And Tumor Cells in Kaposi's Sarcoma

Americani Joirnal ofPathology, Vol. 148, No. 5, May 1996 Copyfight X) American Societyfor Investigative Pathology CD40 Antigen Is Expressed by Endothelial Cells and Tumor Cells in Kaposi's Sarcoma Johannes Pammer,* Andreas Plettenberg,t of CD40 by Kaposi sarcoma tumor ceUs might Wolfgang Weninger,t Barbara Diller,t play an important role in thepathogenesis ofthis Michael Mildner,t Aumaid Uthman,t neoplasm. (AmJPathol 1996, 148:1387-1396) Wolfgang lssing,$ Michael Sturzl,§ and Erwin Tschachlert From the Division ofImmunology,t Allergy, and Infectious The CD40 antigen is a 45- to 50-kd transmembrane Diseases, Department ofDermatology, and Institute of glycoprotein that belongs to the nerve growth factor Clinical Pathology,* University of Vienna Medical School, receptor (NGFR)/tumor necrosis factor receptor Vienna, Austria, and the Department of Otolaryngology,t (TNFR)-superfamily.1 2 Members of this superfamily Grosshadern Clinic, Ludwig Maximilians University, are intimately involved in the regulation of cell sur- Munich, and the Max Planck Institute ofBiochemistry,j vival and include the T cell activation antigen CD27, Martinsnied, Germany the lymphocyte activation antigen CD30, the low af- finity NGFR, the FAS antigen CD95, the TNFRs CD12012 and the lymphotoxin-f3 receptor.3 CD40 was initially described on B cells and certain carci- The CD40 antigen is a member of the tumor ne- nomas.4'5 It has since become clear that it is also crosis factor receptor/nerve growth factor re- expressed on dendritic cells,6 monocytes,7 thymus ceptor superfamily and is involved in ceUprolif- epithelia," and normal basal epithelial cells of the eration, differentiation, and survival. Using nasopharynx,9 ectocervix,10 and skin.11 The pres- different monoclonal antibodies, wefound CD40 ence of this membrane antigen on endothelial cells expression by immunohistochemistry on CD31- has been reported in the past,12 but only very re- and CD34-positive Kaposi's sarcoma spindle cently have the regulatory and functional aspects of ceUs in aU tumors of 18 HIV-1 seropositive and 4 CD40 expression on human umbilical vein endothe- HIV-1 seronegative patients. Western blot analy- lial cells (HUVECs) and human aortic endothelial sis of tumor lysates detected a 48- to 50-kd gly- cells been studied.13,14 coprotein corresponding to the CD40 antigen ex- Stimulation of CD40 by either monoclonal antibod- pressed by B lymphocytes. CD40 expression was ies (MAbs) or its natural ligand induces DNA repli- also detectable in one offour cultures ofspindle cation in resting B lymphocytes, leads to sustained cells derivedfrom Kaposi sarcoma tissue. Treat- proliferation of these cells in the presence of certain ment ofthe CD40-positive spindle ceUs but not of cytokines, and can drive B cell differentiation and the CD40-negative ones with interferon-y up-reg- aggregation.1 Activation of monocytes via the CD40 ulated CD40 surface expression. Besides on Ka- antigen leads to the secretion of interleukin (IL)-6, posi sarcoma tumor ceUs, CD40 was distinctly IL-8, and TNF7 and triggering of CD40 on HUVECs present on vascular endothelial ceUs in areas increases expression of the leukocyte adhesion mol- within and adjacent to the tumors and in benign ecules E-selectin, vascular adhesion molecule-1 and inflammatory lesions such as granulation tissue intercellular adhesion of HIV-1-negative patients. In contrast, CD34- molecule-i 13 Besides their negative endothelia of thin waUed vessels, most likely lymphatics, werepredominantly CD40 neg- Supported by grant P01437-MED from the Austrian Science Foun- ative. Only faint or no CD40 expression was dation and by the European Concerted Action: Oncogenesis in HIV found on endothelial cells in normal skin. We Infection. conclude from our data that expression of the Accepted for publication December 18, 1995. CD40 antigen by endothelial ceUs is Address reprint requests to Dr. Erwin Tschachler, Division of up-regulated Immunology, Allergy, and Infectious Diseases, Department of Der- during tissue inflammation. As signaling through matology, University of Vienna Medical School, Wghringer GCurtel CD40 is able to increase ceU survival, expression 18-20, A-1090 Vienna, Austria. 1387 1388 Pammer et al AJP May 1996, Vol. 148, No. 5 important regulatory role within the immune system, and fistulas of the skin with formation of granula- CD40 and other members of the NGFR/TNFR family tion tissue, and samples of normal skin were ob- have recently been implicated in Epstein-Barr virus tained from the files of the Institute of Clinical (EBV)-associated growth transformation.15 Based on Pathology and the Department of Dermatology at the evidence that CD40 and other proteins of the the University of Vienna. For immunostaining of NGFR/TNFR family associate with a newly identified cryostat sections and for Western blot analysis, 6 cellular protein that interacts with the EBV latent in- KS lesions were biopsied under local anesthesia fection membrane protein (LMP-1), it has been sug- after informed consent was obtained from patients gested that this interaction may interfere with recep- with AIDS. The CD40-positive B cell line R17 and tor-associated signal transduction and constitutively the CD40-negative T cell line H9 were cultured promote cell growth.15 under standard conditions in RPMI 1640 medium Kaposi's sarcoma (KS) is a tumor of probable vas- supplemented with 10% fetal bovine serum. cular origin16-18that has aroused considerable interest of late, as it is the most frequent neoplasm seen in Immunohistochemistry patients with acquired immune deficiency syndrome For immunostaining of paraffin sections, anti-CD31 (AIDS). However, despite intensive research over the (JC/70A, Dako, Glostrup, Denmark), anti-CD34 past few years,17'18-20 the origin of KS tumor cells has (QBend/10, Novocastra Laboratories, Newcastle not been clarified and it is even a matter of contention upon Tyne, UK), and anti-CD40 (BB-20, Serotec, whether KS is a real neoplasm or represents a reactive Kidlington, UK) MAbs or corresponding isotype- proliferative process.18'21 Suspected pathogenic matched control antibodies were applied. Incubation mechanisms regarding this tumor include cytokine with the primary antibodies was followed by a dysregulation22 23 and the participation of the HIV-1 tat streptavidin-biotin complex alkaline phosphatase gene product24 in the activation and growth of spindle system (super sensitive detection system, Biogenex, cells, as well as the potential involvement of a trans- San Ramon, CA). For some experiments an avidin- missible agent.18'25 Recently, noncellular DNA se- biotin staining technique (Vector Laboratories, Bur- quences homologous to EBV sequences have been lingame, CA) was used. identified in KS tissue.26 Although these herpesvirus- Immunoperoxidase staining of cryostat sections like sequences can be detected in practically all sam- was performed as described earlier29 with anti-CD40 ples of AIDS-associated and non-AIDS-associated KS MAbs BB-20 and G28-5 (a kind gift of J. A. Ledbet- analyzed by several groups,26-28 a role for this possi- ter, Seattle, WA). ble new herpesvirus in KS pathogenesis remains to be established. Western Blotting While screening for endothelial markers in normal and inflamed skin, we found distinct expres- For Western blotting, biopsies of KS or H9 and R17 sion of the CD40 antigen on endothelial cells in cells were lysed in NP40 lysis buffer as previously granulation tissue of chronic skin ulcers and fistu- described.29 Glycoproteins were enriched on a lating processes. Because of our interest in the lentil-lectin column (Pharmacia, Uppsala, Sweden) histogenesis and pathology of KS, we analyzed and eluted with 2 column volumes of 1 mol/L the CD40 expression in these tumors by immuno- a-methyl-mannopyranosid (Sigma Chemical Co., histochemical and biochemical methods. In our St. Louis, MO). After quantification, glycoproteins present study we evaluated KS at various stages were electrophoresed through an 8 to 18% gradi- and found strong expression of the CD40 antigen ent gel and transferred onto nitrocellulose mem- on both endothelial and spindle cells. branes (Schleicher & Schuell, Dassel, Germany). The immunoreaction was performed with either the BB-20 MAb or an appropriate isotype-matched Materials and Methods (IgG1; Coulter, Hialeah, FL) control antibody and Tissues and Cells an 1251-labeled sheep anti-mouse Ig antiserum (Amersham, San Jose, CA) as described.29 We analyzed a total of 26 biopsies of KS derived from 18 HIV seropositive and 4 HIV seronegative Tissue Culture and Immunostaining of patients. By routine histology, the biopsies were Cultured KS-Derived Cells classified as patch (n = 1), plaque (n = 9), and nodular KS lesions (n = 16). Formalin-fixed, par- Cultures of spindle cells derived from KS tissue were affin-embedded specimens of KS (n = 20), ulcers established by explant culture method (M7/2, M12/4) CD40 Antigen Expression in Kaposi's Sarcoma 1389 AJP May 1996, Vol. 148, No. 5 or by enzymatic dissection (M7Col 12, M 12T8) of KS distributed uniformly over the tumors (Figure 1) with biopsies from skin of two male patients with AIDS the exception of one sample in which CD40-negative (M7, Mi2). Cells were maintained in Dulbecco's min- areas within the tumor were detectable (Table 1, imal essential medium with 10% fetal bovine serum patient 16). On serial sections we could confirm that as previously described.30'31 KS spindle cells were CD40-positive tumor cells (Figure 2A) comprised the characterized by cytochemical staining and were majority of anti-CD31-positive (Figure 2B) and anti- positive for Ulex europaeus antigen-I and CD34-positive (Figure 2C) KS spindle cells. The BMA120.3°31 Polymerase chain reaction for the same staining pattern, ie, anti-CD40 reactivity of KS detection of KS-associated herpesvirus (KSHV) tumor cells, was obtained when frozen sections of six sequences in these cell cultures using the protocol different tumors derived from patients with AIDS published by Chang et al26 yielded negative re- were stained with two different anti-CD40 MABs sults.

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