Distribution of ICAM-3-bearing cells in normal human tissues ...

American Journal of Pathology, Vol. 143, No. 3, September 1993 Copynight © Amercan Society for Investigative Pathology

Distribution of ICAM-3-Bearing Cells in Normal Human Tissues Expression of a Novel Counter-Receptor for LFA- 1 in Epidermal Langerhans Cells

Agustin Acevedo,* Miguel A. del Pozo,t Alicia G. Arroyo,t Paloma Sanchez-Mateos,t Roberto Gonzalez-Amaro,t and Francisco Sanchez-Madridt Departments ofPathology* and Immunology,t Hospital de la Princesa, Universidad Aut6noma de Madrid, Spain

The LFA-1 integrin mediates its function in leukocyte intercellular interactions by recognition of at least one of its three identified counter-receptors, intercelular adhesion molecules I (ICAM-1), 2, and 3. The ICAM-1 molecule is expressed in an inducible-dependent manner by endothelial and epithelial ceUs, as weU as by other ceU types, including leukocytes. On the other hand, ICAM-2 is constitutively expressed mainly by endothelial cells. We have studied the tissue distribution of the ICAM-3 molecule by immunohistochemical staining of lymphoid and nonlymphoid organs. Only ceUs from the leukocyte lineage were found bearing the ICAM-3 antigen, showing a pattern of expression clearly distinctfrom those of ICAM-1 and ICAM-2. Interestingly, we have found that ICAM-3 is expressed by epidermal dendritic Langerhans cells as assessed by double immunostaining with antibodies specificfor CDI. In contrast, staining of skin sections with anti-ICAM-1 and ICAM-2 antibodies showed an undetectable expression ofthese two molecules on Langerhans cells. However, CD1+ Langerhans ceUs localized in the paracortical area of dermatopathic lymph nodes expressed both ICAM-1 and ICAM-3 antigens. Our results indicate that ICAM-3 is the main LFA-1 counter-receptor in human resident epidermal Langerhans cells. ICAM-3 may have an important role in T-ceU antigen stimulation driven by Langerhans ceUs during skin immune reactions. (Am JPathol 1993, 143:774-783)

The LFA-1 (CD11a/CD18) adhesion molecule is a member of the 12 leukocyte integrin subfamily.1

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LFA-1 plays a crucial role in the cellular interactions of immune response such as lymphocyte activation and proliferation, or leukocyte extravasation and migration to sites of inflammation.1' 2 The LFA-1 integrin mediates its function in intercellular adhesion by interaction with at least one of its two well-characterized counter-receptors, the intercellular adhesion molecules 1 and 2 (ICAM-1 and -2).1-6 Cloning and sequencing studies revealed that ICAM-1 and ICAM-2 molecules belong to the Ig gene superfamily, bearing 5 and 2 Ig-like domains, respectively.4-6 ICAM-1 is an inducible molecule highly expressed by cytokineactivated endothelial and epithelial cells, macro-phages, and activated lymphocytes,7 whereas ICAM-2 is constitutively expressed by the vascular endothelium and most leukocytes.8 9 A recent report has described the identification of a novel third counter-receptor for LFA-1, ICAM-3.10 Moreover, studies on the molecular cloning of ICAM-3 have shown that transfected cells with complementary DNAs encoding the ICAM-3 molecule were able to support leukocyte attachment in a CD1 1a/CD18dependent manner. 11,12 The cell distribution of ICAM-3 has not been studied in detail, but its expression seems to be restricted to myeloid and lymphoid cells.10 The differential expression of ICAMs on different cell types, at distinct stages of cell activation or maturation, may constitute one important mechanism that regulates the cell-cell interactions mediated by LFA-1. Langerhans cells (LCs) are a type of dendritic antigen-presenting cells found in the epidermal layer of the skin. They are derived from precursor cells origSupported by grants from FISS-INSALUD (91/0259) and from Fundaci6n Ramdn Areces (to FSM). Accepted for publication May 11, 1993. Address reprint requests to Dr. Francisco Sanchez-Madrid, Servicio de Inmunologia, Hospital de la Princesa, C/Diego de Ledn 62, 28006 Madrid, Spain.

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inating in the bone marrow, which migrate toward the epidermis, where they differentiate into LCs.13'14 The phenotypic profile of LCs includes numerous cell surface glycoproteins common to other dendritic mononuclear, macrophage/monocyte, or lymphoid cells such as the HLA-class 11 molecules, the CD45 leukocyte common antigen, the CD4 T helper lymphocyte marker, or the activation antigen CD6915-19; they also bear the CD1 HLA class I-like molecules as a characteristic antigenic marker.2'22 LCs play a critical role in antigen presentation to T lymphocytes which is essential for the induction of an immune response against cutaneously contacted antigens.23 25The binding of LCs to T cells in the course of an efficient T-cell activation involves the participation of adhesion and accessory molecules. The role of the adhesion molecule receptor/ligand pairs such as LFA-1/ICAM-1 or CD2/LFA-3 in the generation of a T-cell response induced by cultured LCs has been reported.26 LFA-1, LFA-3, and ICAM-1 are not present on freshly isolated human LCs;27'28 however, LCs show an up-regulated expression of ICAM-1 and LFA-3 as they become more potent antigenpresenting cells, either in tissue culture or "in vivo" after migrating to lymph nodes.23 In this report, we have studied the tissue distribution of ICAM-3 in normal lymphoid and nonlymphoid organs and found that its expression was restricted to myeloid and lymphoid cells. Interestingly enough, we have identified the expression of ICAM-3 on epidermal dendritic LCs, whereas the expression of ICAM-1 and ICAM-2 molecules was undetectable on these cells.

Materials and Methods Monoclonal Antibodies Anti-ICAM-3 HP2/19, TP1/24, and TP1/25 monoclonal antibodies (mAbs) were obtained from fusions with splenocytes of mice immunized with the human leukemic JM T-cell line (HP2/19)29 and human peripheral blood lymphocytes (PBLs) activated for 24 hours with a combination of PMA and antiCD3 mAb.30 The anti-ICAM-3 CBR-IC3/1 mAb has been described10 and was generously provided by Dr. T. A. Springer (Center for Blood Research, Boston, MA). The anti-CD11a TS1/11, antiCD18 TS1/18, and anti-CD1a NA1/34 have been described.31'32 The anti-ICAM-1 RR1/133 and the anti-ICAM-2 6D59 mAbs were kind gifts from Dr. R. Rothlein (Boehringer Ingelhein, Ridgefield, CT) and Dr. P. Nortamo (University of Helsinki, Helsinki, Finland), respectively. mAbs were purified from ascites

fluids with protein A coupled to Sepharose (Pharmacia Fine Chemicals, Uppsala, Sweden). The monoclonal Ig (IgG1,K) from the P3X63 myeloma cell line was used as negative control.

Cells and Tissues PBLs were isolated from whole blood by FicollHypaque centrifugation, and they were activated by culturing the cells for 48 h in the presence of 0.5% phytohemagglutinin (PHA, Pharmacia). Cells were cultured in RPMI 1640 (Flow Laboratories, Irvine, Scotland) containing 10% fetal calf serum (Flow). Neutrophils were isolated from fresh normal human blood by Ficoll-Hypaque density gradient centrifugation (Pharmacia) followed by sedimentation at 1 x g in 1.3% dextran (Sigma Chemical Co., St. Louis, MO) at room temperature. The neutrophilenriched fraction was further purified by hypotonic lysis of erythrocytes, giving a purity higher than 98%. Human tonsils were obtained from children who underwent tonsillectomy. Normal thymuses were from children who had had open heart surgery. Normal samples of spleen, lymph nodes, lung, liver, kidney, and skin were from adult patients who had undergone an exploratory or therapeutic surgery. Two dermatopathic lymph nodes were also included in the study. All tissues were immediately snap frozen in liquid nitrogen and OCT compound (Ames Company, Miles Laboatories, Elkhart, IN) and stored at -80 C. Bone marrow smears were prepared from healthy donors who had undergone a bone marrow harvest for bone marrow transplantation.

Purification of ICAM-3 by mAb Affinity Chromatography ICAM-3 was purified from detergent lysates of human neutrophils by affinity chromatography using the anti-ICAM-3 TP1/24 mAb coupled to CNBractivated Sepharose CL-4B (Pharmacia) at 2 mg/ ml. Human neutrophils were lysed in a buffer containing 20 mmol/L Tris-HCI (pH 8.0) and 0.5% Triton X-100. Lysates were centrifuged at 2500 x g for 60 minutes, and the supernatant was passed in series through columns of CNBr-activated glycinequenched Sepharose CL-4B and anti-ICAM-3 TPi/ 24-Sepharose CL-4B. The TP1/24-Sepharose column was washed with 50 mmol/L ethanolamine (pH 10), 0.5 mol/L NaCI, and 1% n-octyl-f-Dglucopyranoside. Then, proteins bound to the column were eluted with 50 mmol/L ethanolamine

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(pH 11), 0.5 mol/L NaCI, and 1% n-octyl-p3-Dglucopyranoside. Eluates were immediately neutralized with 2 mol/L Tris-HCI (pH 6.7). Fractions containing the eluted ICAM-3 were identified by sodium dodecyl sulfate (SDS)-7% polyacrylamide gel electrophoresis (PAGE), and proteins were visualized by silver staining.

Radiolabeling, Immunoprecipitation, and Electrophoresis Immunoaffinity-purified ICAM-3 (0.5 pg) or PBL, and JM leukemic T cells were radioiodinated with chloroglycoluril (lodogen, Pierce Chemicals Co., Rockford, IL). Cells were lysed with 1% Triton X-100. For immunoprecipitation, equal amounts of 1251-labeled cell lysates or immunoaffinity-purified ICAM-3 was incubated with 100 p1 of mAb-containing culture supernatants. To isolate immune complexes, the anti-mouse K-chain 187.1 mAb31'34 was added followed by protein A from Staphylococcus aureus coupled to Sepharose (Pharmacia). Immunoprecipi-

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tates were processed as described,31 and samples were subjected to SDS-10% PAGE and autoradiography.

Immunohistochemical Staining of Human Tissue Sections Immunoperoxidase Staining Acetone-fixed 4-pm-thick frozen sections were incubated with mAb culture supernatants for 30 minutes at room temperature. Subsequently, sections were incubated with a peroxidase-conjugated rabbit anti-mouse IgG (Dakopatts, Copenhagen, Denmark). Each incubation was followed by three washes with Tris saline (TBS) isotonic buffer (pH 7.6). Then, sections were developed with Graham-Karnovsky solution containing 3,3'diaminobenzidine hydrochloride (0.5 mg/ml) and hydrogen peroxide. The reaction was stopped by washing with TBS. Sections were counterstained with Carazzi's hematoxylin, dehydrated, and mounted by routine methods.

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Figure 1. Specificity of novel anti-ICAM-3 mAb. (A) Immunoprecipitation by anti-ICAM-3 mAbfrom human activated PBL. 125I-labeled cell lysates of human PHA-activated Tlymphocytes were precipitated with HP2/19 (Lane 1), anti-ICAM-3 CBR-IC3/1 (Lane 2), TP1/24 (Lane 3), TP1/25 (Lane 4), anti-CD1la (LFA-1) TS1/11 (Lane 5), and anti-CD18 TS1/18 mAb (Lane 6). Precipitation with control P3X63 mAb is also included (Lane 7). Immune complexes were isolated, and reduced samples were subjected to SDS-7% PAGE and autoradiography. (B) Immunoprecipitation by antiICAM-3 mAb of immunoaffinity-purified ICAM-3. The ICAM-3 antigen was purified from human neutrophils by immunoaffinity chromatography. A sample containing purified ICAM-3 was electrophoresed under reducing conditions on a SDS-7% PAGE and visualized by silver staining (Lane 1). Purified ICAM-3 was radiolabeled andprecipitated with thefollowing mAbs: P3X63 (Lane 2), anti-ICAM-3 CBR-IC3/1 (Lane 3), TP1/24 (Lane 4), and HP2/19 (Lane 5). Immune complexes were isolated, and reduced samples were subjected to SDS-796 PAGE and autoradiography.

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Alkaline Phosphatase Anti-Alkaline Phosphatase Method Sections were incubated with mAb culture supernatants for 30 minutes at room temperature. Subsequently, they were incubated with a rabbit antimouse IgG antiserum (DAKO, Copenhagen, Denmark) followed by an incubation with the alkaline phosphatase anti-alkaline phosphatase complex (DAKO). Each incubation was followed by three washes in TBS. Finally, the alkaline phosphatase reaction was developed by incubating the sections with a Tris-HCI (50 mmol/L, pH 8.4) buffer solution containing 0.2 mg/ml of Naphtol AS-MX Phosphate (Sigma) and 1 mg/ml of Fast Red (Sigma) with 10-5 mol/L levamisole as endogenous alkaline phosphatase inhibitor. Double Immunoenzymatic Staining Double immunostaining was done by the sequential method proposed by Mason et al.35 After the development of diaminobenzidine reaction, the sections were saturated with nonspecific mouse immunoglobulin from the P3X63 myeloma line, washed, and then incubated with the second mAb in the same conditions. Subsequently, an alkaline phosphatase anti-alkaline phosphatase method was developed as described above using Fast Blue salt instead of Fast Red. The reaction product was a dark blue precipitate that contrasted with the brown colour of diaminobenzidine reaction. Sections were mounted in buffered gelatin for microscopic examination. Controls were performed by substituting the first, second, or both mAbs for the P3X63 myeloma Ig.

Quantitative Analysis of LC To estimate the number of LC stained with each mAb, we used a method previously described.36 Briefly, a minimum of 5 mm of linear epidermis was evaluated. LCs were counted by using a reticulate ocular, considering positive those cells with a visible nucleus. Dendrites were not counted. The number of LCs is expressed as the number per 1 mm of linear epidermis.

Results Reactivity of Three Novel mAbs to the ICAM-3 Molecule We selected three mAbs (HP2/19, TP1/24, and TP1/ 25) that immunoprecipitated a 135-kda polypeptide

Table 1. Tissue Distribution of ICAM-3

Lymphoid organs Thymus Medullary thymocytes Cortical thymocytes Epithelial cells Hassall's corpuscles Others Lymph node and tonsil B-cell area Mantle cells Germinal center B cells Germinal center T cells Dendritic cells and macrophages T-cell area Lymphocytes Dendritic cells and macrophages Postcapillary venules Sinusoids (lymph node) Epithelial cells (tonsil) Others

+

+

+

Spleen B-cell area Mantle cells Germinal center B cells Germinal center T cells Others T-cell area Lymphocytes Others Red pulpe cells

+

Bone marrow Red cells Mature granulocytes White cell precursors Megacariocytes Lymphocytes Nonlymphoid organs Skin Keratinocytes Dendritic epidermal cells Dendrocytes Skin appendages Dermal lymphocytes Others (blood vessels, etc.) Liver Hepatocytes Kupffer cells Sinusoidal lymphocytes Biliary epithelium Others Kidney Glomerular structures Tubular epithelium Glomerular and interstitial mononuclear cells Others Lung Alveolar cells Bronchial cells Interstitial mononuclear cells Others

from 1251-cell surface labeled PHA-activated PBL (Figure 1A, Lanes 1, 3, and 4, respectively). This antigen had a molecular size identical to that of ICAM-3, precipitated by the recently described antiICAM-3 CBR-IC3/1 mAb (Figure 1A, Lane 2). To ascertain the specificity of these mAbs, we purified the 135-kda molecule from human neutrophils by affinity

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Figure 2. Immunoperoxidase staining of totsil with anti-ICAM-3 HP2/19 (A), antt-ICAM-3 CBR-IC3/1 (B), and anti-ICAM-1 (C), mAbs

(X250).

chromatography using a TP1 /24-Sepharose column (Figure 1B, Lane 1). The purified antigen was radioiodinated and precipitated by the anti-ICAM-3 CBRIC3/1 as well as by the TP1/24 and HP2/19 mAbs (Figure 1B, Lanes 3-5, respectively) and TP1/25 (data not shown). These results demonstrated that these mAbs recognize the ICAM-3 molecule.

Tissue Distribution of ICAM-3 The expression of ICAM-3 has previously been found to be restricted to cells of the leukocyte lineage by immunofluorescence flow cytometry studies.10 We have performed immunohistochemical studies of tissue sections from different lymphoid and nonlymphoid organs with the anti-ICAM-3 HP2/19 mAb. The staining patterns obtained in different tissues with this mAb are summarized in Table 1. A similar pattern of staining was obtained with the different anti-ICAM-3 mAbs described above, including the CBR-IC3/1 mAb (Figure 2 and data not shown).

In liver, kidney, and lung, only small mononuclear cells located in blood vessels and interstitium were stained with the anti-ICAM-3 mAb. There was no staining of epithelial cells, mesenchymal cells, or extracellular matrix. In thymus, the expression of ICAM-3 was restricted to medullary thymocytes (Table 1). In normal lymph node and tonsil, the staining was confined to paracortical lymphocytes (T-cell zone) and follicular mantle cells (B cells, p,/8+) (Figure 2A and B). Follicular center B cells, dendritic cells, and macrophages were negative. The expression pattern of ICAM-3 in tonsil was compared to that of ICAM-1 and found to be different. ICAM-1+ structures comprised the vascular endothelium in postcapillary venules as well as dendritic cells in both paracortex and follicles (Figure 2C). No lymphoid cells were ICAM-1 +. Immunostaining of bone marrow smears with anti-ICAM-3 mAb showed reactivity in myeloid mature cells and lymphocytes, but not in other lineages such as erythroid cells, megakariocytes, and platelets (Table 1).


Figure 3. Immunoenzymatic staining of epidermal LC. Skin sections were stained with a double immunoenzymatic method, combining in thefirst step the indirect immunoperoxidase method (brown) and, in the second, the alkaline phosphatase anti-alkaline phosphatase method (blue). (A) first mAb, anti-ICAM-3 HP2/19 mAb; second mAb, P3X63 (x250). (B) first mAb, P3X63; second mAb, anti-CDla NA 1/34 (x250). (C) first mAb, anti-ICAM-3 HP2119; second mAb, anti-CDla mAb NA1134 (X500). (D) first mAb, P3X63; second mAb, P3X63 (x500).

Expression of ICAM-3 by Langerhans Cells Immunohistochemical staining of skin sections with anti-ICAM-3 mAb showed reactivity with dendritic epidermal cells and dermal lymphocytes (Figure 3A). The ICAM-3+ cells were regularly distributed along the epidermis and showed large cytoplasmic projections that were similar to those observed in CD1 + LCs (Figure 3B). To evaluate more accurately the nature of the ICAM-3+ dendritic cells, double immunostaining was performed, combining the ICAM-3 with the other well-characterized markers of LCs such as CD1, CD45, or HLA-DR antigens. The majority of CD1 + dendritic epidermal cells co-expressed the ICAM-3 molecule, as demonstrated by double staining with mAb specific for the two antigens (Figure 3C). Quantitative estimation showed that from the to-

tal number of LCs bearing the CDla antigen, 72% expressed the ICAM-3 molecule. Similar results were obtained when anti-ICAM-3 mAb was combined with mAb specific for CD45 or HLA-DR (data not shown). The ICAM-3 staining pattern (Figure 4A) was compared with that of the ICAM-1 and ICAM-2 antigens in serial sections of skin (Figure 4B and C, respectively). No detectable staining of dendritic epidermal cells was found with anti-lCAM-1 and antiICAM-2 mAbs as opposed to the strong reactivity with anti-ICAM-3 mAbs. To assess the expression of ICAM-3 and ICAM-1 antigens on "in vivo" activated LCs, immunoperoxidase staining was performed in two dermatopathic lymph nodes, a well-known process with a high content of LCs in paracortical zones. Serial sections of these areas showed the presence of LCs bearing CDia and ICAM-3 antigens, as well as detectable levels of ICAM-1 (Figure 5).

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immunoaffinity-purified ICAM-3 molecule from human neutrophils. Immobilized purified ICAM-3 was able to support T-cell attachment in an LFA-1dependent manner (our unpublished observations), further demonstrating its function as an LFA-1 counter-receptor. We have found that ICAM-3 expression is restricted to cells from the myeloid and lymphoid lineages, in accordance with a previous cell distribu-

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Figure 4. Immunoperoxidase staining of skin by anti-ICAM-3 (A), anti-ICAM-1 (B), and anti-ICAM-2 (C) mAbs (X250).

Discussion This study reports on the expression of ICAM-3, a recently described counter-receptor for LFA-1, on different lymphoid and nonlymphoid tissues by immunohistochemical techniques. We have obtained three novel anti-ICAM-3 mAbs that recognize an identical cell surface glycoprotein on T cells as the one defined by the previously described CBR-IC3/1 anti-ICAM-3 mAb.10 Moreover, the different antiICAM-3 mAbs were able to precipitate the

tion study by immunofluorescence flow cytometry.10 We observed ICAM-3 expression on certain cell subsets from lymphoid organs, whereas nonlymphoid cell types from either lymphoid or nonlymphoid organs lack ICAM-3 expression. ICAM-3 staining was preferentially localized on medullary T cells in the thymus, as well as on lymphocytes in T-cell areas and mantle B cells of secondary lymphoid organs. Remarkably, a strong staining for ICAM-3 was found on epidermal dendritic cells corresponding to CD1 + resident LCs. The tissue distribution of ICAM-3 differs from that of the two other described counter-receptors for LFA-1, ICAM-1, and ICAM-2, thus suggesting a specialization for different functional aspects of LFA-1-mediated leukocyte interactions. Despite basal levels of ICAM-1 expression that can be found on endothelial and epithelial cells, the expression of this ligand is essentially inducible and regulated during inflammation and immunity,7 thus playing a role in cell recruitment and migration toward inflamed tissues.1 2 By contrast, ICAM-2 is constitutively expressed by resting endothelium, allowing circulation of LFA-1+ lymphocytes through normal vessels.8'9 On the other hand, ICAM-3 molecule is highly expressed by resting leukocytes, and this fact may allow both homotypic and heterotypic leukocyte intercellular interactions (T cell-T cell, T cell-B cell, antigen presenting cell-T cell), which are crucial in the initiation of immune responses. In this context, we have found that homotypic leukocyte adhesion can be initiated through the ICAM-3 molecule (our unpublished observations). Epidermal LCs are the best-characterized nonlymphoid dendritic cells. Phenotypic analysis of LCs has demostrated that they bear numerous functionally relevant leukocyte-restricted antigen markers such as the CR3-complement receptor,37 the CD45 leukocyte-specific tyrosine phosphatase, 17 the CD4 T helper lymphocyte molecule,18 and the CD69 lymphocyte activation antigen.19 In addition, LCs also express the CD1 immature thymocyte antigen,14'20-22 major histocompatibility complex


Figure 5. ExpreNssion of ICAM- I and ICAM-3 by LC in dermatopathic lymphadenopaihy. Serial sections were stained with anti-ICAM- I (A), anti-ICAM-3 (B), anzd anti-CD a (C) mAbs. The paracortical area is sbhuon (X250).

class 11 molecules,14 as well as a number of other less well-characterized antigens.23'24'38 A number of studies have addressed the expression and function of several adhesion receptors on LCs. The involvement of LFA-1/ICAM-1 and CD2/ LFA-3 adhesion pathways has been clearly established in the interaction of T cells with cultured LCs required for an optimal T-cell activation.2326 Additionally, epidermal LCs express j1 integrins, mainly VLA-4, VLA-5, and VLA-6, that mediate their adhesion to laminin and fibronectin.3940 Very interestingly, our results show that the LFA-1 counter-receptor, ICAM-3, is expressed by dendritic epidermal LCs but is absent from other dendritic cells from different lymphoid organs. Although a definitive (immunoelectromicroscopic) proof for the LC nature of ICAM-3-reactive epidermal dendritic cells should be desirable, our data are indicative that ICAM-3 is constitutively expressed on resident LCs. By contrast, LCs appear to be negative when skin tissue sections are stained with antibodies specific for the two other described LFA-1 counterreceptors, ICAM-1 and ICAM-2. However, some dis-

crepancies have been reported regarding the expression of ICAM-1 by resident LCs. Whereas ICAM-1 is hardly detectable or negative on LCs by immunohistochemical staining, it has been visualized by immunoelectronmicroscopy.4144 On the other hand, it has recently been reported that HLADR' LCs acquire the ICAM-1 antigen after culturing for 12 hours and that the LFA-1/ICAM-1 interaction is functionally required for an efficient LC-driven stimulation of alloreactive T cells.45 Interestingly enough, ICAM-3 expression was not detected on dendritic cells of the parafollicular cortex of normal lymph nodes. This is an unexpected finding since it has been proposed that such cells are derived from epidermal LCs. It is possible that ICAM-3 is down-regulated during the normal migration of LCs of lymph nodes. It is also feasible that the low number of ICAM-3+ dendritic cells in normal lymph nodes is not detectable by the conventional techniques employed by us. On the other hand, our results show that in inflammatory conditions the expression of ICAM-3 on lymph node LCs is indeed conserved and that an up-regulated expression of

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ICAM-1 occurs. It will be interesting to define the conditions that regulate the expression of ICAM-3 on LCs. LCs, like other dendritic cells, are motile and efficiently cluster and activate T cells. The LCs are specialized to mediate several physiological components of immunity, and they can migrate to local nodes as part of a cutaneous immune response.23'25 Our finding on the abundant expression of ICAM-3 by resident epidermal LCs is of interest in terms of the potential function of this counter-receptor at the initiation phase of LC-leukocyte interactions taking place during localized skin immune reactions. Furthermore, we entertain the possibility that the expression of ICAM-1 and ICAM-3 is reciprocally regulated on resident epidermal and activated LCs. Our observations that CD1 + LCs localized in the paracortical zone of dermatopathic lymph nodes, a condition with a high content of activated LCs, express both ICAM-1 and ICAM-3 suggest that both adhesion molecules are important in T-cell stimulation driven by LCs. This is in agreement with previous results showing the small variation in the level of ICAM-3 expression during the activation process of T lymphocytes.10 Therefore, the possibility that ICAM-3 acts as an "accessory" molecule involved in T-lymphocyte activation induced by epidermal LCs is currently being addressed. It is conceivable that ICAM-3 may be one of several cell surface molecules that may be employed by LCs to enhance T-cell activation in the localized skin immune response.

References 1. Springer TA: Adhesion receptors of the immune system. Nature 1990, 346:425-434 2. Larson RS, Springer TA: Structure and function of leukocyte integrins. Immunol Rev 1990, 114:181-217 3. Marlin SD, Springer TA: Purified intercellular adhesion molecule-1 (ICAM-1) is a ligand for lymphocyte function-associated antigen 1 (LFA-1). Cell 1987, 51: 813-819 4. Staunton DE, Marlin SD, Stratowa C, Dustin ML, Springer TA: Primary structure of intercellular adhesion molecule 1 (ICAM-1) demonstrates interaction between members of the immunoglobulin and integrin supergene families. Cell 1988, 52:925-933 5. Simmons D, Makgoba MW, Seed B: ICAM, an adhesion ligand of LFA-1, is homologous to the neural cell adhesion molecule NCAM. Nature 1988, 331:624-627 6. Staunton DE, Dustin ML, Springer TA: Functional cloning of ICAM-2, a cell adhesion ligand for LFA-1 homologous to ICAM-1. Nature 1989, 339:361-364

7. Dustin ML, Rothlein R, Bhan AK, Dinarello CA, Springer TA: Induction by IL-1 and interferon, tissue distribution, biochemistry, and function of a natural adherence molecule (ICAM-1). J Immunol 1986, 137: 245-254 8. de Fougerolles AR, Stacker SA, Schwarting R, Springer TA: Characterization of ICAM-2 and evidence for a third counter-receptor for LFA-1. J Exp Med 1991, 174:253-267 9. Nortamo P, Salcedo R, Timonen T, Patarroyo M, Gahmberg CG: A monoclonal antibody to the human leukocyte adhesion molecule intercellular adhesion molecule-2: cellular distribution and molecular characterization of the antigen. J Immunol 1991, 146:25302535 10. de Fougerolles AR, Springer TA: Intercellular adhesion molecule 3, a third adhesion counter-receptor for lymphocyte function-associated molecule 1 on resting lymphocytes. J Exp Med 1992, 175:185-190 11. Fawcett J, Holness CLL, Needham LA, Turley H, Gatter KC, Mason DY, Simmons DL: Molecular cloning of ICAM-3, a third ligand for LFA-1, constitutively expressed on resting leukocytes. Nature 1992, 360:481484 12. Vazeux R, Hoffman PA, Tomita JK, Dickinson ES, Jasman RL, St John T, Gallatin WM: Cloning and characterization of a new intercellular adhesion molecule ICAM-R. Nature 1992, 360:485-488 13. Katz SI, Tamaki K, Sachs DH: Epidermal Langerhans cells are derived from cells which originate in the bone marrow. Nature 1979, 282:324-326 14. Murphy GF: Cell membrane glycoproteins and Langerhans cells. Hum Pathol 1985, 16:103-112 15. Stingl G, Romani N, Wolff K: Langerhans's cells: a distinctive member of bone marrow-derived dendritic cells. Advances in Dermatology. Edited by JP Callen, MV Dahl, CE Golitz, JE Rassmussen, SJ Stegman. Chicago, 1987, pp 269-281 16. Rowden G, Lewis MG, Sullivan AK: la antigen expression on human epidermal Langerhans' cell. Nature 1977, 268:247-248 17. Flotte TJ, Murphy GB, Bhan AK: Demonstration of T200 on human Langerhans' cells surface membranes. J Invest Dermatol 1984, 82:535-537 18. Wood GS, Warner NL, Warnke RA: Leu-3/T4 antibodies react with cells of monocyte/macrophage and Langerhans' lineage. J Immunol 1983, 131:212-216 19. Bieber T, Rieger A, Stingi G, Sander E, Wanek P, Strobel J: CD69, an early activation antigen on lymphocytes, is constitutively expressed by human epidermal Langerhans cells. J Invest Dermatol 1992, 98:771-776 20. Murphy GB, Bhan AK, Sato S, Mihm MC, Harrist TJ: A new immunologic marker for human Langerhans cells. N Engl J Med 1981, 304:791-792 21. Murphy GB, Bhan AK, Sato S, Harrist TJ, Mihm MC: Characterization of Langerhans cell by the use of monoclonal antibodies. Lab Invest 1981, 45:465-472


22. Fithian E, Kung P, Goldstein G, Rubenfeld M, Fenoglio C, Edelson R: Reactivity of Langerhans cells with hybridoma antibody. Proc Natl Acad Sci USA 1981, 78: 2541-2544 23. Steinman RM: The dendritic cell system and its role in immunogenicity. Annu Rev Immunol 1991, 9:271-296 24. King PD, Katz DR: Mechanisms of dendritic cell function. Immunol Today 1990, 11:206-211 25. Streilein JW, Grammer SF, Yoshikawa T, Demidem A, Wermeer M: Functional dichotomy between Langerhans cells that present antigen to naive and memory/ effector T lymphocytes. Immunol Rev 1990, 117:159183 26. Teunissen MBM: Functional role of adhesion molecules LFA-3 and ICAM-1 on cultured human epidermal Langerhans cells in antigen-specific T-cell activation. J Invest Dermatol 1992, 99:77S-79S 27. Teunissen MBM, Wormmeester J, Rongen HAH, Kapsenberg ML, Bos JD: Conversion of human epidermal Langerhans cells into interdigitating cells is not associated with functional maturation. Eur J Dermatol 1991, 1:45-54 28. Romani N, Lenz A, Glassel H, Stossel H, Stanzl U, Majdic 0, Fritsch P, Schuler G: Cultured human Langerhans cells resemble lymphoid dendritic cells in phenotype and function. J Invest Dermatol 1989, 93: 600-609 29. Sanchez-Madrid F, de Landazuri MO, Morago G, Cebrian M, Acevedo A, Bernabeu C: VLA-3: a novel polypeptyde association within the VLA molecular complex: cell distribution and biochemical characterization. Eur J Immunol 1986, 16:1343-1349 30. Cebrian M, Yague E, Rincon M, L6pez-Botet M, de Landazuri MO, Sanchez-Madrid F: Triggering of T cell proliferation through AIM, an activation inducer molecule on activated human lymphocytes: J Exp Med 1988, 168:1621-1637 31. Sanchez-Madrid F, Nagy J, Robbins E, Simon P, Springer TA: A human leukocyte differentiation antigen family with distinct a-subunits and a common ,B-subunit: the lymphocyte function-associated antigen (LFA-1), the C3bi complement receptor (OKM1/ Mac-1) and the p150.95 molecule. J Exp Med 1983, 158:1785-1803 32. Sanchez-Madrid F, Toribio ML, Gambon F, de Landazuri M: Cell surface molecular changes on the activation of human thymocytes. J Immunol 1985, 135:

3938-3943 33. Rothlein R, Dustin ML, Marlin SD, Springer TA: A human intercellular adhesion molecule (ICAM-1) distinct from LFA-1. J Immunol 1986, 137:1270-1274 34. Ware CF, Reade JL, Der DL: A rat anti-mouse kappa chain-specific monoclonal antibody, 187.1.10; purifi-

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

cation, immunochemical properties and its utility as a general second-antibody reagent. J Immunol Methods 1984, 74:93-98 Mason DY, Abdulaziz Z, Falini B, Stein H: Double immunoenzymatic labeling. Immunocytochemistry. Edited by J Polak, S Van Noorden. Bristol, PSG Wright, 1983, p 113 Harrist TV, Muhkbauer JG, Murphy GF, Mihm MC, Bhan AK: T6 is superior to la (HLA-DR) as a marker for Langerhans cells and Indeterminate cells in normal epidermis: a monoclonal antibody study. J Invest Dermatol 1983, 80:100-103 Stingl G, Wolff-Schreiner EC, Pichler WJ, Gschnait F, Knapp W, Wolff K: Epidermal Langerhans' cells bear Fc and C3 receptors. Nature 1977, 268:245-246 Zhou L-J, Schwarting R, Smith HS, Tedder TF: A novel cell-surface molecule expressed by interdigitating reticulum cells, Langerhans cells, and activated lymphocytes is a new member of the Ig superfamily. J Immunol 1992, 149:735-742 Staquet MJ, Levarlet B, Dezutter-Dammbuyant C, Schmitt D: Human epidermal Langerhans cells express 13 integrins that mediate their adhesion to laminin and fibronectin. J Invest Dermatol 1992, 99:12S14S Le Varlet B, Dezutter-Dammbuyant C, Staquet MJ, Delorme P, Schmitt D: Human epidermal Langerhans cells express integrins of the ,11 subfamily. J Invest Dermatol 1991, 96:518-522 Lange-Wantzin GL, Ralfkiaer E, Lisby S, Rothlein R: The role of intercellular adhesion molecules in inflamatory skin reactions. Br J Dermatol 1988, 119:141145 Nickoloff BJ: Role of interferon-e in cutaneous trafficking of lymphocytes with emphasis on molecular and cellular adhesion events. Arch Dermatol 1988, 124: 1835-1843 Singer KH, Tuck DT, Sampson HA, Hall RP: Epidermal keratinocytes express the adhesion molecule intercellular adhesion molecule-1 in inflamatory dermatoses. J Invest Dermatol 1989, 92:746-750 De Panfilis G, Manara GM, Ferrari C, Torresani C: Adhesion molecules on the plasma membrane of epidermal cells. II. The intercellular adhesion molecule-1 is constitutively present on the cell surface of human resting Langerhans cells. J Invest Dermatol 1990, 94: 317-321 Simon JC, Cruz PD, Tigelaar RE, Sontheimer RD, Bergstresser PR: Adhesion molecules CD11a, CD18 and ICAM-1 on human epidermal Langerhans cells serve a functional role in the activation of alloreactive T cells. J Invest Dermatol 1991, 96:148-151