mixed cellularity, ICAM-1 reactivity was often associated with a strong staining for HLADR, and with a weak and infrequent staining for ELAM1NCAM-1. Immunostaining of serial sections revealed that ICAM-1, VCAM-1, and ELAM-1 could be present on the same blood vessel. ELAM-1 is involved in neutrophil adhesion.34 Thus, we have determined the neutrophil content (number of neutrophils/5 oil fields in paraffinembedded, H&E stained sections) in the cellular areas of nodular sclerosis and in mixed cellularity. It was found that the number of neutrophils present in 33 consecutive cases of nodular sclerosis (mean, 39 ± 59) was significantly higher (P < 0.001) than that of 32 cases of mixed cellularity (mean, 3 ± 7).

Discussion The tissue distribution of ICAM-1 and VCAM-1 in human lymph nodes was previously investigated.1' 78,19 ICAM-1 was detected in vascular endothelial cells, and in several types of antigen presenting cells including macrophages, FDRCs, IDRCs, and Hodgkin's cells.1'19 This pattern of distribution supports the observation that ICAM-1 has at least two important functions: it mediates the adhesion and extravasation of circulating leukocytes,1'20 and contributes to maintaining intercellular adhesion between macrophage/reticular cells and lymphocytes during antigen presentation.21'22 Immunoreactivity for VCAM-1 was described in activated endothelial cells, macrophages, FDRCs, and in some reticular cells interpreted as IDRCs.7,8 Expression of VCAM-1 in endothelial cells is involved in the adhesion of circulating lympho-

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t ~tp A,

o~. 54; ^***t

o~ * .* 51). NE = not evaluated.

NE +

++ ++

+++ +++ +++ ++

+

+

+/++++/+/+++ + ++

VCAM-1

+ +

+-

NE NE NE

++ 1-5; +, 6-10; + +,11-50; + ++

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present study, we have found that there are two distinctive patterns of vascular activation in Hodgkin's disease. In the histologic variant nodular sclerosis, blood vessels were ICAM-1 +/ELAM-1 +NCAM-1 + and HLA-DR-, whereas in the mixed cellularity type, they were ICAM1 + /HLA-DR + and ELAM-1 -NCAM-1 -. Furthermore, the biological relevance of ELAM-1 expression in nodular sclerosis was supported by the presence of numerous perivascular neutrophils. These findings may indicate that cytokine-induced expression of adhesion molecules has a role in the pathogenesis of HD lesions, and that differing patterns of cytokine production exist in nodular sclerosis versus mixed cellularity. The great variability in the capacity of cytokine secretion among different Hodgkin's cell lines37 supports our interpretation.

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