Search

Several hybridoma cell lines secreting NP-specific, murine IgE antibodies were generated by fusion of P3-X20 (γ,κ) tumour cells with spleen cells from (BALB/c x C57Bl/6)F1 (CB6F1) mice previously immunized with NP-ovalbumin. Four subclones (designated NP-ε-3.57, NP-ε-15,88, NP-ε-91,58 and NP-ε-95,31) were propagated in vivo and milligram quantities of the corresponding IgE antibodies were purified from ascitic fluid by gel filtration, ion exchange chromatography and affinity chromatography. Immunological analyses and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) indicated that NP-ε-15,88, NP-ε-91,58 and NP-ε-95.31 all possessed λ1 (or possibly λ3) light chains; and that NP-ε-3,57 possessed λ2 light chains; NP-&elipson;-95,31 also expressed the P3-X20 derived, MOPC-21 κ light chain. Radioallergosorbent test (RAST) titration curves, generated from the interaction of the four monoclonal IgE antibodies with NP-BSA attached to paper discs (NP-BSA-P) were found to be non-overlapping. Measurements of the relative amounts of NP-ε-aminocaproic acid (NP-CAP) and 4-hydro-3-iodo-5-nitrophenylacetyl-ε- aminocaproic acid (NIP-CAP) that were required to inhibit by 50% the binding of the 4 IgE antibodies to NP-BSA-P indicated that these antibodies were all heteroclitic, since their affinity for NIP appeared to be higher than their affinity for NP. These results, in conjunction with other findings reported in the literature, suggested that the V regions of NP-specific IgE antibodies are similar to the V regions of NP-specific IgM and IgG antibodies, produced by the same mouse strains. Finally, in vitro histamine release measurements demonstrated that two of these monoclonal IgE antibodies could mediate antigen induced histamine release from passively sensitized rat peritoneal mast cells. [ABSTRACT FROM AUTHOR]

A factor found in the serum of patients with leprosy that inhibits the growth of mitogen-stimulated normal peripheral blood lymphocytes has been studied. The inhibitor, previously identified as an IgG, has been shown to act by blocking the recruitment of lymphocytes into growth. It was not cytotoxic and did not inhibit the rate of growth of those lymphocytes that had been stimulated. The inhibitory activity was less potent if the serum was added after mitogen stimulation. The inhibitor, which could be absorbed by activated but not resting lymphocyte cultures, appeared to act by inhibition of an early event preceding the release of IL-2. The inhibition of mitogen stimulation was overcome by the addition of purified IL-2, although the inhibitor did not block the action of IL-2 on a long-term cultured IL-2-dependent cell line. [ABSTRACT FROM AUTHOR]

In the autologous Theileria mixed leucocyte reaction (MLR), irradiated Theileriaparva-infected cells induce proliferative responses in autologous peripheral blood mononuclear leucocytes (PBM)II irrespective of the immune status of the donor animal. In this paper we have analysed the cellular basis of this response in naive and immune cattle to determine the Thelleria specificity of the response. The magnitude of proliferation is dependent on two parameters, namely the presence or absence of monocytes in the responder population, and the phenotype of the parasitized stimulator cells, both of which appeared to be independent of the immune status of the donor animal. Monocyte-depleted responders invariably gave stronger proliferative responses but generated cytotoxicity from immune cattle that tended to be less genetically restricted. Marked differences were observed in the stimulatory capacity of cloned parasitized T-cell and non-T cell lines. At least part of this variation was associated with differences in the capacity of the parasitized cells to secrete soluble suppressive factors and possibly also stimulatory factors. Two observations indicated that, in immune cattle, part of the proliferative response in the autologous Theileria MLR is parasite-specific. First, stimulator cells fixed with glutaraldehyde stimulated proliferative responses in monocyte-depleted PBM from immune animals but not naive animals. Second, in autologous Theileria MLRs with intact PBM, genetically restricted cytotoxic cells were generated from immune but not naive animals. While inonocytes seem not to be required for induction of the parasite-specific component of the response, their absence from the assay when viable stimulator cells are utilized appears to enhance the non- specific component of the proliferative and cytotoxic responses. [ABSTRACT FROM AUTHOR]

Lymphocyte migration into inflammatory sites involves a change from a spherical, non-motile phenotype to an irregular, constantly shape-changing, motile phenotype. We have previously shown that lymphocytes are maintained in the non-motile state by the constitutive activity of protein kinase C (PKC). In this paper we have attempted to identify the PKC isotype which regulates these morphological changes by three different approaches. (a) Motile and non-motile T- cell lines were compared for expression of the α, βI, βII, γ, δ, ε, η, ζ and θ isotypes by Western blotting. There was no obvious correlation of isotype expression with motility. (b) Two different PKC inhibitors, one specific for classical isotypes, Go6976 and the other GF109203X, which inhibits both classical and non-classical isotypes were compared for induction of motility in non- motile lymphocytes. Only GF109203X induced motility implying that a non-classical isotype is involved. (c) Non-motile lymphocytes were chronically treated with the PKC activator bryostatin and the time courses of induction of motility and downregulation of PKC isotypes were compared. Induction of motility correlated better with downregulation of ε, η and θ than with α or β. It is concluded that the data fit best with the involvement of a non-classical PKC isotype in regulating lymphocyte motility although no association with a particular isotype was found. [ABSTRACT FROM AUTHOR]

This paper describes a model for investigation of the functional implications of B-cell activation for antigen presentation. Mixed lymphocyte cultures were used to assess the ability of freshly isolated B cells, mitogen-activated B cells and Epstein Burr virus (EBV)-transformed B-cell lines to stimulate the activation and proliferation of allogeneic T cells under a variety of experimental conditions. It was found that resting B cells presented antigen poorly, while activated cells were highly immunogenic. Paraformaldehyde fixation completely eliminated antigen presentation by resting B cells, despite constitutive expression of class II MHC antigens. However, fixation had little effect on antigen presentation by activated B cells that expressed B7-1 and B7-2 in addition to class II major histocompatibility complex (MHC) molecules. Arrest of B-cell activation by serial fixation after treatment with F(ab′)2 fragments of goat anti-human IgM produced cells with variable antigen-presenting capacity. Optimal antigen presentation was observed for cells fixed 72hr after the initiation of B-cell activation. Although both BT-1 and B7-2 antigen expression increased after B-cell activation, it was found that the rate of T-cell proliferation correlated most closely with B7-2 expression. Stimulation of T cells by fixed activated B lymphocytes could be blocked by antibodies directed at class II MHC molecules, indicating involvement of the T-cell antigen receptor. In addition, T-cell proliferation was inhibited by antibodies specific for B7-1 and B7-2 and by the fusion protein CTLA4-Ig, demonstrating a requirement for CD28 signal transduction. The sole requirement of B7 family expression for antigen presentation by B lymphocytes was shown by demonstration of T-cell stimulation by fixed resting B cells in the presence of CD28 antibody as a source of artificial costimulation. [ABSTRACT FROM AUTHOR]

This paper investigates the Vβ usage of lymph node cells from mice immunized with TNP and of cell lines made from them. In cell lines stimulated weekly with TNP in vitro for 1 month, about 87% of the cells were Vβ8+ and further analysis showed that these cells were actually Vβ.2+. This was also true for the cells that proliferated in lymph nodes in response to TNP 4 days after primary immunization, i.e. proliferation occurred mainly in the Vβ+, and in particular in the Vβ8.2+, population while much less proliferation occurred when the Vβ8- or Vβ8.2- T-cell populations are used. This was not due to non-specific damage during separation, as the response to concanavalin A and alloantigen was intact. In a separate series of experiments, mice were acutely depleted of Vβ8+ T cells by treatment with F23.1 or a control monoclonal antibody (mAb) in vivo given before immunization. Treatment with the relevant mAb virtually abolished the response to TNP. In contrast, SJL mice, which lack the gone segment coding for the Vβ8 family and several other Vβ chains, made a normal proliferative and delayed-type hypersensitivity (DTH) response to TNP. This poses the problem, which may he important in the study of the T-cell repertoire, of why acute removal of Vβ8+ T cells, which are dominantly used in the response to TNP, does not allow T cells using other chains to substitute in the response, while the absence of this population over a long period of time, because of a deletion in the genome, allows the use of T cells hearing other Vβ chains. [ABSTRACT FROM AUTHOR]

The role of perforin in cytotoxicity is controversial. This paper characterizes a novel monoclonal antibody (anti-Phu) against human perforin, using murine cell lines transfected with human perforin cDNA. The antibody specifically stains human pefforin in transfected mouse CTLL-2. Anti-Phu blocked granule-mediated haemolysis in an in vitro assay using intact granules isolated from the natural killer (NK)-like human cell line YT, indicating that perforin is a major granule component causing lysis of red blood cells (RBC) in this assay. Inhibition of haemolysis by anti-Phu demonstrated that the antibody binds to undenatured protein as well as fixed perforin molecules. However, the antibody did not inhibit lysis by an allospecific T-cell clone or by YT cells. This could be due to an extremely tight contact between effector and target cell, preventing the antibody from interfering with perforin function by stearic hindrance. Physiologically this may reduce lysis of bystander cells. The anti-Phu antibody is a useful tool for further studies of perforin-induced cytotoxicity in vitro and in vivo. [ABSTRACT FROM AUTHOR]

This paper describes attempts to isolate and characterize glycosaminoglycan (GAG)-binding molecules on the surface of lymphocytes and lymphoma cell lines and relate their expression to splenic and lymph node homing capacity. Initial binding studies with radiolabelled GAG and rosetting studies with GAG-coupled erythrocytes revealed that there are receptors on lymphocytes for the major classes of GAG (i.e. hyaluronic acid, chrondroitin sulfates, heparin), but lymphocytes bind heparin much more avidly than other GAG species. Analysis of the binding of solubilized radiolabelled cell-surface molecules to immobilized GAG revealed cell-type specific expression of GAG-binding molecules. Thus, each of four lymphoma cell lines tested gave a characteristic pattern of GAG-binding molecules, some molecules being unique to a particular cell line and others being shared by some of the lines. Similarly, splenocytes expressed at least 10 distinct GAG-binding molecules with molecular weights (MW) ranging from 10,000 to 100,000, whereas thymocytes expressed additional GAG-binding proteins of 190,000 and 250,000 MW. Furthermore, splenocytes differed from thymocytes by possessing a unique family of cell-surface molecules which reacted with each GAG. Immunoprecipitation studies demonstrated that the GAG-binding molecules on splenocytes did not correspond to any of the cell-surface antigens tested, notably the cell adhesion molecules MEL-14, CD11/CD18 and CD44, although CD8 bound weakly to heparin. Four lymphoma cell lines with well-characterized migration properties were examined for GAG-binding molecules which may control lymphocyte migration. It was found that no one GAG-binding protein could be correlated with the entry of cells into a particular lymphoid organ. Nevertheless, the role of GAG-binding molecules in the subsequent positioning of lymphocytes within lymphoid organs requires further investigation. [ABSTRACT FROM AUTHOR]

Summary: Transmissible cancers are malignant cells that can spread between individuals of a population, akin to both a parasite and a mobile graft. The survival of the Tasmanian devil, the largest remaining marsupial carnivore, is threatened by the remarkable emergence of two independent lineages of transmissible cancer, devil facial tumour (DFT) 1 and devil facial tumour 2 (DFT2). To aid the development of a vaccine and to interrogate how histocompatibility barriers can be overcome, we analysed the peptides bound to major histocompatibility complex class I (MHC‐I) molecules from Tasmanian devil cells and representative cell lines of each transmissible cancer. Here, we show that DFT1 + IFN‐γ and DFT2 cell lines express a restricted repertoire of MHC‐I allotypes compared with fibroblast cells, potentially reducing the breadth of peptide presentation. Comparison of the peptidomes from DFT1 + IFNγ, DFT2 and host fibroblast cells demonstrates a dominant motif, despite differences in MHC‐I allotypes between the cell lines, with preference for a hydrophobic leucine residue at position 3 and position Ω of peptides. DFT1 and DFT2 both present peptides derived from neural proteins, which reflects a shared cellular origin that could be exploited for vaccine design. These results suggest that polymorphisms in MHC‐I molecules between tumours and host can be 'hidden' by a common peptide motif, providing the potential for permissive passage of infectious cells and demonstrating complexity in mammalian histocompatibility barriers. [ABSTRACT FROM AUTHOR]

Examines alloantigens of human lymphoid cell lines. Purification of membrane glycoproteins; Antigens involved in reaction with alloantisera; Specificity of alloantigents to B cell type cell lines.

Diffuse large B-cell lymphoma (DLBCL), the most common type of malignant lymphoma, accounts for 30% of adult non-Hodgkin lymphomas. Epstein-Barr virus (EBV) -positive DLBCL of the elderly is a newly recognized subtype that accounts for 8-10% of DLBCLs in Asian countries, but is less common in Western populations. Five DLBCL-derived cell lines were employed to characterize patterns of EBV latent gene expression, as well as response to cytokines and chemotaxis. Interleukin-4 and interleukin- 21 modified LMP1, EBNA1 and EBNA2 expression depending on cell phenotype and type of EBV latent programme (type I, II or III). These cytokines also affected CXCR4- or CCR7-mediated chemotaxis in two of the cell lines, Farage (type III) and Val (type II). Further, we investigated the effect of EBV by using dominant-negative EBV nuclear antigen 1(dnEBNA1) to eliminate EBV genomes. This resulted in decreased chemotaxis. By employing an alternative way to eliminate EBV genomes, Roscovitine, we show an increase of apoptosis in the EBV-positive lines. These results show that EBV plays an important role in EBV-positive DLBCL lines with regard to survival and chemotactic response. Our findings provide evidence for the impact of microenvironment on EBV-carrying DLBCL cells and might have therapeutic implications. [ABSTRACT FROM AUTHOR]

H60a is a minor histocompatibility antigen expressed in BALB and 129/Sv but not C57BL/6 mouse strains. The majority of CD8 T cells in C57BL/6 mice responding to BALB.B splenocytes are specific for H60a. Interestingly, H60a is expressed constitutively on tumour cells, but its nature as a tumour rejection antigen, as a parallel to its function as a transplant rejection antigen, has not been studied. In this report, we show that tumour cells that constitutively express H60a at the cell surface can be recognized by H60a-specific T cells. Furthermore, when H60a-expressing sarcoma cell lines are transplanted into C57BL/6 mice, H60a-specific T cells can be found at high percentages among the tumour-infiltrating CD8 T cells. These findings were seen in C57BL/6 but not F1 (C57BL/6 × 129) mice (which express H60a), suggesting that endogenous tolerance mechanisms suppress the antigenic properties of H60a. Our findings have implications for the generation of tumour vaccines against human natural killer group 2D ligands, such as MHC class I chain-like gene A, that are also transplantation antigens. [ABSTRACT FROM AUTHOR]

Dendritic cells (DC) generated from MUTZ-3, an immortalized acute myeloid leukaemia-derived cell line, have potential application as a model for the study of human DC, and as a tool with which to stimulate immunotherapeutic responses to cancer. However, the relationship of MUTZ-3 DC to their non-transformed counterparts remains incompletely understood. Immunoselected CD14+ MUTZ-3 cells were used to generate a homogeneous population of DC (M3DC). These cells had a cell surface phentoype and morphology characteristic of conventional monocyte-derived DC (MDDC). Whole genome transcriptome comparison of M3DC and MDDC however, revealed extensive differences between these two cell types. Functional ontology-based data analysis revealed three enriched clusters of genes downregulated in M3DC, with functions in pathogen recognition, DC maturation and cytokine/chemokine signalling. Downregulation of protein expression was confirmed for several of these genes. The molecular differences were accompanied by a profoundly impaired phenotypic and functional response of M3DC to microbial stimulation. The immortalized phenotype of MUTZ-3 therefore reflects not only deregulated proliferative capacity, but substantial perturbation of normal antigen-presenting cell function. These results have important implications for studies using MUTZ-3 as a model of MDDC or for cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Annexin-1 (ANXA1) is a glucocorticoid-regulated protein that modulates the effects of bacterial lipopolysaccharide (LPS) on macrophages. Exogenous administration of peptides derived from the N-terminus of ANXA1 reduces LPS-stimulated inducible nitric oxide synthase (iNOS) expression, but the effects of altering the endogenous expression of this protein are unclear. We transfected RAW264.7 murine macrophage-like cell lines to over-express constitutively ANXA1 and investigated whether this protein modulates the induction of iNOS, cyclooxygenase-2 (COX-2) and tumour necrosis factor-α (TNF-α) in response to LPS. In contrast to exogenous administration of N-terminal peptides, endogenous over-expression of ANXA1 results in up-regulation of LPS-induced iNOS protein expression and activity. However, levels of iNOS mRNA are unchanged. ANXA1 has no effect on COX-2 or TNF-α production in response to LPS. In experiments to investigate the mechanisms underlying these phenomena we observed that activation of signalling proteins classically associated with iNOS transcription was unaffected. Over-expression of ANXA1 constitutively activates extracellular signal regulated kinase (ERK)-1 and ERK-2, components of a signalling pathway not previously recognized as regulating LPS-induced iNOS expression. Inhibition of ERK activity, by the inhibitor U0126, reduced LPS-induced iNOS expression in our cell lines. Over-expression of ANXA1 also modified LPS-induced phosphorylation of the ERK-regulated translational regulation factor eukaryotic initiation factor 4E. Our data suggest that ANXA1 may modify iNOS levels by post-transcriptional mechanisms. Thus differential effects on iNOS expression in macrophages are seen when comparing acute administration of ANXA1 peptides versus the chronic endogenous over-expression of ANXA1. [ABSTRACT FROM AUTHOR]

B-cell linker protein (BLNK) is a component of the B-cell receptor (BCR) as well as of the pre-BCR signalling pathway, and BLNK–/– mice have a block in B lymphopoiesis at the pro-B/pre-B cell stage. A recent report described the complete loss or drastic reduction of BLNK expression in approximately 50% of human childhood pre-B acute lymphoblastic leukaemias (ALL), therefore we investigated BLNK expression in human pre-B ALL cell lines. One of the four cell lines tested, HPB-NULL cells, was found to lack BLNK expression, and we used these human pre-B ALL cell lines that express and do not express BLNK to investigate the intracellular signalling events following pre-BCR cross-linking. When pre-BCR was cross-linked with anti-μ heavy-chain antibodies, significant phosphorylation of intracellular molecules, including Syk, Shc, ERK MAP kinase, and AKT, and an activation of Ras were observed without regard to deficiency of BLNK expression, suggesting that BLNK is not required for pre-BCR-mediated activation of MAP kinase and phosphatidyl-inositol 3 (PI3) kinase signalling. By contrast, phospholipase C-γ2 (PLC-γ2) phosphorylation and an increase in intracellular Ca2+ level mediated by pre-BCR cross-linking were observed only in the BLNK-expressing cells, indicating that BLNK is essential for PLC-γ2-induced Ca2+ influx. Human pre-B cell lines expressing and not expressing BLNK should provide an in vitro model for investigation of the role of BLNK in the pre-BCR-mediated signalling mechanism. [ABSTRACT FROM AUTHOR]

Summary The mechanisms by which tumour cells escape recognition by the immune system or subvert antitumour effector responses remain poorly understood. In the course of investigating the potential of costimulatory signals in anticancer immunotherapy strategies, we have observed that HeLa cells (a human cervical carcinoma cell line) cocultured with peripheral blood lymphocytes (PBL) acquire the capacity to inhibit PBL proliferation in response to interleukin-2 (IL-2). This immuno-inhibitory phenotype was further shown to result from induction of the tryptophan-catabolizing enzyme, indoleamine 2,3-dioxygenase (IDO), by interferon-γ (IFN-γ) secreted from cocultured allo-reactive PBL. This enzyme has recently been shown to be a critically important modulator of immunological responses, most notably through the capacity to protect allogeneic concepti from alloreactive maternal lymphocytes. While the cytostatic consequences of IDO activity in tumour cells has received attention, the data presented in this report support the hypothesis that IDO activity may also act to impair antitumour immune responses. [ABSTRACT FROM AUTHOR]

We raised anti-VH315 antibodies by immunization of rabbits with VH315 fragments, the variable portions of the immunoglobulin heavy chains of MOPC315 myeloma protein. Inhibition radioimmu-noassay using various immunoglobulins as inhibitors showed that the anti-VH315 antibodies specifically reacted with the variable portions of the heavy chains of MOPC315 myeloma protein. When the variable region (VH) gene of the heavy chains was cloned and sequenced from the cells producing the heavy chains detected by the anti-VH315 antibodies, the VH gene was closely related (82% homology at amino acid level) to the VH gene of MOPC315, When we examined the frequency with which the variable region(s) detected by the anti-VH315 antibodies were expressed in eight Ig- null Abelson virus-transformed cell lines (DJ/DJ or VDJ-/DJ). which were able to generate functional V regions during culture, only one cell line, ATB-1, produced a small number of intracytoplasmic/μ-positive cells (VH315+ cells) stained by the anti-VH315 antibodies. The percentage of the total number of the VH315+ cells to the total number of intracytoplasmic μ-positive cells was 0·91% in AT8-1. In the remaining seven cell lines, no VH315+ cells were detected. In the present study we estimate, for the first time at the individual cell level, the frequency of the utilization of the heavy chain variable region(s) identical or closely related to that of MOPC315 in the functional V region formation during early B-cell development. [ABSTRACT FROM AUTHOR]

Antigen-specific sheep T-cell lines have been generated in vitro from peripheral blood mononuclear cells (PBMC). PBMC prepared from antigen-primed animals were cultured in the presence of ovalbumin (OVA) or purified protein derivative (PPD). After 5 days of culture, activated antigen- specific cells were expanded by further culture in the presence of recombinant human interleukin-2 (IL-2). Cell lines generated after two cycles of antigen stimulation followed by expansion with IL-2 show a proliferative response to antigen only in the presence of autologous antigen-presenting cells (APC) and recognize only the antigen used in the original stimulation. An OVA-specific cell line was found to be capable of recognizing a synthetic peptide corresponding to amino acid residues 323-338 of OVA. The cell lines also responded by proliferation in an allogeneic mixed leucocyte reaction (MLR). Cell-surface phenotyping shows that the cell lines comprise both CD4- and CD8-positive cells. [ABSTRACT FROM AUTHOR]

We have analysed homotypic adhesion induced by the phorbol ester TPA in EBV-immortalized Down's syndrome (trisomy 21) and normal lymphoblastold cell lines. Our results show that the trisomy 21 cells aggregated more readily than normal cells. The aggregation of the two types of cell was blocked effectively by the addition of monoclonal antibodies to the α and β chains (CD11a, CD18) of lymphocyte function-associated antigen (LFA-), but not by monoclonal antibodies to LFA-3 and the α chain of p150,95 (CD11c). Since TPA did not increase the expression of CD18 on trisoiny 21 cells, we conclude that the increased adhesiveness is the result of over-expression of CD18 as a result of gene dosage. [ABSTRACT FROM AUTHOR]

Mononuclear cells play an important role in the elimination of immune complexes (IC). In the presence of complement (C) the binding and degradation of IC by mononuclear cells is enhanced at least two-fold. The enhancement of binding is caused by a synergistic interaction of the IC with cellular Fc and complement receptors (R). In the present study we have investigated the contribution of the complement receptors CRI and CR3 of human monocyte cell line U937 on the complement- mediated binding and degradation of immune complexes and soluble aggregates of IgG (AIgG) bearing C3b or iC3b. It was found that deposition of C3b on AIgG enhanced the binding of AIgO to U937 cells at least two-fold. The C3b-mediated enhancement of binding was abolished by anti-CR1. iC3b-bound to AIgG also enhanced the binding of AIgO to the cells. This binding was only partially reduced by anti-CR3 antibodies, but the combination of anti-CR1 and anti-CR3 fully abolished the iC3b-mediated enhancement of binding. These results suggest that both CR1 and CR3 contribute to the complement-mediated binding and degradation of soluble IC by mononuclear phagocytes. [ABSTRACT FROM AUTHOR]

A new cell line has been produced by fusing human cervical keratinocytes with HeLa cells. This cell line secretes eosinophil-activating activity upon stimulation with tumour necrosis factor (TNF). About one-third of the eosinophil-activating activity co-purifies with eosinophil-activating factor (EAF) from mononuclear cell supernatants. The purification procedure indicates that it resembles EAF in molecular weight and acidity. It also resembles EAF in its effect on eosinophils. Not only does it enhance the cytotoxic activity of eosinophils to antibody-coated schistosomula of Schistosoma mansoni, but it also increases the oxidative activity of eosinophils, as measured by reduction of nitroblue tetrazolium, and changes the morphology of eosinophils, affecting the distribution of F-actin in the cell. [ABSTRACT FROM AUTHOR]

A subpopulation of human lymphoid cells called natural killers is able to lyse certain normal and neoplastic targets in an in vitro cytotoxicity assay. The molecules which enable them to recognize sensitive cells, or permit turnout cells to escape remain unknown. In the studies described here we have compared some of the plasma membrane characteristics of a NK-sensitive human leukaemia cell line (K562) with those of a partially resistant subclone derived from it (K562-Clone I). Gel electrophoresis of cell-surface proteins radiolabelled by lactoperoxidase-catalysed iodination, periodate-borohydride tritiation, or biosynthetically by incubation with [3H]fucose did not reveal any reproducible differences between the sensitive and resistant lines. However, analysis of glycolipids showed that Clone I incorporated significantly more fucose than did the parental line, and that it synthesized a minor population of complex structures not found in the original K562. A subclone of Clone I(Clone I-Con ArI), made resistant to the toxic effects of concanavalin A, became sensitive once again to NK, and showed the parental glycolipid profile. These results suggest that the Clone I line, selected for resistance to NK, may have altered one or more of its intermediate oligosaccharides or pathways of fucose incorporation into glycolipid, and points to one process by which a tumour cell might modulate its surface to escape recognition by natural killers. [ABSTRACT FROM AUTHOR]

The capacity of cloned murine Ia-positive BK-BI-2.6.C6 T cells to present protein antigens to antigen-reactive long-term cultured T-cell lines was investigated. Antigen recognition by T-line cells on presenting BK-BI-2.6.C6 T-accessory cells resulted in efficient production of lymphokines. While antigen-dependent T cells with transient interleukin-2 receptor (IL-2R) expression were not induced to proliferate, T cells with constitutive IL-2R expression proliferated in response to the secreted IL-2. Although antigen presentation by BK-BI-2.6.C6 T cells resulted in a slight induction of IL-2R expression on responding T cells, as measured by flow cytometry, this augmentation was much smaller than that induced by antigen-presenting spleen cells. Thus the inability of antigen-presenting T-accessory cells to stimulate proliferation of responding T cells with transient IL-2R expression appears to reflect a lack of signal(s) necessary for the induction of IL-2R up to a level critical for initiation of cell division. This test system represents an ideal model to investigate the nature of signals required, in addition to triggering of the T-cell antigen receptor, for the induction of IL-2R. [ABSTRACT FROM AUTHOR]

In order to explore the role of glutathione in cell-mediated cytotoxicity, we have examined the effect of the sulphydryl-reactive and glutathione-depleting agent 2-cyclohexene- 1 -one on antibody- dependent cellular cytotoxicity, spontaneous cell-mediated cytotoxicity, and cell-mediated lympho- lysis by human peripheral blood mononuclear cells. 2-Cyclohexene- 1 -one significantly inhibited (P < 0.001) both antibody-dependent and spontaneous cell-mediated cytotoxicity using three different cell-line targets, at three different killer: target cell ratios (10: 1,25: 1 and 50: 1). Using K-562 cell-line targets, spontaneous cell-mediated cytotoxicity was inhibited by 2-cyclohexene-1-one with an ID50 of 0.71 × 10-4 M-3.98 × 10- M, while antibody-dependent cellular cytotoxicity was less sensitive to inhibition, and required slightly higher concentrations of 1.48 × 10-4 M-3.98 × 10-4 M to achieve 50% inhibition. Similar results were seen with human colon tumour cell-line and Chang liver cell-line cells as targets. Maximal inhibition occurred when 2-cyclohexene-1-one was added to the cytotoxicity assay 60 mm prior to, at the start of, or within the first 60 mm of a 4-hr assay; inhibition of cytotoxicity occurred with pretreatment of effector cells; and no inhibition of cytotoxicity was observed with pretreatment of target cells. Both the allogeneic mixed leucocyte reaction and cell- mediated lympholysis were also significantly inhibited (P < 0.001) by 2-cyclohexene-1-one. These studies demonstrate that 2-cyclohexene-1-one is an effective inhibitor of cell-mediated cytotoxicity and suggest that glutathione, specific glutathione-protein interactions, or protein-bound sulphydryl groups are involved in allowing cells to carry out cytolysis. [ABSTRACT FROM AUTHOR]

A simple and highly reproducible bone marrow culture system for the generation of cytolytically active NK cells from immature precursors in the bone marrow is described. The NK cells can be generated with various sources of IL-2, including Con A-conditioned medium, supernatants from IL- 2-producing cell lines and recombinant IL-2. Neither IL-1, IL-3 nor α/β interferon induced significant cytotoxicity in bone marrow cells. Identification of the cytotoxic cells as NK cells was based on their phenotypic characteristics (aGMI+, Thy 1±, Ly 1-2-. Ia-, RlL-2±, H2+), as well as their spectrum of target specificity. The deliberate addition of peripheral blood mononuclear cells as a source of mature NK cells and elimination of cells expressing markers specific for mature NK cells indicated that the generated NK cells were descendants of precursors of NK cells harboured in the bone marrow and not derived from mature NK cells contaminating the bone marrow preparations. Thus, it was shown that not only functionally active NK cells but also their precursors are highly dependent on IL-2 for differentiation and growth. This culture system should be helpful in studying the origin of NK cells in relation to other cell lineages as well as the regulation of the maturation of NK cells from their precursors. [ABSTRACT FROM AUTHOR]

The requirement for activity of the enzyme ADP-ribosyl transferase (ADPRT) and changes in singleastrand DNA breaks were assessed during the initial stimulation of quiescent murine splenic lymphocytes with mitogen alone, the stimulation of activated blasts with IL-2-containing medium and, for comparison, the serum stimulation of quiescent fibroblasts and the induction of haemoglobin synthesis in an erythromyeloid cell line K562. Inhibitors of ADPRT, at concentrations previously found to have no effect on the proliferation of lymphoblastoid cell lines, blocked the stimulation of spleen cells by Con A or LPS; non-inhibitory analogues had much less effect. No early increase in ADPRT activity after mitogenic stimulation was detectable. The rejoining of single- strand breaks was observed after stimulation of splenic lymphocytes with Con A, but not consistently with LPS. Conversely, ADPRT inhibitors had only little effect on the IL-2-induced stimulation of Con A blasts, and no effect on the stimulation of fibroblasts or K 562. Neither were any changes in strand breaks associated with these systems. These findings implicate ADPRT activity and the rejoining of strand breaks in the early mitogenic response as being distinct from later IL-2 activation and changes from quiescence to growth in other cell types. [ABSTRACT FROM AUTHOR]

When the serum content of tissue culture medium is reduced from 10% to 1%, the capacity of T cells to proliferate in response to antigen within that medium is dramatically reduced. Physiological concentrations of platelet-derived growth factor (PDGF) or epidermal growth factor (EGF) are able to partially replace the requirement for serum, in that they are able to increase antigen-driven T-cell proliferation at a serum concentration of 1%. Neither growth factor is mitogenic for T cells in the absence of antigen, and neither is able to act synergistically with T-cell growth factor (TCGF) or IL-2) in the absence of antigen. Antigen-presenting cells (APC) pulsed with antigen in the presence of PDGF or EGF are able to stimulate antigen-specific T-cell proliferation to a greater extent than antigen-presenting cells pulsed in the absence of exogenous PDGF or EGF. Both growth factors increase the expression of MHC Class Il antigens on antigen-presenting cells. [ABSTRACT FROM AUTHOR]

Two PPD-reactive T-cell lines and two clones derived from them have been characterized. The lines were maintained for a period of 10–12 weeks in 11–2 containing medium. The clones were derived from the uncloned lines by the limiting dilution method and maintained in culture for 12 weeks. The cloning efficiency was 1%, Both the cloned and the uncloned lines were highly reactive to tuberculin in a proliferation assay and produced migration inhibition factors following antigenic stimulation. Both these functions were dependent on the addition of antigen-presenting cells and genetically regulated by Class II molecules of the MHC. Each uncloned line and the clones derived from them were restricted by just one of he DR alleles of the autologous host. An analysis of ell types involved in antigen presentation showed that macrophages and Epstein-Barr virus-transformed B-cell lines induced both proliferation and MIF secretion in the T-cell lines and clones cultured with PPD. Phenotypic studies indicate that the cells are Sheep E+, OKT4+, OKT8- and HLA-DR+. [ABSTRACT FROM AUTHOR]

Mononuclear phagocytes can synthesize a cytotoxin, similar to that in turnout necrosis serum, which is cytotoxic to certain tumour cell lines in vitro. This study has investigated whether susceptibility and resistance to the cytotoxin can be explained in terms of the amount of cytotoxin receptor expressed on the tumour cell surface, Binding of cytotoxin has been quantitated both by direct adsorption and competitive inhibition assays with cytoxin-resistant or -susceptible tumour lines and with sublines of susceptible lines, selected for resistance to the cytotoxin. For both rabbit and human cytotoxin, there was no correlation between cytotoxin adsorption by turnout cell lines and their cytotoxin susceptibility, suggesting that resistance to the cytotoxin is expressed at a post-receptor stage. Preliminary studies on the cytotoxin receptor of K562 cells have shown that it is probably not the transferrin receptor, and that protein but not carbohydrate is essential for its function. [ABSTRACT FROM AUTHOR]

Seven Epstein-Barr virus (EBV)-transformed B cell lines were derived from circulating lymphocytes of two atopic and two non-atopic individuals, two preparations of cord blood lymphocytes and one tonsillar lymphocyte preparation. All the cell lines contained a significant proportion of cells expressing FccR as detected by rosette formation with IgE-coated bovine erythrocytes (E-IgE) and by flow cytometry using IgE-linked to fluorescent microspheres. None of the cell lines displayed FcR for IgA, IgM or IgG. The cell-free supernatants (CFS) of EBV-transformed cells contained IgE-binding factors (IgE-BFs) detected by their ability to inhibit the binding to RPMI 8866 cells of either E-IgE or IgE-linked to microspheres. Whereas these CFS enhanced the synthesis of IgE and suppressed the synthesis of IgG by purified B lymphocytes isolated from the blood of allergic donors and cultured in the absence of stimulant, their effect on the synthesis of IgA or IgM was not predictable. CFS significantly enhanced the secretion of IgE by the U266 myeloma cell line without interfering with secretion of IgM, IgG or IgA by EBV-transformed cells. These data are in accord with similar properties of RPMI 8866 cells and suggest that B lymphocytes might play a regulating role in the IgE synthesis. [ABSTRACT FROM AUTHOR]

Human monocytes can kill or inhibit the growth of certain tumour cell lines. Amongst the possible mediators is a cytotoxin synthesized in readily detectable amounts by endotoxin-stimulated monocytes. A neutralizing antiserum to the cytotoxin has been used to assess the cytotoxin's contribution to monocyte killing of turnout cells. The antiserum was tested for possible inhibition of monocyte killing of three tumour cell lines—L929, K562 and A549. Inhibition was complete with L929, partial with K562 and insignificant with A549. Thus the contribution of the cytotoxin to monocyte killing of turnout cells depends upon the tumour line under test. Antibody against endotoxin-induced cytotoxin also neutralized cytotoxin induced in monocytes by other agents including BCG, Corynebacterium parvum, pokeweed mitogen and zymosan. Cytotoxin could be quantitatively removed from monocyte supernatants by Sepharose-bound, anti-cytotoxin antibody. Recovery of the cytotoxin from the immunoadsorbent was difficult because of its lability in the solutions commonly used for desorption. The best recovery has been achieved with 2 M urea. 2 M MgCl2 giving a 25% yield and a forty-fold increase in purity in a single step. [ABSTRACT FROM AUTHOR]

Receptors for IgE of rat basophilic leukaemia (RBL) cells, maintained in different laboratories were isolated by means of various anti-receptor antisera, and characterized by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. Most antisera were shown to react with a receptor designated H and which, as shown previously, could be isolated by IgE-Sepharose but not by a combination of IgE and anti-IgE. Only two highly purified anti-receptor antibody preparations, which had been purified by adsorption to and elution from rat basophilic leukaemia cells, reacted primarily with a second kind of receptor molecule which had previously been designated R. Some indirect evidence was obtained which suggests that H is a highly immunogenic molecule. It was confirmed that the apparent molecular weight of H-like molecules varied on different RBL cell lines. [ABSTRACT FROM AUTHOR]

Receptors for IgE of rat basophilic leukaemia (RBL) cells, maintained in different laboratories were isolated by means of IgE-Sepharose or IgE and anti-IgE, and characterized by SDS-polyacrylamide gel electrophoresis. All cell lines were found to be associated with a receptor molecule (R) which could be isolated either with IgE-Sepharose or IgE and anti-IgE and a second receptor (H) which could only be isolated with the aid of IgE-Sepharose. The relative amounts of these two molecules, as isolated from surface iodinated cells, varied from one RBL cell line to the other and their apparent molecular weights were not identical on all cell lines. Since comparisons were made on the same gel using receptors isolated from cells labelled with different isotopes of iodine, differences in molecular weight must be considered as being intrinsic and not due to methodological variations. These results provide an explanation why differences were observed among receptors for IgE as characterized in various laboratories. In spite of the fact that the various RBL cell lines originated from the same chemically-induced tumour they have, over the years, undergone changes which are reflected in the receptors for IgE. [ABSTRACT FROM AUTHOR]

Two populations of human blood lymphocytes one forming rosettes with sheep erythrocytes, the other non-rosetting are cytolytic in vitro for several long term cultured tumour-derived cell lines. A particular breast cancer-derived target cell (MDA-157) is only killed by the non-rosetting effector. Rosetting cells from normal donors infected or immunized with influenza virus augment cytolytic activity on MDA-157 targets by non-rosetting effector cells. Similar augmenting activity can be induced by incubating the rosetting population with sources of immune (gamma), but not leucocyte (alpha) interferon in vitro. This augmentation of cytolytic activity does not require compatibility at the major histocompatibility locus between the augmenting and effector populations. [ABSTRACT FROM AUTHOR]

Spent culture media of all the human cell lines tested have been found to contain the antigenic activity present on the ll,000-Dalton HL-A common portion fragment of the HL-A antigen molecule that appears to be a characteristic, invariant portion of HL-A antigen molecules. From the culture medium of one of these lines, RPMI 1788, a lymphoid cell line, the substance carrying HL-A common activity was isolated, which was shown to be identical to the HL-A common portion fragment with respect to molecular size, electrophoretic mobility, isoelectric focusing patterns, and certain antigenic characteristics. By an isolation procedure involving differential ultrafiltration, gel filtration, and column electrophoresis, 8 litres of the culture medium yielded 1.5-2.0 A280 units of the substance representing 15-20 per cent of the HL-A common antigenic activity originally present. A single protein band with a Rf of 0.47 was obtained by disc electrophoresis. The molecular size was shown to be about 11,000 Daltons by gel filtration and by sodium dodecyl sulphate acrylamide gel electrophoresis. Upon isoelectric focusing two bands were obtained which corresponded exactly to those obtained with HL-A common portion fragment prepared from papain-solubilized HL-A antigen preparations by acid dissociation. The isoelectric point of the major band was 5.0. The reactions of this substance with rabbit antisera against human lymphoid cell membrane and against the substance were essentially identical to the reactions of HL-A common portion fragment with these same antisera. [ABSTRACT FROM AUTHOR]

Mast cells are known to adhere to laminin, although there is limited information on the characteristics of this event. To further examine this adhesive interaction, we thus determined the adherence of murine mast cell lines and primary bone marrow cultured mast cells (BMCMC) to murine laminin (mLN), human placental laminin-1 (hLN), merosin (laminin-2) and various laminin fragments, concentrating on activating stimuli, the involvement of integrins, and the effect on mast cell activation. Murine mast cells were found to adhere to both mLN and hLN and to merosin, not only following exposure to phorbol 12-myristate 13-acetate (PMA), but also after Fc∊RI aggregation or addition of stem cell factor (SCF). Adhesion to laminin was partially inhibited by soluble E8 and PA22-2, both fragments of laminin that promote mast-cell adhesion when bound on surfaces. Mast-cell lines and BMCMC consistently expressed high levels of α6 integrin. Antibody to α6 blocked spontaneous and inhibited activated mast-cell adhesion to hLN, and inhibited mast-cell adhesion to mLN and its fragment E8. Mast-cell adhesion to both laminin isoforms increased Fc∊RI-mediated mast-cell secretion. These observations demonstrate that mast- cell attachment to laminin is promoted by physiological stimuli, is mediated principally by α6 integrin, and results in enhanced cell activation. [ABSTRACT FROM AUTHOR]

Exogenous prolactin (PRL) has been shown to synergize with low-dose interleukin-2 (IL-2) and induce the proliferation and lymphokine-activated killer (LAK) maturation of natural killer (NK) cells. PRL itself can also generate LAK activity. Here we show that its local production occurs during, and is necessary for, LAK development. IL-2-stimulated peripheral blood mononuclcar cells (PBMC) and purified NK cells were exposed to anti-human (h)PRL antiserum, and residual LAK activity was measured on day 7 against the promyelocytic leukaemia cell line HL-60. Inhibition of LAK activity was much more evident in PBMC compared with NK cell cultures (47% decrease, P = 0·013 and 18·5% decrease, P = 0·048, respectively). Up-modulation of a 32S-methionine-labelled 27000 MW protein was detected in the lysates and supernatants of IL-2-stimulated PBMC immunoprecipitated with an anti-PRL antiserum. By contrast, the cytoplasmic PRL immunoreactivity observed in freshly isolated NK cells and in IL-2-stimulated, but not unstimulated, NK cell cultures was not associated with PRL gene activation, and can thus be referred to internalized PRL. Preferential re-uptake of externally derived PRL by IL-2-stimulated NK cells was also indicated by up-modulation of the PRL receptor. These data, as a whole, indicate that the PRL promotion of LAK differentiation is mainly mediated by paracrine secretion, with a minor contribution from internalized PRL. [ABSTRACT FROM AUTHOR]

Circulating lymphocytes are rounded, non-motile cells which on contact with cytokines, specialized or activated endothelium, acquire a constantly shape-changing, polarized morphology which enables migration into appropriate sites. The biochemical mechanisms which regulate this switch are not understood but the various stimuli may have a common final pathway. In this study we show that protein kinase C (PKC) inhibitors of the bisindolylmaleimide type (GF 109203X, Ro 31-8220, CGP 41 251) induce resting, spherical lymphocytes to change rapidly (<30 min) into polarized, locomotory cells. This phenomenon was seen with diverse populations of blood T lymphocytes, tonsillar B cells and Jurkat and Molt4 T-cell lines. Consistent with this, down- regulation of PKC by chronic treatment (44 hr) with bryostatin also induced the polarized phenotype in blood lymphocytes and non-motile Molt4 cells. Conversely, treatment of a spontaneously motile subline of Molt4 cells with various PKC activators caused a reversion to the non-motile phenotype within minutes. PKC activation must be sufficient to overcome the effects of a constitutively active phosphatase because bisindolylmaleimide induction of motility could be prevented by pretreatment of the cells with a phosphatase inhibitor, calyculin A. It is concluded that, in resting lymphocytes, chronic activation of a PKC offsets the action of a constitutively active phosphatase and the net result is maintenance of the non-motile state. Agents which alter the kinase/phosphatase balance in favour of dephosphorylation result in induction of the locomotory phenotype. [ABSTRACT FROM AUTHOR]

Lipopolysacchiride (LPS), known as one of the potent activators of macrophages, has inhibitory effects on the proliferation of normal macrophages and macrophage-like cell lines. We report here that LPS dose- and time-dependently suppressed the tritiated thymidme ([³H]TdR) incorporation into the acid-insoluble fraction with a significant inverse correlation to the tumour necrosis factor-α (TNF) production in the J774.1 macrophage cell line. Among the three tested enzymes involved in DNA synthesis, only thymidine kinase (TK) activity decreased progressively in parallel with the decline in [³H]TdR incorporation, reaching 97% inhibition within 12 hr of LPS treatment, while changes in the activities of other two enzymes, DNA polymerase α and thymidylate synthase (TS), were less significant. On the other hand, LPS inhibited the cell proliferation only incompletely, as judged by 62% inhibition of cell growth at 36 hr. Even in the experiments done in a TdR-free medium, cell growth was inhibited by LPS to the same extent, suggesting that TK was not directly involved in the proliferation of J774 cells. LPS also inhibited the conversion of TdR to thymidine monophosphate (TMP) in murine peritoneal exudate inacrophages (PEM). Thus LPS-induced suppression of TdR salvage related to TNF production is common in both normal and neoplastic macrophages, and therefore may be of potential importance in the process of macrophage activation. [ABSTRACT FROM AUTHOR]

Despite profound knowledge about the molecular structure of the γδ T-cell receptor (TCR), the physiological function of γδ T cells remains enigmatic. Participation of these cells in complex immune reactions, however, is suggested by the appearance of γδ T cells in sites of infectious and autoimmune-induced inflammations. Only a few in vitro models of γδ T-cell stimulation have been established: besides a reactivity in the presence of microbial ligands, human γδ T cells proliferate upon in vitro challenge with cells from an allogeneic B-lymphoblastic cell line (B-LCL). We present data here demonstrating that this reactivity is not confined to allogeneic B-LCL. Autologous B-LCL are also very strong stimulators for γδ T cells; more important, autologous B cells can stimulate γδ T cells after a period of mitogen-activation but not in a resting state. This activation seems to address a subgroup of γδ T cells, as the percentage of Vδ1+ cells is increased after stimulation. Activated γδ T cells, on the other hand, are able to exert an influence on B cells by inhibiting the secretion of IgG in coculture experiments. These data define a simple regulatory circle of B cells and γδ T cells in vitro and propose a model for γδ T-cell function which could explain many in vivo observations of γδ T-cell activation. [ABSTRACT FROM AUTHOR]

CD43 (leucosialin, sialophorin) is the major sialoprotein of nearly ail circulating leucocytes and has important biological activities in cellular differentiation and activation. Recently, the expression of CD43 has also been demonstrated on mast cells and basophils by flow cytometry. In order to further characterize mast cell/basophil leucosialin we have investigated CD43 on the human mast cell line HMC-1, the human basophilic precursor cell line KU-812, and the human promonocytic cell line U-937. The apparent molecular weights (MW) were 123,000 (HMC-1 and KU-812) and 144,000 (U-937) by Western blot analysis. Expression of CD43 on HMC-1 was down-regulated after stimulation with phorbol myristate acetate (PMA). Three monoclonal antibodies (mAb) specific for human CD43 induced homotypic mast cell line (HMC-1) aggregation in a semi-quantitative assay, a phenomenon that has not been described before with mast cells. Monoclonal antibodies specific for seven other surface antigens and an irrelevant mAb of the same isotype had no effect. The level of aggregation was dependent on anti-CD43 mAb concentration, time and temperature. Anti-leucosialin-induced aggregation of HMC-1 cells was completely inhibited by mAb against CD11a (LFA-1) and CD18 (β2-chain). Monoclonal antibody to CD54 (ICAM-1) partially inhibited anti-CD43-induced homotypic aggregation, while antiCD11b (CR3), anti-CD11c (p 150, 95) and a control mAb had no inhibitory effect. We conclude that mast cell line CD43 antigen expression is differentially regulated during cell activation, and speculate that anti-CD43-induced homotypic aggregation of HMC-1 cells is closely associated with modulation of β2-integrins. [ABSTRACT FROM AUTHOR]

We have previously reported a method for inducing natural suppressor (NS) cells by long-term culture of normal adult mouse spleen cells. The NS cells were further identified as mucosal or immature mast cells by morphology, cytochemistry, histochemistry and function. A cloned immature mast cell line was also confirmed to have NS activity. As NS cells, the cell line suppressed non-specifically the plaque-forming cell (PFC) response. The NS cell-free supernatant was partially enriched by chromatography and some fractions suppressed the PFC response and thymocyte proliferation. Heat treatment of the fractions failed to deplete the suppressive activity. The fractions were confirmed, by immunoblotting analysis, to contain transforming growth factor (TGF)-β. Recombinant human TGF-β was also able to suppress the PFC response and thymocyte proliferation. Neutralizing anti-TGF-β reversed the suppression by both human TGF-β and the fraction. From the above results, it is clear that mast cells displayed NS activity, at least partially, through the release of TGF-β. [ABSTRACT FROM AUTHOR]

Cell line PER-117 is a T-cell receptor negative human T-cell line that can be induced to express a functional interleukin-2 receptor (IL-2R). Recombinant interleukin-1 (IL-I) as well as certain combinations of inducer substances could be shown to stimulate the expression of the p55 (α)-chain of the IL-2R in PER-1 I7 cells: The synergistic increases in 1L-2 Rα expression were demonstrated at the cell surface as well as at the mRNA level. The results suggested that in PER-1 I7 cells 1L-1 appears to induce expression of the α-chain by pathways that are different to activation via protein kinase C (PKC), and that drug-induced cyclic AMP (cAMP) activation did not substitute for IL-1. We found that the regulation of mRNA for IL-2Rβ (p75) differed significantly from that seen for IL-2Rα. Moreover, the requirements for IL-2Rα induction determined for this cell line differ from other human cell lines, which may reflect that there are distinct requirements for activation depending on the stage of differentiation and/or lineage of the cells. The PER-1I7 cell line provides a unique model to examine further the mechanism leading to induction of a functional IL-2R at an early stage of human T-cell differentiation. [ABSTRACT FROM AUTHOR]

We have studied activation-induced changes in intracellular calcium [Ca2+ ]1, interleukin-2 (IL-2) secretion, and clonal abortion of the human leukaemic T-cell line Jurkat and three T-cell receptor (TcR)/CD3 receptor negative clones deficient for the TcRα, TcRβ and CD3γ chains respectively, as well as three transfectant clones reconstituted with the appropriate TcR/CD3 cDNA. For activation, the cells were exposed to anti-TcR/CD3, anti-CD2 and anti-major histocompatibility complex (anti-MHC) class I monoclonal antibodies (mAb) respectively. Cellular activation by these mAb leading to an increased IL-2 secretion was preceded by a rise in [Ca2+]1 and was relatively dependent on the expression of the a TcR/CD3 complex. In contrast, anti-MHC class I mAb-induced clonal abortion in Jurkat T cells may occur without previous fluctuations in [Ca2+]1 and appeared to be independent of TcR/CD3 expression. The present observations suggest the existence of different secondary messenger systems operating in Jurkat cells following activation via the TcR/CD3, CD2 and the MHC class I pathways, respectively. [ABSTRACT FROM AUTHOR]

The biochemical analysis of murine FcγRI has been difficult because this receptor is co-expressed with other Fc receptors on the cell surface of myeloid cell and all precipitate together with immune complexes. To overcome this problem and to study cells which expressed FcγRI and only this Fc receptor, stable transfection of Chinese hamster ovary (CHO) cells (FcγRI‾) with FcγRI cDNA was performed thereby permitting biochemical characterization of FcγRI in isolation from other Fc receptors. Studies of FcγRI+CHO cells showed the mature FcγRI to be a 70,000 MW single-chain receptor on the cell surface. The 70,000 MW molecule was also identified on the FcγRI+ cell lines, J774 (a macrophage-like cell line), and 18.81 (a pre-B-cell line). FcγRI was shown to be N-linked glycosylated and after deglycosylation to have a protein core of 49,000 MW. FcγRI was found to be phosphorylated and after PMA stimulation, the level of phosphorylation was increased; serine residues in the cytoplasmic tail were identified as the phosphate acceptors. Thus FcγRI can now be definitely characterized as a 70,000 MW phosphoprotein. [ABSTRACT FROM AUTHOR]

The active metabolite of cholecalciferol, 1,25-dihydroxycholecalciferol (1,25-DHCC), is a mononuclear phagocyte product with immunoregulatory properties which can influence not only surrounding T cells but also other mononuclear phagocytes; and which acts in an autocrine fashion. In this study we have used the U937 cell line as a starting point model to investigate further the comparative role of 1,25-DHCC and phorbol myristate acetate (PMA) upon growth, differentiation and phenotype in the mononuclear phagocyte system, and have correlated our findings with changes in 1,25-DHCC metabolism and receptor expression. Both 1,25-DHCC and PMA inhibit growth and differentiate U937 cells in a dose-dependent fashion. When used together, however, at Iow doses of PMA, 1,25-DHCC protects against the PMA-induced growth inhibition. At high concentrations of both compounds there is a decrease (1,25-DHCC) or an increase (PMA) in 1,25-DHCC receptor expression, with either 24-OHase (1,25-DHCC) or 1-OHase (PMA) synthesis. If the compounds are used in combination the receptor levels are equivalent to controls, and both enzymes are produced. The phenotype of the 1,25-DHCC-induced cell shows light adherence, class I+, increase in CD4 and CD14 and decrease in CD71. The PMA-induced cell is tightly adherent, class 1+, and strongly positive for CD13 with a concomitant decrease in both CD4 and CD71. These findings suggest another role for 1,25-DHCC in the mononuclear phagocyte system, as a potential mitogenic agent. They also suggest that 1,25-DHCC may act at both membrane and nuclear levels within this model of the mononuclear phagocyte pathway and demonstrate one possible way in which physiological peripheral macrophage heterogeneity might be induced, i.e. due to the nature of the signals which are implicated during differentiation. The presence of increased CD4 and decreased CD13 on the surface of 1,25-DHCC-differentiated cells, and vice versa on PMA-differentiated cells, illustrates how this may then be reflected in functional mononuclear phagocyte heterogeneity, which may in turn be reflected in differential peripheral function. [ABSTRACT FROM AUTHOR]

Intravenous injection of purified CD4+ CD8- T cells from thymus, spleen or lymph nodes of adult dm2 donor mice (H-2d, Ld-, IgMa) into young C. B-17 scid/scid (scid) mice (H-2d, Ld+, IgMb) partially and selectively reconstituted the splenic CD4+ T-cell compartment of recipient scid mice. This was demonstrated by cytofluorographic analyses, histological examinations, growing donor-derived CD4+ T-cell lines in vitro from spleens of transplanted scid mice, and serial passage of Ld-, CD3+ CD4+ CD8- T-cell lines through young scid recipients for more than 1 year. Host-derived IgMb appeared in sera of all CD4+ T-cell-transplanted young scid mice, while none of the sex- and age-matched, non-transplanted control scid mice was Ig+. B-cell leakiness was most efficiently induced by transfer of low numbers of adult CD4+ CD8- thymocytes: transfer of 10³ purified CD4+ CD8 thymocytes efficiently rescued scid-derived B cells, while transfer of 2 × 107 non-fractionated thymocytes from the same donor mouse was inefficient. Serum antibodies of scid mice with T-cell induced B-cell leakiness stained congenic 'double-positive' (CD4+ CD8+) thymocytes and immunoprecipitated protein bands with an apparent MW of 14,000, 28,000, 43,000, 65,000 and 80,000 from 125I-labelled thymocytes. These data indicate that repopulation of peripheral lymphoid organs with CD4+ T cells is always accompanied by partial co-reconstitution of the B-cell system, and raises the question of the interdependence of these two lymphocyte subsets. [ABSTRACT FROM AUTHOR]

Tumour necrosis factor (TNF) and interferon-gamma (IFN-γ) induce differentiation of human myeloid cell lines along the monocytic lineage. In this study we investigated the effects of TNF and IFN-γ on the expression of the CD4 protein and messenger RNA (mRNA) in the two myeloid cell lines, ML3 and HL-60. We observed that CD4 antigen expression on ML3 cells is almost undetectable and that TNF and IFN-γ induced CD4 antigen expression on these cells. HL-60 cells express surface CD4 antigen at high density and treatment with TNF and IFN-γ caused a decrease of CD4 expression. We also investigated the expression of CD4 mRNA in ML3 and HL-60 cells. ML3 constitutively express, albeit at low levels, CD4 mRNA. TNF induced CD4 mRNA in ML3 cells and IFN-γ synergistically potentiated the effect of TNF, thus indicating that the enhanced expression of the CD4 protein on ML3 cells is due, at least in part, to an enhanced accumulation of the CD4 mRNA. CD4 mRNA is constitutively expressed in HL-60 cells at high levels. TNF and IFN-γ, alone or in combination, did not cause any significant change of CD4 mRNA expression in HL-60 cells, thus indicating that decrease of surface CD4, which accompanies differentiation with these cytokines, is likely due to alterations of the CD4 protein synthesis and/or transport to the plasma membrane. These results provide evidence that myeloid cell lines are heterogeneous in expression of CD4, and that in ML3 cells, which constitutively express Iow levels of CD4 mRNA and undetectable amounts of surface CD4, the predominant effect of the two cytokines is to induce both CD4 mRNA and protein. [ABSTRACT FROM AUTHOR]

The structural genes of human complement regulatory proteins are clustered on chromosome 1 at position q3.2. Human chromosome 1 was transferred into a mouse fibroblast cell line, A9 [designated as A9(neo-1)], and the surface expression of its gene products participating in complement regulation, namely C3b/C4b receptor (CR1, CD35), decay-accelerating factor (DAF, CD55), membrane co- factor protein (MCP, CD46) and C3d/EB virus receptor (CR2, CD21), were assessed using respective monoclonal antibodies by flow cytometry. CR1 became positive within 7 days of culture. MCP appeared in a small population of cell by Day 3 and, together with DAF, began to increase on Day 7. CR2 appeared on Day 14. The order of the expression was CR1> DAF=MCP> CR2. On Day 42, however, all became negative except for MCP, which was markedly diminished. These human regulatory proteins were specifically associated with the presence of human chromosome 1, since none of them were expressed on human chromosome 12-transferred A9 cells [A9(neo-12)]. Intact A9 and A9(neo-12) cells activated human complement via the alternative pathway. The activation of this pathway was suppressed in the A9(neo-l) cells that expressed CR1, DAF and MCP. Slight protective activity was still observed in the 42-day cultured A9(neo-1) cells expressing only trace MCP. These results suggest that human complement regulators, expressed via the transferred human chromosome 1, can protect heterologous cells from complement, overcoming their ability to activate the human alternative pathway. [ABSTRACT FROM AUTHOR]

The MCP-5 murine mast cell line, as well as primary bone marrow-derived cultured mast cells (BMCMC), are demonstrated to bind to fibronectin, a ubiquitous adhesion protein of the extracellular matrix. BMCMC required activation by phorbol myristate acetate (PMA) to adhere to fibronectin, whereas MCP-5 displayed spontaneous adherence. The binding of both MCP-5 and BMCMC was dose dependent, with maximal adhesion at a fibronectin concentration of 20 μg/mi. The 120,000 molecular weight (MW) proteolytic fragment of fibronectin containing the RGDS cell attachment site was able to substitute for the native fibronectin molecule in promoting mast cell attachment. Mast cell adhesion to fibronectin, in addition, could be inhibited by the RGDS peptide alone. These data suggest that, in addition to the previously described mast cell-laminin interactions, mast cells also adhere to fibronectin, thus providing further insight into their tissue localization and possible roles in processes such as wound healing and fibrosis. [ABSTRACT FROM AUTHOR]