Heterogeneous profiles of a factor that renders neutrophils cytotoxic obtained from a concanavalin A-stimulated spleen cell culture in partial purification process

Concanavalin A (Con A)-stimulated rat spleen cells were cultured in a serum-free conditioned medium. This culture supernatant contained a certain factor(s) that renders neutrophil cytotoxic for various tumor cells. The factor was tentatively termed neutrophil-activating factor (NAF). Rat NAF was partially purified from the serum-free culture supernatant by using ion exchange chromatography of DEAE-Sephadex A-50, gel filtration of Sephadex G-100, and affinity chromatography of Con A-Sepharose 4B. NAF activity was eluted in broad fractions by the ion exchange chromatography and the gel filtration. Moreover, on the Con A column, some NAF activities were bound to the column, but other activities passed through the column. These results showed the heterogeneity or polydispersity of NAF activity in both molecular size and charge-based separation properties. Monoclonal antibodies were produced by fusing BALB/c myeloma cells (P3-X63 Ag8.653) with spleen cells from syngeneic mice immunized with partially purified NAF (pNAF) obtained from the gel filtration. Absorbent beads which were linked with one monoclonal antibody (ANAF-10) partially absorbed NAF activity from supernatants of a Con A-stimulated spleen cell culture. Further purification of pNAF was performed with the use of affinity chromatography of ANAF-10-linked Sepharose. Through these procedures, the NAF activity was concentrated about 10,000-fold. Heterogeneity of NAF activity, however, did not disappear in even this affinity chromatography. On the other hand, 125I-labeled material of the final product migrated to one major band corresponding with an m.w. of about 20,000 as determined by SDS-PAGE analysis, and NAF activity was detected in the same band.     

A mitogenic factor, released by stimulated human mononuclear cells and distinct from interleukin 2 (IL 2), B cell growth factor (BCGF), and interleukin 1 (IL 1)

Two lymphocyte mitogenic factors, interleukin 2 (IL 2) and blastogenic factor (BF), are generated concomitantly in human mixed lymphocyte cultures (MLC). The latter mitogenic factor is directly mitogenic for unstimulated lymphocytes, whereas the former mitogenic factor acts only on previously activated lymphocytes. Both factors had a m.w. range, as determined by gel filtration, of 18,000 to 30,000. Thus, these two factors were inseparable on the basis of m.w. size. However, BF and IL 2 were separable during ion exchange chromatography on the DEAE cellulose and phenyl-Sepharose chromatography. In addition, BF activity in the supernatants of MLC reached a maximum after day 5, whereas IL 2 activity peaked at day 3, thus distinguishing BF from IL 2 kinetically. These results clearly indicate that BF activity is mediated by molecules distinct from IL 2. The biochemical relationship between B cell growth factor (BCGF) and BF was also examined. Because BF was readily separable from BCGF by Con A-Sepharose chromatography, BF is distinguishable from BCGF. No augmentation of PHA-stimulated C3H mouse thymocyte proliferation was associated with the preparation of partially purified BF, demonstrating that BF and IL 1 are distinct molecules. Taken together, these results indicate that BF is clearly distinct from IL 2, BCGF, and IL 1. BF-containing MLC supernatants have direct mitogenic activity on both T and B cells. Both T and B cell blastogenic activities copurified during ammonium sulfate precipitation, gel filtration, DEAE cellulose ion exchange chromatography, and hydrophobic chromatography. Thus, these two activities appear to be biochemically inseparable. Monoclonal anti-Tac, that has been suggested to recognize the receptor for human IL 2, was highly inhibitory to the T cell response to the phenyl-Sepharose preparations of BF (IL 2-free). In contrast, this antibody had minimal or no effect on BF-induced B cell proliferation. However, when MLC supernatants were absorbed with a cloned IL 2-dependent T cell line, only IL 2 activity, but not BF activity, was removed, demonstrating that BF and IL 2 have different binding specificities. The precise mechanism(s) by which anti-Tac inhibits BF-induced proliferation of T cells is unknown at present. Additionally, during the course of these experiments, we observed that Con A-Sepharose chromatography could be used as a simple one-step method of separating BCGF from IL 2.     

Occurrence of glycoprotein glycosidases in mature seeds of mung bean (vigna radiata)

The presence of nine different glycosidases was demonstrated in the crude extract of mature mung bean seeds. N-Acetyl β-D-glucosaminidase, α-D-galactosidase and β-D-glucosidase were each resolved into two respective active forms by gel filtration. The other glycosidases showed single forms only. The apparent MWs of the glycosidases were determined. The glycosidases were absorbed to Con A-Sepharose column, with the exception of a small percentage of α-galactosidase and α-mannosidase which were eluted unretarded. The bound enzymes displayed varying affinities for the immobilized lectin, indicating differences in glycosylation. With the exception of β-galactosidase and invertase, all the glycosidase activities were detected in the protein bodies isolated from the seeds.     

Functional characteristics of histamine receptor-bearing mononuclear cells. II. Identification and characterization of two histamine-induced human lymphokines that inhibit lymphocyte migration

Although functional histamine receptors have generally been restricted to those human T lymphocytes expressing suppressor cell functions, more recent evidence suggests that histamine receptor-bearing human T lymphocytes are functionally heterogeneous and capable of other immunomodulatory activities. Lymphocyte chemoattractant factor (LCF) is a cationic sialoprotein with an apparent m.w. of 56,000, whose production is limited to histamine-type 2 receptor-bearing human T cells. LCF is selectively chemokinetic for T lymphocytes, and presumably contributes to the recruitment of unsensitized effector lymphocytes at inflammatory sites. In addition to LCF, Sephadex G-100 gel filtration of histamine-induced lymphocyte supernatants revealed two regions of migration inhibitory activity for human blood T and rat splenic lymphocytes. These regions corresponded to m.w. of 70,000 to 80,000 (LyMIF75K) and 30,000 to 40,000 (LyMIF35K). LyMIF75K had a single pI of 7.5 to 8.0, and its biologic activity was sensitive to trypsin but not to neuraminidase or heat (56 degrees C). LyMIF35K had a single pI of 8.5 to 8.8, and its biologic activity was sensitive to neuraminidase and heat but not to trypsin. These LyMIFs therefore appeared to be distinct from one another and physicochemically different from other migration inhibitory lymphokines. All three lymphokine activities appeared within 4 hr of incubation. The minimum concentration of histamine required to stimulate production of the LyMIF was 10(-6) M. Lymphocytes that did not adhere to a histamine affinity matrix were unable to produce either LyMIF upon subsequent stimulation with histamine or concanavalin A (Con A). Lymphocytes incubated with histamine and diphenhydramine produced LCF but neither LyMIF, whereas cells incubated with histamine in the presence of cimetidine produced both LyMIF but not LCF. These data suggest that a subset of lymphocytes defined by the presence of histamine-type 1 receptors are capable of producing two distinct species of lymphocyte migration inhibitory activity. These cells may contribute to the immobilization of effector T lymphocytes chemokinetically attracted to certain inflammatory sites.     

Separation and purification of lymphocyte chemotactic factor (LCF) and interleukin 2 produced by human peripheral blood mononuclear cells

Two T-cell chemotactic factors, lymphocyte chemotactic factor (LCF) and interleukin 2 (IL-2), were separated and characterized from culture supernatants of concanavalin A-stimulated human peripheral blood mononuclear cells. LCF was purified approximately 7800-fold to homogeneity from culture supernatant using gel filtration and high-performance liquid chromatography (HPLC). LCF was found to be distinct from both IL-2 and interleukin-1. Sephadex G-100 gel filtration of crude supernatants from concanavalin A-stimulated mononuclear cells showed two molecular weight regions of T lymphocyte chemotactic activity. A 10,000- to 25,000-Da region contained both IL-2 and LCF and a 45,000- to 75,000-Da region contained only a high molecular weight form of LCF. Both high and low molecular weight species of LCF eluted with 40-44% acetonitrile from a reversed-phase C18 HPLC column. IL-2 present only in the low molecular weight region eluted from the C18 column with 65-75% acetonitrile. The migration of T lymphocytes to IL-2 was totally inhibited by anti-interleukin 2 receptor antibody while the response of T cells to LCF was unaffected. LCF eluting off the C18 column was purified to homogeneity by two subsequent cycles of gel filtration HPLC. The resultant protein showed a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular weight of 10,500. The data presented here demonstrate that IL-2 and LCF are distinct lymphocyte chemotactic factors and although they are not readily separable from crude supernatants by molecular sieve chromatography, they can easily be distinguished by reversed-phase HPLC.     

Role of prostaglandin E2 in the induction of nonspecific T lymphocyte suppressor activity

Activated human monocytes and concanavalin A (Con A)-activated T lymphocytes are known to suppress T and B lymphocyte proliferation and B cell maturation into immunoglobulin-producing cells. We have now shown that monocyte suppressive activity is predominantly mediated through release of prostaglandin E2 (PGE2), which is active only in the presence of a "short-lived," radiosensitive T lymphocyte subset. PGE2, at high concentration, can activate T suppressor lymphocytes (TS), which display the same characteristics as Con A-activated TS lymphocytes. Moreover, Con A activation of TS lymphocytes was obtained only in the presence of PGE2, as specific anti-PGE2 antiserum or indomethacin prevented TS activation; this suggested a double signal as a prerequisite for activation of the nonspecific TS cell subset. We propose that TS lymphocytes modified by Con A become sensitive to small amounts of PGE2 produced by monocytes that must be present during the Con A-stimulated activation phase of suppressive cells.     

The identification, purification and characterisation of an inhibitor of collagenase (20K) produced by neoplastic epithelial cells

A rat carcinoma cell line (T2/H7) constitutively synthesised interstitial collagenase. When these cells were incubated with 12-O-tetradecanoylphorbol 13-acetate (TPA) they secreted an inhibitor of collagenase, which resulted in a net decrease of collagenolytic activity being detected in conditioned medium. Using reverse zymography, the Mr of the inhibitor was found to be 20,000 which suggests that it may be the rat homologue of inhibitor of metalloproteinase 2 (IMP2; TIMP-2), as it inhibited both the gelatinolytic and collagenolytic activities of rat collagenase. The inhibitor was separated from collagenase by filtration through a YM30 membrane. The inhibitor was purified further by sequential chromatography on heparin-Sepharose and Con A-Sepharose. It bound to heparin-Sepharose in 75 mM NaCl and was eluted with 300 mM NaCl. It did not bind to Con A-Sepharose, suggesting that it was a non-glycosylated molecule. The inhibitor was resistant to treatment with either trypsin, APMA or heat.     

Murine IgA binding factors (IgA-BF) suppressing IgA production: characterization and target specificity of IgA-BF

Chemical and functional properties of IgA binding factor(s) (IgA-BF) from both murine Con A-activated spleen cells and Fc gamma R+, Fc alpha R+ T hybridoma cells (T2D4) were studied. IgA-BF produced from the cells after preculture with IgA were purified with IgA-Sepharose. Purified IgA-BF inhibited the binding of IgA to Fc alpha R+ L5178Y T lymphoma cells, and class-specifically suppressed in vitro IgA synthesis of the pokeweed mitogen (PWM)-stimulated murine spleen cells. Both IgA-specific suppressive activity and IgA binding activity of the factor(s) were co-fractionated between BSA and OVA in gel filtration analysis. SDS-PAGE analysis of IgA-BF biosynthetically labeled with [35S]methionine showed a specific band on 56,000. Suppressive activity of IgA-BF was absorbed with lentil-lectin-Sepharose and was eluted with 0.2 M alpha-methyl-D-mannoside. The suppressive activity obtained from T2D4 cells (H-2k) and BALB/c Con A blasts (H-2d) was absorbed with the corresponding anti-H-2 and anti-I-A column and recovered in the acid-eluate. The activity was not absorbed with the unrelated anti-H-2 column. Despite the presence of MHC products, IgA-BF from both cell sources equally suppressed IgA-specific responses of BALB/c (H-2d), C3H/He (H-2k), and C57BL/10 (H-2b) spleen cells. They also suppressed IgA production as well as IgA synthesis of PWM-stimulated culture of human peripheral blood lymphocytes without affecting IgM and IgG responses. Suppression of murine and human IgA responses both in mouse and human were mediated by the molecules having the same Ia products, suggesting that there is no MHC, as well as species restriction, for the interaction between IgA-BF and their target cells. IgA-specific suppressive activity was absorbed with human B blastoid cells bearing surface IgA (Dakiki) but not with those bearing surface IgG (CESS) or murine and human T cell line cells (BW5147, L5178Y, HPB-ALL, and MOLT4), indicating that IgA-BF interact with B cells bearing IgA to suppress their differentiation.     

Biological and biochemical characterization of bovine interleukin 2. Studies with cloned bovine T cells

Bovine peripheral blood lymphocytes stimulated with the T cell mitogen concanavalin A (Con A) secrete a lymphokine with biological properties similar to T cell growth factor (TCGF) or interleukin 2 (IL 2) from other species. The material supports proliferation of Con A-derived T cell blasts, limiting dilution cloning of T cell blasts, and continuous growth of T cell clones for over 6 mo in vitro. A quantitative microassay with the use of TCGF-dependent, Con A-unresponsive cloned T cells was used to determine the biological activity during purification of IL 2. A single peak of activity with an apparent m.w. of 25,000 to 28,000 was recovered after gel filtration. This material eluted from DEAE-Sephacryl between 135 and 165 mM NaCl. After isoelectric focusing, high pressure liquid chromatography, and gel electrophoresis under reducing conditions, peak IL 2 activity was associated with proteins having m.w. of 20,000 and 23,000.     

Separation of human lymphocyte subpopulations by density gradient electrophoresis. I. Different mobilities of T (T mu, T alpha) and B lymphocytes from human tonsils

T and B lymphocytes from human tonsils were separated by density gradient electrophoresis on the basis of their surface charge. The high-mobility cell fractions were found to be highly enriched in T lymphocytes with only very small proportions of B cells. In contrast, the low-mobility fractions were predominantly B lymphocytes, and had only 10 to 30% contamination of T cells. The intermediate-mobility fractions contained both T and B lymphocytes in approximately equal proportions. IgM-bearing lymphocytes, as well as cells with receptors for mouse erythrocytes, the Fc portion of IgG, and complement were found in the intermediate- and low-mobility fractions. T lymphocytes, prepared by E rosetting, were also electrophoresed by this method and found to be of higher mobility as compared with peripheral blood T lymphocytes. T cells with Fc receptors for IgM (Tμ) or IgA (Tα) were found to be considerably heterodisperse with regard to surface charge and were present in all fractions. The separated cell fractions were treated in vitro with various concentrations of concanavalin A and thereafter examined for Tμ, Tγ, and Tα phenotypes. Low concentrations of Con A (2.5 μg/ml) had no effect on cell surface phenotypes. However, higher concentrations of Con A (20μg/ml) significantly reduced the numbers of T cells having IgM receptors (Tμ), but failed to alter the expression of the Tγ phenotype. The latter finding contrasts to that observed with T cells from the peripheral blood where high concentrations of Con A increase the proportions of the Tγ cells. This study demonstrates that density gradient electrophoresis can be used for the separation and study of lymphocyte subpopulations from human tonsils.     

Production of lymphocyte-activating factor (Interleukin 1) by macrophages activated with colony-stimulating factors

Murine peritoneal exudate macrophages incubated with medium conditioned by L929 cells were stimulated to produce lymphocyte-activating factor (LAF, Interleukin 1). This stimulatory activity was partially neutralized by antiserum prepared against partially purified L cell colony-stimulating factor (CSF) and comigrated upon gel filtration with the myeloproliferative activity. LAF-inducing activity of three different L cell CSF preparations, including one purified to homogeneity, was dependent upon the concentration of CSF. A minimum of 1,000 to 3,000 units of CSF activity was required to stimulate macrophagfes to produce LAF. Concanavalin A (Con A) stimulated splenic supernatants also contained CSF and LAF-inducing activities that co-eluted upon gel filtration. LAF-inducing activities co-eluting with the two Con A CSF peaks (apparent m.w. of 25,000 and 35,000) were effective at minimum dilutions containing 1,000 to 3,000 units of CSF activity correlating in potency with L cell-derived CSF. Based on these data, it is proposed that CSF, whether of L cell or lymphoid origin, not only has myeloproliferative activities but also is capable of stimulating macrophages to produce LAF.     

Human T-lymphocyte chemotactic activity: nature and production in response to antigen

Previous studies have shown that supernatants from concanavalin A-stimulated human peripheral blood mononuclear cells and isolated Leu-2 suppressor/cytotoxic T cells are chemotactic for Leu-3 helper/inducer T cells. The current study shows that lymphocyte chemotactic factor (LCF) is also produced following antigen (tetanus toxoid) challenge of mononuclear cells obtained from recently immunized human donors. LCF was detected in 24-hr supernatants from mononuclear cells challenged with tetanus and was produced maximally at 48 hr. Tetanus toxoid challenge of mononuclear cells obtained from individuals whom had not received a tetanus immunization for 7 to 10 years prior to testing showed little or no production of LCF. Serial studies of these individuals following a tetanus booster immunization showed that LCF was produced by antigen-challenged mononuclear cells obtained 1-5 days postimmunization, was produced optimally 5-15 days postimmunization, and was still produced by antigen-challenged mononuclear cells obtained 6 weeks later. Fractionation of mononuclear cells from immunized donors into glass wool nonadherent lymphocytes, T lymphocytes, and non-T lymphocytes showed that tetanus-toxoid-induced LCF was produced by nonadherent lymphocytes and T cells but not non-T cells. Further fractionation of T lymphocytes into Leu-2 and Leu-3 T-cell subpopulations showed that LCF production by antigen-challenged isolated subpopulations was limited to the Leu-2 suppressor/cytotoxic T-cell subset. Characterization of both Con A and tetanus toxoid-induced LCF by gel filtration on Sephadex G-100 demonstrated the presence of two peaks of LCF corresponding to molecular weights of approximately 14,000-17,000 and 40,000-50,000.     

Cytotoxic activity of lymphocytes. VII. cellular origin of alpha-lymphotoxin.

To detect the cellular origins of alpha-lymphotoxin (alpha-LT), we cultured various subpopulations of human blood lymphocytes separated by erythrocyte-rosetting techniques with various mitogens. T cell-enriched subpopulations responded to PHA by increased 3H-thymidine uptake into DNA and large amounts of alpha-LT production. SPL and Con A-Sepharose stimulated DNA synthesis in T cell-enriched cultures if the macrophage content was greater than 1.5%; however, alpha-LT production was not induced by these two mitogens even when reconstituted with 10% macrophages. B and/or null cell-enriched populations severely depleted of T cells (less than 0.7% did not respond to PHA, SPL, or Con A-Sepharose. However, reconstitution to 5 or more percent in E-RFC allowed all three mitogens to stimulate DNA synthesis and alpha-LT production. The LT made by all cell populations 5 and 7 days after stimulation were equally neutralized by a heterologous antiserum to alpha-LT. These results show that human T and B and/or null cells, when appropriately stimulated, can produce alpha-LT.     

Early increase in lymphocyte cyclic nucleotide phosphodiesterase activity upon mitogenic activation of human peripheral blood mononuclear cells.

Cyclic nucleotide phosphodiesterase activity of human peripheral blood mononuclear cells was significantly increased following a short (30 min) incubation with the mitogenic lectin Concanavalin A. Con A stimulated phosphodiesterase activity to the same extent whatever the subcellular compartment (homogenate, cytosol or pellet). Further separation of the Con A-activated mononuclear cells into lymphocyte-enriched and monocyte-enriched populations showed that the Con A-induced increase of phosphodiesterase activity exclusively affected the lymphocyte-enriched population. In lymphocytes, cyclic GMP phosphodiesterase activity was more importantly enhanced by Con A (+275%) than cyclic AMP phosphodiesterase activity (+75%). The increase of both activities occurred as early as from 10 min of Con A incubation and proved to be maximal with Con A doses of 2.5 and 5 micrograms per 10(6) cells, lower and higher doses being less effective. Inhibition experiments with reference inhibitors suggested that, among the high affinity phosphodiesterase isoforms, the cyclic GMP-inhibited enzyme might be more selectively enhanced by Con A than the cyclic AMP-specific, Rolipram-sensitive one. The non-mitogenic lectin Helix pomatia hemagglutinin, was not able to enhance cyclic nucleotide phosphodiesterase activity of human mononuclear cells whereas anti-CD3 monoclonal antibody, although being less effective than Con A, exhibited a significant stimulatory effect. Putting together these results suggest that the early increase in phosphodiesterase activity might be a normal step involved in the mitogenic activation of human lymphocyte.     

Certain characteristics of human lymphocyte migration inhibitory factors (LyMIF) and the effect of ampicillin on their appearance in unstimulated and PHA-stimulated cultures

Supernatants of human blood mononuclear cells stimulated with PHA, contained factors inhibitory for in vitro migration of human lymphocytes and granulocytes. After ultrafiltration of supernatants through Amicon PM-10 some stimulatory activity appeared in the bottom fraction. Sephadex G-100 gel filtration chromatography of supernatants showed three zones of lymphocyte migration inhibitory activity: In the range of molecules of m.w. 35 000-90 000, heat-stable; Factors of m.w. from several to from ten to twenty thousand daltons, heat unstable; Low molecular weight substances, resistant to heat. The possible relationship of these factors to lymphotoxins, soluble lymphocyte T receptors for SRBC, lymphocyte chemotactic factor and prostaglandins is discussed. Ampicillin in doses of 10, 20 and 50 micrograms/ml potentiated both the development of lymphocyte migration inhibitory factors and the production of factors with an opposing effect (stimulating lymphocyte migration).     

Beta-1,4-mannosyl-glycoprotein beta-1,4-N-acetylglucosaminyltransferase III activity in human B and T lymphocyte lines and in tonsillar B and T lymphocytes

beta-1,4-mannosyl-glycoprotein beta-1,4-N-acetylglucosaminyltransferase III (GlcNAc-T III) catalyzes the incorporation of a "bisecting" N-acetylglucosamine (GlcNAc) residue in beta 1-4 linkage to the beta-linked mannose of the core of asparagine linked-protein bound oligosaccharides (N-glycans). The activity of GlcNAc-T III was determined in Triton X-100 extracts of four human Epstein-Barr virus (EBV)-infected B-cell lines, in four T-cell lines originally established from lymphocytes of patients with acute lymphatic leukemia, and in human tonsillar B and T lymphocytes. The four EBV-transformed B-cell lines showed appreciable GlcNAc-T III activities (ranging from 3.4 to 19.0 nmol.h-1.mg protein-1), while the tonsillar resting B lymphocytes had much less activity (0.68 nmol.h-1.mg protein-1). The four T-cell lines and the tonsillar T lymphocytes had negligible GlcNAc-T III activities (ranging from 0.02 to 0.25 nmol.h-1.mg protein-1). Enzyme product was identified by high resolution proton nuclear magnetic resonance spectroscopy and methylation analysis. This is the first demonstration of GlcNAc-T III activity in human lymphocytes. The presence of GlcNAc-T III in B-cell lines correlates with the reported occurrence of bisecting GlcNAc residues in the oligosaccharides of human immunoglobulins G, A1, M, and D, tonsillar class II antigens, and membrane glycoproteins from B lymphocytes. The negligible GlcNAc-T III activity of the four human T-cell lines and of tonsillar T lymphocytes agrees with the reported absence of bisected structures in N-glycans from human T lymphocyte membrane glycoproteins.     

Production of a human T lymphocyte chemotactic factor by T cell subpopulations

Concanavalin A-stimulated human peripheral blood mononuclear cells release a lymphocyte chemotactic factor. This lymphocyte chemotactic factor is produced optimally after 24 to 48 hr of culture and is not found before 3 hr of culture, which suggests that the factor is synthesized de novo and is not preformed and secreted after Con A stimulation. This is further supported by experiments showing that the protein synthesis inhibitors cycloheximide and puromycin totally prevent the production of the chemotactic factor. Experiments using cultured and uncultured T lymphocytes as responding cells show that cultured T cells respond more efficiently than uncultured T cells to this factor. Furthermore, the lymphocyte chemotactic factor preferentially stimulates T lymphocyte locomotion as compared to peripheral blood non-T lymphocyte migration. Fractionation of mononuclear cells into glass nonadherent lymphocytes, monocyte-enriched preparations, T lymphocytes, and non-T lymphocytes shows that lymphocyte chemotactic factor is produced by Con A-stimulated, glass nonadherent lymphocytes and T cells but not by monocytes or non-T lymphocytes. Further fractionation of T lymphocytes into Leu-2 and Leu-3 T cell subpopulations shows that the production of T lymphocyte chemotactic factor can be attributed to the Leu-2 suppressor/cytotoxic T cell subset. The generation of a T lymphocyte chemotactic factor by Leu-2 T cells may represent a means of recruiting other T cells to the site of its release.     

Production of monoclonal antibodies against a cell surface concanavalin a binding glycoprotein

Concanavalin A-binding (Con A)-binding cell surface glycoproteins were isolated, via Con A-affinity chromatography, from Triton X-100-solubilized Chinese hamster ovary (CHO) cell plasma membranes. The Con A binding glycoproteins isolated in this manner displayed a significantly different profile on sodium dodecyl sulfate-polyacrylamide gels than did the Tritonsoluble surface components, which were not retarded by the Con A-Sepharose column. [¹²⁵I]-Con A overlays of the pooled column fractions displayed on sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE) demonstrated that there were virtually no Con A receptors associated with the unretarded peak released by the Con A-Sepharose column, whereas the material which was bound and specifically eluted from the Con A-Sepharose column with the sugar hapten α-methyl-D-mannopyranoside contained at least 15 prominent bands which bound [¹²⁵I]-Con A. In order to produce monoclonal antibodies against various cell surface Con A receptors, Balb/c mice were immunized with the pooled Con A receptor fraction. Following immunization spleens were excised from the animals and single spleen cell suspensions were fused with mouse myeloma P3/X63-Ag8 cells. Numerous hybridoma clones were subsequently picked on the basis of their ability to secrete antibody which could bind to both live and glutaraldehyde-fixed CHO cells as well as to the Triton-soluble fraction isolated from the CHO plasma membrane fraction. Antibody from two of these clones was able to precipitate a single [¹²⁵I]-labeled CHO surface component of ～265,000 daltons.     

Purine metabolic enzymes in lymphocytes. I. Adenosine deaminase and purine nucleoside phosphorylase activities in mouse lymphocyte subpopulations

The distribution of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activities in lymphoid organs and lymphocyte subpopulations in mice, and the effect of phytohemagglutinin P (PHA-P) and concanavalin A (Con A) on the enzyme activities were studied. ADA activity was distributed equally in cells from all organs used and no mouse strain differences were observed. In contrast, PNP activity varied with the mouse strain, being highest in C57BL/6 mice and lowest in BALB/c mice, and with the organ in ICR mice, being high in peripheral blood lymphocytes and spleen lymphocytes, low in mesenteric lymph node cells and absent or very weak in thymus cells. T and B lymphocytes were prepared from spleen of ICR mice. High ADA activity was found in both T and B lymphocytes, whereas PNP activity in the T lymphocytes was about one-third of that in the B lymphocytes. PNP activity in thymus cells was increased to the normal level of T lymphocytes in the spleens by cultivation without stimulant. The development of PNP activity in thymus cells was partially inhibited by Con A but was not affected by PHA-P. ADA activity in thymus cells was enhanced by in vitro stimulation with PHA-P but not with Con A. In contrast, in spleen lymphocytes the development of ADA activity was enhanced by stimulation with PHA-P and Con A, and that of PNP activity was enhanced by PHA-P but not by Con A.     

Cell-mediated immune responses (CMIR) in human rhinosporidiosis

Cell mediated immune responses (CMIR) to Rhinosporidium seeberi in human patients with rhinosporidiosis have been studied. With immuno-histochemistry, the cell infiltration patterns in rhinosporidial tissues from 7 patients were similar. The mixed cell infiltrate consisted of many plasma cells, fewer CD68+ macrophages,a population of CD3+ T lymphocytes, and CD56/57+ NK lymphocytes which were positive for CD3 as well. CD4+ T helper cells were scarce. CD8+suppressor/cytotoxic-cytolytic cells were numerous. Most of the CD8+ cells were TIA-l+ and therefore of the cytotoxic subtype. CD8+ T cells were not sub-typed according to their cytokine profile; 1L2, IFN-γ (Tcl); IL4, ILS (Tc2).In lympho-proliferative response (LPR) assays in vitro, lymphocytes from rhinosporidial patients showed stimulatory responses to Con A but lymphocytes from some patients showed significantly diminished responses to rhinosporidial extracts as compared with unstimulated cells or cells stimulated by Con A, indicating suppressor immune responses in rhinosporidiosis. The overall stimulatory responses with Con A suggested that the rhinosporidial lymphocytes were not non-specifically anergic although comparisons of depressed LPR of rhinosporidial lymphocytes from individual patients, to rhinosporidial antigen with those to Con A, did not reveal a clear indication as to whether the depression was antigen specific or non-specific. The intensity of depression of the LPR in rhinosporidial patients bore no relation to the site, duration, or the number of lesions or whether the disease was localized or disseminated. Rhinosporidial extracts showed stimulatory activity on normal control lymphocytes, perhaps indicating mitogenic activity. These results indicate that CMIR develops in human rhinosporidiosis, while suppressed responses are also induced. This revised version was published online in June 2006 with corrections to the Cover Date.