Expression of the CD30 antigen in non-lymphoid tissues and cells.

Originally, expression of the CD30 antigen was shown to be typical of the tumour cells of Hodgkin's disease (HD) and of anaplastic large cell lymphomas (ALCLs). In reactive lymphoid tissue, CD30 is expressed only in a small population of activated lymphoid blasts. Since then, several reports have been published describing CD30 expression in non-lymphoid tissues and malignancies, such as embryonal carcinomas (ECs), seminomas, cultivated macrophages, two histiocytic neoplasms, decidual cells, and mesotheliomas. As CD30 detection is important in the differential diagnosis of HD and ALCL, the expression of CD30 in different non-lymphoid tissues was re-evaluated by immunohistology and in situ hybridization. Extra-lymphoid CD30 expression was found in 48/51 cases of EC or EC components of germ cell tumours, in decidual cells of 1/10 cases, in activated mesothelium in 16/28 pleural and peritoneal effusions, and in small foci of tumour cells in 2/8 mesotheliomas. CD30 expression was not confirmed in 27 germ cell tumours of the testis without an EC component nor in cultivated macrophages and 17 histiocytic malignancies. The knowledge of these CD30 expression patterns is important for the immunohistological differential diagnosis of anaplastic tumours. The absence of CD30 expression in reactive and neoplastic macrophages does not favour the concept that HD and ALCL are derived from these cells.

Antiproliferative effect of human interleukin-4 in human cancer cell lines: studies on the mechanism.

Interleukin-4 (IL-4) plays an important role in activating the immune system against malignant cells. The human interleukin-4 receptor (hIL-4R) is not only expressed by hematopoietic cells but also on a large number of tissue specimens which include colon, breast and lung carcinomas. In this study we report that rhIL-4 has an antiproliferative effect on 2 out of 3 non-small cell lung carcinoma (NSCLC) cell lines in vitro as measured by human tumor cloning assays (HTCA). In comparison, rhIL-4 had no effect on the growth of small cell lung carcinoma cell lines (SCLC) in vitro. The response towards the cytokine is correlated with expression of at least 1500 high affinity receptors/cell for hIL-4 on the responsive cell lines. Xenotransplanting the human lung tumor cell lines into nude mice followed by 12 days of systemic treatment of the mice with rhIL-4 revealed a significant growth retardation of the IL-4R positive NSCLC cell lines when compared with the controls, whereas the growth of the IL-4R negative SCLC cell lines was unaffected also in vivo. Studies of possible mechanisms involved in the antiproliferative effect of rhIL-4 showed that rhIL-4 does not induce apoptosis or modulation of the transcription factor c myc in the responsive NSCLC cell lines. Additionally, the expression of the epidermal growth factor receptor (EGFR), which is discussed as mediating autocrine/paracrine growth stimulation of NSCLC, is unaffected by rhIL-4. However, we have observed that rhIL-4 inhibited G1-S-phase cell cycle progression. We conclude that rhIL-4 has an antiproliferative effect on the growth of some NSCLC in vitro and in vivo. The mechanisms involved remain to be further elucidated.

Recombinant human interleukin 4 has antiproliferative activity on human tumor cell lines derived from epithelial and nonepithelial histologies.

Interleukin 4, a T cell-derived 20-kDa glycoprotein, plays an important role in regulating the immune response of B cells, T cells, and macrophages against infections and malignant cells. For this reason recombinant human interleukin 4 (rhIL-4) has entered early clinical trials in cancer patients. In the present study we report that rhIL-4 has an antiproliferative effect on five of nine cell lines derived from human colon tumors, head and neck tumors, and glioblastomas as measured by a decrease of colony formation in human tumor cloning assays. All of the cell lines with in vitro responsiveness express at least 100 high-affinity receptors for human interleukin 4 per cell on their cell surface, whereas the nonresponsive tumor cell lines lack expression of high-affinity receptors for human interleukin 4 on their cell surface. In the next series of experiments we have xenotransplanted some of the responsive cell lines into athymic nude mice. Subsequently, the animals were treated s.c. twice daily with 0.5 mg/m2 rhIL-4 or control vehicle for at least 12 days. There was a clear growth inhibition of these xenotransplanted tumors in the mice treated with rhIL-4. Histology of the tumors in both groups revealed no marked infiltration with murine hematopoietic and lymphocytic cells as evaluated by staining with a rat anti-mouse CD45 antibody. We conclude that rhIL-4 has a direct therapeutic activity on the growth of some human epithelial and nonepithelial tumor cell lines which, along with its regulatory function on hematolymphopoietic cells, makes this cytokine an interesting candidate for experimental tumor therapy.

CD30 antigen in embryonal carcinoma and embryogenesis and release of the soluble molecule.

The expression, serological detection, and possible functional role of the CD30 antigen in Hodgkin's disease and anaplastic large cell lymphoma is well documented. In embryonal carcinoma (EC), the expression of this cytokine receptor has been demonstrated only by immunohistology. Because the CD30 monoclonal antibody Ki-1 was found to cross-react with an unrelated molecule, we examined by in situ hybridization testicular germ cell neoplasms for the presence of CD30-specific transcripts. CD30 mRNA was detectable in the tumor cells of 9 of 9 cases of EC or mixed germ cell tumors with an EC component but in no other nonlymphoid tumors. Thus, the CD30 transcript expression pattern proved to be identical to the immunostaining pattern seen with the CD30-specific monoclonal antibody Ber-H2. By Northern blot analysis, CD30 transcripts could be demonstrated in the EC cell line Tera-2. Employing a highly sensitive second generation sandwich enzyme-linked immunosorbent assay, we could detect the soluble CD30 molecule in 8 of 8 sera from patients with a diagnosis of EC but not in 8 of 10 sera from patients with other testicular germ cell tumors. In fetal tissue, no CD30-expressing germ cells or epithelial cells could be observed. Thus, the cellularly expressed CD30 marker for testicular neoplasms of EC type. Moreover, the serum levels of soluble CD30 antigen seem to be a promising parameter for monitoring patients with EC.

The human OX40 homolog: cDNA structure, expression and chromosomal assignment of the ACT35 antigen.

Tissue distribution and expression on mitogen and virally stimulated lymphocytes render the ACT35 molecule a human lymphocyte activation antigen which as yet could not be clustered. Expression cloning of the ACT35 antigen from a pCDM8 library of the HUT-102 cell line revealed strong homology of the cDNA and its encoded protein sequence with the formerly described rat OX40 antigen. The 1.4-kb nucleotide sequence and the deduced 277-amino acid sequence of the single transmembrane protein were 65% and 63% identical, in human and in rat, respectively. Conservation included one N-linked glycosylation site and one protein kinase C phosphorylation site. When expressed in COS-1 cells, the cDNA presented properties comparable to the native ACT35 antigen and the rat OX40 molecule (relative molecular mass 48,000). Thus, the ACT35 protein corresponds to the hitherto unknown human OX40 antigen and is, therefore, another member of the tumor necrosis factor/nerve growth factor receptor (TNFR/NGFR) family. After applying fluorescence in situ hybridization, the human ACT35/OX40 gene could be mapped to chromosome band 1p36 and is, thus, linked to the genes for TNFR II and CD30.

Recombinant human interleukin-4 inhibits growth of some human lung tumor cell lines in vitro and in vivo.

Cytokines play an important role in activating the immune system against malignant cells. One of these cytokines, interleukin-4 (IL-4) has entered clinical phase I trials because of its immunoregulatory potency. In the present study we report that recombinant human (rh) IL-4 has major direct antiproliferative effects on one human lung cancer cell line (CCL 185) in vitro as measured by a human tumor cloning assay (HTCA), tritiated thymidine uptake, and counting cell numbers and marginal activity in a second cell line (HTB 56) in the HTCA. This activity could be abolished by neutralizing antibody against rhIL-4. The biological response of the tumor cells to the cytokine is correlated with expression of receptors for human IL-4 on both the mRNA level and the protein level. The responsive cell line, CCL 185, secretes IL-6 after being incubated with rhIL-4. On the other hand, neutralizing antibodies against IL-6 showed no influence on the growth modulatory efficacy of rhIL-4 in this cell line. Furthermore, CCL 185 does not show detectable production of IL-1, tumor necrosis factor alpha or interferon gamma after incubation with rhIL-4. Thus, the response to rhIL-4 is not mediated through autocrine production of these cytokines triggered by rhIL-4. In a next series of experiments some of the cell lines were xenotransplanted to BALB/c nu/nu mice. Subsequently, the mice were treated for 12 days with two doses of 0.5 mg/m2 rhIL-4 or control vehicle subcutaneously per day. Treatment with rhIL-4 yielded a significant inhibition of tumor growth versus control in two of the non-small cell lung cancer cell lines being responsive in vitro (CCL 185, HTB 56). Histology of the tumors in both groups showed no marked infiltration of the tumors with murine hematopoietic and lymphocytic cells consistent with the species specificity of IL-4. In contrast, no tumor growth inhibition was found in the small cell lung cancer cell lines (HTB 119, HTB 120) being nonresponsive in vitro. We conclude that rhIL-4 has direct antiproliferative effects on the growth of some human non-small cell lung cancer cell lines in vitro and in vivo, which together with its regulatory effects on various effector cell populations makes this cytokine an interesting candidate for further investigation in experimental cancer treatment.

PG-M1: a new monoclonal antibody directed against a fixative-resistant epitope on the macrophage-restricted form of the CD68 molecule.

A new anti-macrophage monoclonal antibody (PG-M1) was produced by immunizing BALB/c mice with fresh spleen cells from a patient with Gaucher's disease. PG-M1 reacts strongly with a fixative-resistant epitope of an intracytoplasmic molecule, selectively expressed by virtually all macrophages of the human body. Although attempts to immunoprecipitate the molecule recognized by PG-M1 have failed so far, the reactivity of the antibody with COS-1 and WOP cells transfected with a human complementary DNA clone encoding for the CD68 antigen suggests that PG-M1 is a new member of the CD68 cluster. However, unlike other CD68 antibodies (KP1, EBM11, etc.), which react with both macrophages and myeloid cells, PG-M1 detects a fixative-resistant epitope on the macrophage-restricted form of the CD68 antigen. In 957 routinely fixed, paraffin-embedded samples, PG-M1 showed a more restricted reactivity with elements of the monocyte/macrophage lineage than the previously described monoclonal antibodies MAC-387 (anti-calgranulins), KP1 (CD68) and Ki-M1P. Among hematological malignancies, PG-M1 only labels acute leukemias of M4 and M5 type and rare examples of malignant histiocytosis/true histiocytic sarcoma. In contrast, acute leukemias of the M1, M2, M3, M6, M7, and L1-L3 types, non-Hodgkin's lymphomas, and Hodgkin and Reed-Sternberg cells of Hodgkin's disease are consistently PG-M1-negative. In the daily diagnostic practice, PG-M1 seems to be particularly valuable for the diagnosis of myelomonocytic or monocytic leukemia and neoplasms of true histiocytic origin in routine paraffin sections.

Interleukin-4 inhibits growth of a human lung-tumor cell-line in-vitro and has therapeutic activity against xenografts of this cell-line in-vivo.

In the present study we report that recombinant human (rh) interleukin 4 (IL-4) has direct, dose-dependent antiproliferative effects on the human lung cancer cell line CCL 185 in vitro as measured by a human tumor cloning assay (HTCA). The biological response of the tumor cells to rhIL-4 is correlated with expression of specific binding sites for human (h) IL-4 on the cell surface. Furthermore, we have xenotransplanted CCL 185 into BALB/c nu/nu mice. Subsequently, the mice were treated for 17 days with twice 0.5 mg/m2 rhIL-4 or control vehicle subcutaneously per day. Treatment with rhIL-4 yielded a significant inhibition of tumor growth versus control. Histology of the tumors in both groups showed no marked infiltration of the tumors with murine hematopoietic and lymphocytic cells consistent with the species specificity of IL-4. We conclude that rhIL-4 has direct antiproliferative effects on the growth of a human lung tumor cell line in vitro and in vivo which together with its regulatory effects on various effector cell populations makes this cytokine a possible candidate for experimental cancer treatment.

Molecular cloning and expression of a new member of the nerve growth factor receptor family that is characteristic for Hodgkin's disease.

In man, Hodgkin's disease (HD) represents the most frequent lymphoma entity whose pathogenesis is still unknown. In order to contribute to the characterization of the molecular mechanisms of this disease, cDNAs coding for the HD characteristic antigen CD30 were cloned from expression libraries of the human HUT-102 cell line using the monoclonal antibodies Ki-1 and Ber-H2. The open reading frame of the cDNA that can be translated from two mRNA species of 2.6 kb, and 3.8 kb, respectively, predicts a 595 amino acid protein with leader, extracellular, single transmembrane, and intracellular domains. When expressed in COS-1 cells, the cDNA presented properties comparable to native CD30 antigen. The CD30 extracellular domain proved to be homologous to members of the nerve growth factor receptor superfamily. Six cysteine-rich motifs could be recognized within the putative ligand-binding domain.