Linkage between monokine production and regulation of the negative surface charge density of human monocytes.

The regulation of the negative surface charge density of human monocytes was investigated with the help of the synthetic glycolipid analogue BAY R 1005. This compound is incorporated into the outer membrane of isolated monocytes during 24 hours of incubation. After this time the electrophoretic mobility (EM) of monocytes is unchanged at 0.95 x 10(-4) (cm2 V-1 s-1) and remains unchanged even under conditions where non-treated monocytes increase their EM up to 1.1 x 10(-4) (cm2 V-1 s-1). In addition BAY R 1005 stops differentiation of monocytes to macrophages, it triggers monokine production and abolishes monocyte suppressor activity and spreading capability. The results show that BAY R 1005 affects intracellular features. In connection with earlier investigations of the regulation of the negative surface charge density of human monocytes (1,2) the study suggests that monokine production and maintenance of the EM of monocytes are linked.

Ciprofloxacin enhances T cell function by modulating interleukin activities.

Ciprofloxacin (CIP) is a quinolone carboxylic acid derivative with a broad spectrum of antibacterial activity. CIP (0.1-30 micrograms/ml) enhanced DNA synthesis of mouse spleen cells and human peripheral blood lymphocytes (PBL) that had been activated with T cell mitogens or with alloantigens. In addition, CIP increased the amount of IL-2 found in the supernatants of phytohaemagglutinin (PHA)-stimulated human PBL. The presence of CIP in the medium (0.3-10 micrograms/ml) increased the levels of IL-1 found in the culture supernatants of adherence-enriched mouse macrophages, human monocyte/macrophages and a human monocytic cell line stimulated with lipopolysaccharide. In contrast there was no effect of CIP on the release of IL-1 by freshly isolated human monocytes or by cells of the keratinocyte line, A431. CIP alone had no influence on the basal release of IL-2 by NOB-1 cells, a T cell line that responds to IL-1 with an increase in IL-2 synthesis, but, in combination with recombinant IL-1, CIP significantly enhanced the release of IL-2 by these cells. The results of this study suggest that CIP modulates the immune response at two levels--the production of IL-2 by activated T cells and the production of IL-1 by activated monocyte/macrophages. However, CIP did not affect the primary antibody response in vitro or in vivo against sheep erythrocytes and ovalbumin respectively. Thus the enhancing action of ciprofloxacin on the immune system appears to be restricted to T cell function and macrophage/T cell interactions.

Isolation of human B-cell subpopulations for pharmacological studies.

Three groups of human peripheral blood B-lymphocytes were separated from each other by countercurrent centrifugal elutriation and free-flow electrophoresis. They differed in their state of maturation and in their capability to produce antibodies in vitro. These B-cell subpopulations were used to study features of a drug such as BAY R 1005. BAY R 1005 is a synthetic glycolipid analogue (GLA), which is supposed to modulate antibody synthesis. Mature, immunoglobulin- (Ig-) secreting B-lymphocytes secreted equal quantities of antibodies in the presence and in the absence of the GLA. BAY R 1005 was found to be without mitogenic activity on resting B-cells and did not induce them to produce antibodies. However, it supported the antibody production of preactivated B-lymphocytes. The in vitro preactivated B-cells were affected via monocytes. Only in vivo preactivated B-lymphocytes increased their antibody production under the direct influence of BAY R 1005.

Pretreatment with cyclosporine and anti-interleukin 2 receptor antibody abrogates the anti-idiotype response in rat recipients of cardiac allografts.

A 10-day course with ART-18, a mouse monoclonal antibody (mAb) directed against the rat interleukin 2 receptor (IL-2R), prolongs the survival of (LEW x BN)F1 cardiac allografts in LEW recipients to approximately 3 weeks (acute rejection = 8 days, P less than 0.001). We examined host responses against ART-18 idiotype (Id) and mouse immunoglobulin in recipients immunomodulated with ART-18 mAb. Treatment with ART-18 elicited high titers of anti-Id antibodies 14 days after transplantation. However, naive rats given ART-18 before transplantation showed strong anti-Id responses as early as day 4 after engraftment, coinciding with abrogation of the treatment effect (graft survival, approximately 10 days). Preimmunization with irrelevant mouse IgG, which elicited high titers of anti-IgG, did not influence the efficacy of ART-18 upon graft survival (17 days). The use of cyclosporin A (CsA) in conjunction with ART-18 prior to transplantation suppressed the anti-Id response and led to dramatic graft prolongation (greater than 58 days), with two of five grafts surviving indefinitely. This striking effect of CsA plus ART-18 pretreatment did not depend upon CsA per se, as grafts were rejected within 12 days in animals pretreated with CsA alone; in both groups CsA trough levels were comparable. Moreover, administration of CsA before transplantation in concert with control IgG (instead of ART-18) prompted rejection within 2-4 weeks. Thus, discrete interaction(s) between anti-IL-2R mAb and CsA prior to engraftment induces partial host unresponsiveness/tolerance to anti-IL-2R mAb treatment following transplantation and suppresses the neutralizing anti-Id responses, which results in long-term/permanent graft acceptance. This study provides a strategy to overcome the anti-Id response mounted by graft recipients that otherwise limits the efficacy of anti-IL-2R mAb treatment.

Mechanism of action of anti-IL-2R monoclonal antibodies. ART-18 prolongs cardiac allograft survival in rats by elimination of IL-2R+ mononuclear cells.

ART-18, a mouse IgG1 mAb recognizing the IL-2 binding domain of the rat p55 subunit IL-2R molecule, prevents graft rejection in various experimental models, although its mechanism of action in vivo, like that of anti-IL-2R mAb generally, remains elusive. These studies were designed to define whether IL-2R+ T effector cells were actually eliminated or their function merely inhibited by comparing directly the in vitro and in vivo efficacy of intact ART-18 and its F(ab)/F(ab')2 fragments. Addition of each mAb preparation profoundly suppressed MLR set up between naive LEW responders and x-radiated BN stimulators, suggesting that mAb fragments retained Ag binding functions in vitro. However, both ART-18 F(ab) and F(ab')2 were ineffectual in vivo as judged by their inability to affect acute (8 days) rejection of (LEW X BN)F1 cardiac allografts in LEW recipients (graft survival ca. 11 and 9 days, respectively, compared to ca. 21 days after therapy with intact ART-18, p less than 0.001). The sera levels of ART-18 and ART-18 F(ab')2 were 4 to 5 micrograms/ml, but only less than 0.5 micrograms/ml of F(ab) could be detected. The therapeutic failure of ART-18 fragments was unrelated to potential host sensitization, as rat antimouse F(ab) or F(ab')2 serum IgG titers remained in the same range as those against intact ART-18. The role of the Fc portion of Ig in the mode of action of ART-18 was then tested further by flow microfluorimetry analysis of host mononuclear spleen cells and immunoperoxidase stains of the graft infiltrate. IL-2R+ cells were abundant in rats treated with ART-18 fragments, comparable to acutely rejecting controls. In contrast, IL-2R expression was abolished in animals undergoing ART-18 therapy. The elimination of IL-2R+ cells is required to prolong cardiac allograft survival in rats after IL-2R targeted treatment with ART-18 mAb.

Biodistribution of anti-interleukin 2 receptor monoclonal antibodies correlates with their therapeutic efficacy following transplantation.

IL-2R-targeted therapy prevents graft rejection in various experimental models and in man. However, the principles of optimal mAb selection remain elusive, as their efficacy in vivo does not always correlate with their characteristics in vitro. ART-18 and OX-39, mouse IgG1 mAbs, define distinct epitopes on the p55 subunit of the rat IL-2R. Treatment of LEW hosts with ART-18 prolongs survival of LBN cardiac allografts up to a month; in contrast, OX-39 never affects acute (8-day) rejection. In this study, we evaluated the biodistribution of 125I-labeled ART-18 and OX-39 administered iv to untreated and heart-grafted rats. ART-18 was cleared from the circulation (half-life time ca. 29 hr) and accumulated in host lymph nodes and spleen to a greater extent than OX-39 (P less than 0.001). In contrast, OX-39 was retained in blood (half-life time ca. 66 hr) and was eventually sequestered in liver, lungs, and kidneys, a pattern comparable to an irrelevant IgG1 (MOPC-21). ART-18 but not OX-39 entered specifically acutely rejecting allografts (allograft:native heart activity ratio = 4.0 and 2.3, respectively, P less than 0.01). The distribution of ART-18 was IL-2R epitope but not mAb isotype specific as tissue accumulation of "hot" ART-18 was comparable in recipients conditioned with "cold" ART-18 of IgG1 or IgG2b isotype, but not in those treated with OX-39. Thus: (1) the biodistribution of anti-IL-2R mAbs is not random; the mAb "effective" in combating rejection (ART-18) penetrates preferentially host lymphoid tissues and the graft itself, whereas the biologically "ineffectual" mAb (OX-39) is retained in the circulation for prolonged periods; (ii) the epitope of IL-2R defined by the mAb is critical; a mAb may be "captured" by unrelated cells expressing a common epitope in vivo before reaching the related targets, and/or some epitopes may be more accessible than others for iv administered mAbs.

A method for the quantitation of interleukin-2 activity.

A bioassay for the determination of interleukin-2 activity is described. We have compared the traditional method of data processing, which involves probit analysis and curve fitting, with a simpler method based on the so-called AUC (area under the curve). The latter method is readily applicable to spreadsheet software and can handle large amounts of data.

Agonistic and antagonistic interactions of anti-interleukin 2 receptor monoclonal antibodies in rat recipients of cardiac allografts.

A panel of five mouse mAbs recognizing 4 distinct epitopes (R1-4) of the rat 55kD IL-2R molecule were tested for their influence on acute rejection (8 days) of (LEWxBN)F1 cardiac allografts in LEW hosts. IL-2R1 targeted therapy with ART-18 (IgG1, inhibits IL-2-dependent responses) prolonged graft survival to ca.21 days. IL-2R2 is recognized by ART-65 and ART-75, mAbs that do not inhibit T cell growth. Treatment with ART-65 (IgG1) but not ART-75 (IgG2a) abrogated acute rejection (ca. 16 days and 9 days, respectively). ART-35, an anti-IL-2R3 mAb (IgG1, does not inhibit T cell function) extended graft survival marginally to ca. 12 days. Finally, therapy with OX-39, (anti-R4 IgG1 mAb, inhibits IL-2 binding, but not IL-2-driven growth) was completely ineffectual. Simultaneous targeting of two IL-2R epitopes increased the therapeutic index synergistically (ART-18 [R1] + ART-65 [R2]--60% permanent graft acceptance), additively (ART-75 [R1] + ART-35 [R3]--graft survival ca. 18 days), or did not improve further graft survival at all (ART-18 [R1] + OX-39 [R4]--graft survival ca. 18 days). Thus, the cellular targeting patterns and isotype of mAbs are crucial: (1) targeting at functionally distinct epitopes controls rejection most effectively; (2) IgG1 and IgG2b mAbs are more influential in vivo than IgG2a, data supported by the studies employing the family of ART-18 isotype switch variants. Treatment with anti-IL-2R mAb did not depress Ts activity as tested both in vitro and in vivo. Sparing of putative Ts by mAb is also shown by thymectomy of graft recipients before or during ART-18 therapy, which shortened graft survival to 13-15 days; thymectomy after ART-18 therapy did not influence graft survival. However, infusion of IFN-gamma recreated classic acute rejection within 10 days in otherwise longstanding cardiac allografts in ART-18 treated hosts. Upregulation of MHC class II antigen and IL-2R expression seems to be primarily responsible for this striking biological effect of IFN-gamma in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

Monitoring of interleukin-2 receptor (IL-2R) expression in vivo and studies on an IL-2R-directed immunosuppressive therapy of active and adoptive adjuvant-induced arthritis in rats.

Recent evidence indicates that adjuvant arthritis (AA) of rats induced by complete Freund's adjuvant (CFA) is an autoimmune disease that is mediated by T cells. This report describes the distribution of activated IL-2 receptor (IL-2R)-bearing cells in spleen, popliteal lymph nodes (PLN) and blood in AA rats and in naive healthy rats using the monoclonal antibody (mAb) ART-18. It was found that in the primary lymph nodes (injected side) two peaks of elevated numbers of IL-2R-positive cells (Day 9/10 with a 40-fold increase; Day 25 with a 75-80-fold increase) occur. The PLN of the non-injected site also show an increase (30-fold) in the number of IL-2R-positive cells on Day 25. This investigation also included the monitoring of soluble IL-2R in the serum of AA rats in comparison to control sera of non-induced rats. The incidence of free IL-2R in the serum of AA rats does not completely correlate with the pattern of the distribution of receptor-bearing cells in PLN; elevated levels of IL-2R were observed at Day 9 and subsequently declined to below control levels. On Day 25, there was no correlation between IL-2R+ cells and soluble IL-2R. ART-18 was not active in suppressing the development of AA, in contrast to the complete inhibition of the passively transferred AA.

Immunosuppressive effects of the macrolide antibiotic bafilomycin towards lymphocytes and lymphoid cell lines.

The effects of bafilomycin macrolide antibiotics on primary lymphocytes and on tumor cell lines were investigated. Bafilomycin A markedly suppressed DNA, RNA, and protein synthesis in splenocyte cultures of several inbred mouse strains. Bafilomycins were also inhibitory towards cultures of concanavalin A- or lipopolysaccharide-activated murine spleen cells, and inhibited the mitogen-induced differentiation of B lymphocytes into immunoglobulin-secreting plasma cells. Corresponding results were obtained in human cell cultures. A hydrolysis product of the bafilomycin molecule was inactive. Bafilomycin also inhibited the growth of various lymphoid cell lines, the B cell line BCL1, the macrophage cell lines J774 and P338D1, and the T cell line EL4. The sensitivity of the tumor cell lines increased when, simultaneously with bafilomycin, mitogens were applied to the cell cultures. The immunosuppressive action of cyclosporin A could be enhanced by bafilomycin, which could be of importance for the elucidation of the molecular mechanism of T cell suppression, and for applied medical research.

Monoclonal antibodies detecting plasminogen activators on the membrane of leukemic lymphoid cells of T-cell origin.

The specificities of six monoclonal antibodies produced against plasminogen activator of the human Bowes melanoma cell line are described. They have been used to detect membrane-bound plasminogen activator on cultured human lymphoid cell lines and in neoplastic human lymphocytic and myeloid cells of leukemic patients. These studies indicate that only certain phenotypic subsets of the T-cell lineage derived from patients with chronic lymphocytic leukemia or with Szezary syndrome express plasminogen activator on their surface membrane.

Cutaneous infiltrates of histiocytosis X contain plasminogen activator-bearing epidermotropic dendritic cells different from Langerhans cells.

Plasminogen activators (PA) play an important role in cell migration and tissue degradation. Considering the strong epidermotropism of atypical mononuclear cells in histiocytosis X (HX) skin infiltrates leading to intraepidermal abscess formation, it was the purpose of this study to look for tissue-type PA (t-PA) and/or urokinase-type PA (u-PA) on HX cells. Four monoclonal antibodies against PA were used, employing the alkaline phosphatase anti-alkaline phosphatase (APAAP) technique on cryostat sections from four patients with HX. Twenty percent to 40% of infiltrating cells in the epidermis expressed the t-PA antigen. t-PA+ cells were present in the follicular centers of human tonsil, absent in normal epidermis and scanty in cutaneous infiltrates from mycosis fungoides and lupus erythematosus. Double labeling with anti-PA and T6 (CD1) or S100 protein revealed some of the HX cells to express both antigens (t-PA+ CD1+ or t-PA+ S100+). We conclude that cutaneous infiltrates of HX contain PA+ dendritic cells which are different from normal Langerhans cells and which may be responsible for the strong epidermal alterations in HX.

Administration of silica or monoclonal antibody to Thy-1 prevents low-dose streptozotocin-induced diabetes in mice.

Multiple injections of low doses of streptozotocin induce an experimental diabetes in mice. We have analyzed in two inbred strains whether the development of hyperglycaemia can be influenced by administration of macrophage-toxic silica particles or by a monoclonal antibody to Thy-1.2. Mice received streptozotocin (30 or 40 mg/kg) on five consecutive days (day 0-day 4) and in addition either silica particles (starting at day 0) or anti-Thy-1.2 (starting at day -2 or -3). In both strains mice receiving streptozotocin alone became hyperglycaemic within two weeks. Additional treatment with silica almost fully prevented diabetes development. Anti-Thy-1.2 administration was similarly effective in C57B1/Ks and partially protective in C57BL/6 mice. Histological analysis of pancreatic islets showed that a large fraction of beta cells had been spared from destruction by this treatment. The data indicate a role for both macrophages and Thy-1 positive cells in the pathogenesis of low-dose streptozotocin-induced diabetes.

Activation of murine B lymphocytes by suramin.

Suramin stimulated DNA synthesis in spleen cell cultures of all inbred strains of mice tested, including, for example, CBA, DBA/2, C57BL/6, and the lipopolysaccharide (LPS)-nonresponsive strain C3H/HeJ. The cells responding to the drugs were removed by passage through nylon wool columns, but they were not eliminated by in vivo treatment of the mice with anti-Thy 1.2 antibody. Spleen cells of homozygous nude mice (C57BL/6 or BALB/c background) were as reactive as those of their heterozygous littermates. Collectively the data show that suramin is a B-cell mitogen in the mouse.

Analysis with monoclonal antibodies of human lymphoid cells forming rosettes with rabbit red blood cells.

Rabbit red blood cells have previously been shown to rosette with a subpopulation of thymocytes and with mitogen activated peripheral lymphocytes but not with unstimulated lymphocytes. Using monoclonal antibodies and double marker assays we studied the phenotype of these cells. In thymus, over 90% of rosetting cells express antigens of immature thymocytes (HTA1, OKT6). A proportion of the rosetting cells shows in addition antigens of mature thymocytes (OKT3, UCHT1). These cells probably correspond to a stage of intrathymic maturation between common and mature thymocytes. Virtually all rosetting cells are T cells and express an antigen related to T cell activation (TAC) when lymphocytes are activated by mitogens like PHA or Con A. Few rosetting cells are Ia positive. Two other antigens (OKT9, OKT10) known to be associated with proliferating and immature cells, are found in variable proportions on rosetting cells. After stimulation with allogeneic lymphocytes, fewer rosettes are detected than after stimulation by mitogens. Cells activated by a soluble antigen (PPD) and forming rosettes with rabbit red blood cells have a helper phenotype (Leu3a positive). Screening of leukaemia cell samples revealed that only cells from patients with T-ALL form rosettes with rabbit red blood cells. Rosette formation is almost totally inhibited by a polyclonal anti-thymocyte serum and two monoclonal antibodies (OKT11A,Lyt3) which have been shown to block rosettes with sheep erythrocytes.