Tibor Diamantstein

Serum levels of soluble CD30 molecule (Ki-1 antigen) in Hodgkin's disease: relationship with disease activity and clinical stage

In all cases of Hodgkins disease (HD) Reed‐Sternberg (RS) cells express the CD30 antigen. It has been recently demonstrated that this molecule can be released from the cell membrane of CD30+ neoplastic cells in a soluble form (sCD30), detectable in culture supernatants and body fluids. In this paper we investigated by an immunoassay the serum levels of sCD30 in 58 patients with HD, in order to define the possible relationship of this molecule to the clinical and pathological findings. sCD30 molecule was found at detectable levels in 24 out of 50 patients (48%) with active disease, whereas it was always absent in control sera and in cases in complete remission. Among the patients with active HD, the incidence and mean values of detectable levels (±SEM) of sCD30 were higher in cases with progressive or relapsing disease (61.5%, mean 458±190 U/ml) as compared to those at presentation (43.2%, mean 116 ± 3 3 U/ml). Among the cases at presentation, detectable levels were observed more often in patients with advanced stages (III+IV: 61%) and constitutional symptoms (‘B’: 61‐5%) than in early stages (I+11: 26%) and without symptoms (‘A’: 33.3%). In addition, higher mean values were found in stages III+IV (182±60 U/ml) and in ‘B’cases (208±73 U/ml) than in stages I+II (65 ± 30 U/ml) or ‘A’patients (64 ± 26 U/ ml).

The interleukin-2 receptor, its physiology and a new approach to a selective immunosuppressive therapy by anti-interleukin-2 receptor monoclonal antibodies.

In this report we have summarized our findings on the IL-2 receptor and our attempts to find an IL-2 receptor targeted immunosuppressive therapy. IL-2 receptors are detectable in two different forms: as monomeric, surface expressed, and as dimeric, presumably non-surface expressed molecules. The dimeric form seems to be non-covalently bound to an as yet undefined 110 KD molecule. The functions of the monomeric versus the dimeric form, as well as that of the noncovalently bound molecule, and their relationship to high and low affinity IL-2 receptors are not yet clear and remain to be elucidated. Upon antigenic or mitogenic stimulation, IL-2 receptors became expressed at the surface of T-lymphocytes. Receptor expression is accompanied by the capacity of the cells to proliferate in response to IL-2. Resting T-cells do not proliferate in response to IL-2. IL-2 dependent proliferation of cells without external stimulation is either due to the presence of a small number of IL-2 receptor bearing cells in the respective population or due to a small number of IL-2 receptors present on the surface of cells. IL-2 itself does not induce IL-2 receptor expression on resting cells but has been shown to up-regulate its own receptor once expressed. In contrast to resting lymphocytes, some leukemic cells and early embryonic thymocytes in the species tested constitutively express IL-2 receptors. The role of such constitutively expressed receptors is not yet clear. As demonstrated in mice, the requirement(s) for induction of IL-2 receptor expression for the helper/inducer subset (Lyt-2+) are different from those of the cytotoxic/suppressor subset (L3T4+). In contrast to Lyt-2+ cells, the accessory cell requirement for L3T4+ cells could not be replaced by cytokines. Whether Lyt-2+ cells require an additional, not yet defined receptor inducing factor (RIF) besides IL-2 in order to become IL-2 receptor positive and to consequently proliferate in response to IL-2, is a matter of controversy. There is evidence that interleukin-1 and some functionally related factors produced by leukemic cells enhance expression and/or function of the IL-2 receptors. IL-2 receptors of the high and low affinity type expressed upon antigenic stimulation are cleared from the cell surface. As demonstrated in this report, the vast majority of them, probably those of low affinity type, are released from the cells continuously. The mechanism of their release and the possible immunoregulatory role of circulating receptors found in the serum of animals is not yet clear.(ABSTRACT TRUNCATED AT 400 WORDS)

Interleukin 2 modifies the bioelectric activity of some neurosecretory nuclei in the rat hypothalamus

Introduction of 15-30 U of interleukin-2 (IL-2) into the 3rd ventricle of Wistar rats was followed by a marked and significant decrease in neuron discharge frequency in the ventromedial nucleus of hypothalamus and a marked increase in the supraoptic and paraventricular nuclei. The monoclonal antibody ART62 partly blocked these effects. The conventional anti-IL-2 receptor monoclonal antibody ART18 had only a non-significant influence on the effects of IL-2. Since the supraoptic and paraventricular nuclei secrete the antidiuretic hormone, their excitation may offer a partial explanation of the considerable water retention observed during IL-2 therapy against neoplasia.

Lack of tumorigenicity of interleukin 4 autocrine growing cells seems related to the anti-tumor function of interleukin 4

Recently, the failure of interleukin 4 (IL4) autocrine growing CT4S cells to grow in vivo has been demonstrated. Because it could not be excluded that the cells produce insufficient amounts of IL4 to support their growth in vivo, subclones were established which are unresponsive to exogenous IL4 and therefore have acquired full growth autonomy. From the fact that the subclones likewise did not give rise to tumors when injected into nude mice, one may conclude that the IL4 production of autocrine growing CT4S prevents their growth in vivo. To test this hypothesis, a retroviral vector containing the IL4 gene under the control of the immunoglobulin heavy chain (Igh) enhancer/promoter was constructed and used to infect the myeloma cell line J558L. An IL4 producing clone was established (J558L-XEPIL4) and the tumor progression in comparison to the parental clone J558L was monitored in nude mice. The IL4 production significantly delayed the growth of J558L-XEPIL4 in vivo. Tumor suppression was much more evident when J558L-XEPIL4 cells were injected into syngeneic BALB/c mice. These results may explain why autocrine growing CT4S do not grow in vivo and suggest the involvement of functional T lymphocytes in the effectiveness of the host dependent anti-tumor action of IL4.

Regulation of interleukin-2 receptor expression and receptor release.

The generation and cell surface expression of IL-2 receptors was monitored by: (i) an ELISA that permits quantitative determination of detergent-solubilized or soluble IL-2 receptors; and (ii) detection of the binding of 125I-labelled recombinant IL-2 and of anti-IL-2 receptor antibodies to receptor bearing cells. Upon lectin stimulation both high and low affinity IL-2 receptors became expressed in parallel at the cell surface. Both high and low affinity receptors were upregulated by IL-2. Upon lectin activation the amount of cell-associated receptors increased and on day 2 of the culture period IL-2 receptors were detectable in the culture supernatant. IL-2 upregulated both high and low affinity IL-2R expression on T-lymphoblasts. IL-2R bearing leukemic cells and T lymphoblasts released IL-2R when cultured in vitro. IL-2R release by T lymphoblasts was enhanced dramatically by IL-2. On the other hand, IL-2-receptor positive leukemic cells released receptors in an IL-2 independent manner. Release of receptors could also be detected in serum-free medium. At least a part of the released receptors could be specifically bound to immobilized pure recombinant IL-2 and to monoclonal anti-IL-2-receptor antibodies. Small but significant amounts of soluble IL-2 receptors were detectable in the sera of normal mice. In sera of mice inoculated with IL-2-receptor positive syngeneic leukemic cells, elevated levels of IL-2 receptors were detectable. Release of IL-2 receptors seems to represent one of the major routes by which the receptors are cleared from the cell surface.

The Mouse High Affinity IL 2 Receptor Complex: I. Evidence for a Third Molecule, the Putative γ-Chain, Associated with the α- and/or γ-Chain of the Receptor*

Low and high affinity receptors for interleukin 2 were investigated on mitogen-activated mouse T lymphocytes and on interleukin 2 (IL2) receptor-bearing T cell lines by chemical crosslinking of 125I-labelled interleukin 2 (125I IL2) to its binding proteins or membrane proteins associated with the IL2 binding sites. In all cells investigated, the crosslinking produced a 65-70 kD complex thought to be one beta-chain molecule (55 kD IL2 receptor) and one IL2. Occasionally, a 120-130 kD band could be seen which is thought to be IL2 bound to beta-chain homodimer. Using low IL2 concentrations (200 pM), high affinity receptor-bearing cells showed specific additional complexes. Either bands of 85 kD, 105 kD and greater than 160 kD or of 90 kD and 115 kD and greater than 160 kD could be found when mouse T blasts or CTLL 16 cells were used, respectively. In the exclusively low affinity receptor-bearing T cell line Eb, only a single band of 70 kD (beta-chain + IL2) was detectable. These results show that in addition to a 85-90 kD complex, as shown already in humans, a third complex of 105-115 kD exists in mice that is specifically associated with high affinity receptors. This is the first indication of a putative gamma-chain of the high affinity IL2 receptor. All IL2-containing complexes of the mouse cells could be precipitated by the newly developed MAb AMT 45-20 raised against the beta-chain of the mouse IL2R and recognizing an epitope distinct from those recognized by the MAb AMT 13 or IL2. This suggests that either 1) all complexes contain the beta-chain, 2) share an antigenic determinant with the beta-chain, or 3) that the higher mw complexes become coprecipitated with the beta-chain.

Immunotherapy of experimental autoimmune encephalomyelitis (EAE): Differential effect of anti-IL-2 receptor antibody therapy on actively induced and T-line mediated EAE of the Lewis rat

Treatment of Lewis rats with monoclonal anti-interleukin-2 receptor (IL-2 R) antibody ART-18 is highly efficient in protecting the recipients from T-line transferred experimental autoimmune encephalomyelitis (tEAE) in vivo. In contrast, ART-18 did not affect the development of EAE actively induced (aEAE) by immunization with myelin basic protein (MBP) in complete Freunds adjuvant (CFA). ART-18 caused a slight delay in the development of aEAE only in combination with a subtherapeutic dose of cyclosporine A (Cy-A), but failed to influence duration or severity of clinical signs. The discrepancy in therapeutic efficiency of ART-18 in tEAE and aEAE could be due to a different intensity of IL-2 R-expression on in vitro- and in vivo-activated MBP-specific T cells. Our results therefore caution against a general therapeutic application of anti IL-2 R-directed therapy in all manifestations of T-cell-mediated autoimmunity.

The role of dipeptidylpeptidase IV positive T cells in wound healing and angiogenesis

In our former investigations we showed that dipeptides like Lys-Pro and others with the structure X-Pro are able to promote wound healing and angiogenesis in several experimental models (implantation of polyvinyl rings, open skin wounds, wound breaking strength). These dipeptides can originate from free amino termini of various polypeptides (collagenopeptides, cytokines, growth factors etc.) by degradation by dipeptidylpeptidase IV (DPP IV, EC 3.4.14.5). This enzyme occurs, among other sources, in exocrine epithelia, hepatocytes, renal tubuli, endothelia, and myofibroblasts. T-lymphocytes also reveal this enzyme activity (CD26 antigen) [1]. T-lymphocytes, monocytes, and macrophages generate numerous polypeptides and other factors capable of stimulating and modulating the proliferation and function of fibroblasts, endothelial cells and other cell types [2]. It is known that in cases of immunosuppressive therapy, disturbances of would healing may occur and that patients suffering from immunodeficiencies, among others, have impaired wound healing. In contrast immunostimulation may improve wound healing. We have shown that Wistar rats, systemically treated by complete Freunds adjuvant produce more granulation tissue than untreated animals and that T cells are involved in this process [3]. Other investigators showed that nude mice have a lower capa-

Prolongation of rat pancreatic islet allograft survival by treatment of recipient rats with monoclonal anti-interleukin-2 receptor antibody and cyclosporin

SummarySince interleukin-2-receptor expressing cells play a role in allograft rejection, we investigated the effect of anti-interleukin-2 receptor monoclonal antibody treatment on graft survival of allografted pancreatic islets. When pancreatic islets obtained from Lewis A-rats (haplotype RT1a) were grafted under the kidney capsules of streptozotocin-diabetic Lewis rats (haplotype RT1u), the recipients relapsed into hyperglycaemia within 11 days (7±1 days). Treatment of the recipient rats with low-dose cyclosporin (1.5 mg/kg body weight) had no effect on allograft survival (9±1 days). The application of anti-interleukin-2 receptor monoclonal antibody (1mg/kg body weight) for 10 days resulted in a prolongation of allograft survival (42.5±15.3,p<0.01). In 3 out of 11 animals a permanent normoglycaemia (>120 days) associated with glucose intolerance was observed. When the recipients were treated for 10 days with cyclosporin and anti-interleukin-2 receptor monoclonal antibody, the allograft survival was also prolonged (45.1±14.6,p<0.01); again 3 out of 11 animals remained permanently normoglycaemic while exhibiting a normal glucose tolerance.

Development of suppressor lymphocytes during acute rejection of rat cardiac allografts and preservation of suppression by anti-IL-2-receptor monoclonal antibody

Suppressor acivity was investigated in rats undergoing acute rejection of heterotopic cardiac allografts. Spleen cells were harvested at 7 days from LEW rats rejecting (LEWxBN) F1 heart grafts and fractionated into their T, T suppressor/cytotoxic, and t helper subpopulations. Transfer of alloimmune unseparated spleen