Lymphokine-mediated activation of human monocytes: neutralization by monoclonal antibody to interferon-gamma

Purified natural and recombinant human immune interferon (IFN-gamma) were found to activate human monocytes from peripheral blood to exert enhanced cytotoxicity against human colon adenocarcinoma HT-29 cells. A marked monocyte activation was observed at low concentrations (1 and 10 U/ml) of IFN-gamma. Marked monocyte activation was also obtained with two lymphokine preparations, produced in peripheral blood mononuclear cell (PBM) cultures induced with phytohemagglutinin (PHA) or by combined stimulation with PHA and 12-O-tetradecanoylphorbol 13-acetate (TPA). The component responsible for macrophage activation in such lymphokine preparations in the past was considered to be "macrophage-activating factor" (MAF). When monoclonal antibody specifically neutralizing IFN-gamma was added to these lymphokine preparations, all MAF activity disappeared, indicating that IFN-gamma is the sole protein showing MAF activity in these preparations.     

Modulation of the activity of sea bass (Dicentrarchus labrax) head-kidney macrophages by macrophage activating factor(s) and lipopolysaccharide

The aim of this study was to establish the requirements for macrophage activating factor (MAF) production by sea bass head-kidney leucocytes and the kinetics of macrophage activation when exposed to MAF-containing supernatants and/or lipopolysaccharide (LPS), a known macrophage stimulant. MAF activity was found in culture supernatants of total head-kidney leucocytes pulsed with 5 microg ml(-1)Con A, 5 or 10 ng ml(-1)PMA and 100 ng ml(-1)calcium ionophore, or 10 microg ml(-1)Con A alone, as assessed by the capacity to prime macrophages for enhanced production of reactive oxygen intermediates (ROI). Mixed leucocyte cultures from two or eight fish showed higher MAF activity after stimulation, indicating that a mixed leucocyte reaction was also important for MAF production. MAF-induced activation of macrophage cultures was highest at 18 h of exposure and was lost by 72 h except for MAF induced by Con A-stimulation alone. LPS primed macrophages for increased ROI production at early incubation times and down-regulated ROI production after 24 h. LPS had no effect in further stimulating the MAF-induced priming effect on production of ROI and down-regulated the MAF-priming by 48 h. Sea bass head-kidney macrophages did not show increased nitrite production when exposed to MAF and/or LPS, which may be related to their differentiation status.     

Biological and Biochemical Characterization of Macrophage Activating Factor (MAF) in Murine Lymphocytes: Physicochemical Similarity of MAF to Gamma Interferon (IFN-γ)

During the course of an investigation designed to separate macrophage activating factor (MAF) activity from interferon (IFN) antiviral activity in the lymphokine-rich fraction (LKF) produced by stimulation of murine splenic cells with concanavalin A (Con A), we found molecular evidence for the similarity of the two activities. MAF activity was expressed as the rate of inhibition of intracellular growth of Salmonella typhimurium in macrophages based on the linear correlation between relative MAF activity and LKF concentration. The antiviral substance in LKF was identified as IFN-γ based on the observation that its activity was inactivated at pH 2 and neutralized with anti-mouse IFN-γ serum but not with anti-mouse IFN-α/β serum. MAF and IFN antiviral activities displayed identical sensitivity to pH 2 and temperature. Further, neither activity was affected by β-mercapto-ethanol, but both were inactivated by guanidine hydrochloride and by sodium dodecyl sulfate, suggesting that the structures related to conformation of the protein of the two molecules may be similar. In affinity chromatography of the LKF on a Con A-Sepharose column, MAF and IFN activities were found in both the nonadsorbing (F I) and adsorbing (F II) fractions. However, the rates of F II of MAF and IFN activities increased proportionally when the sample was applied on a column of higher capacity, suggesting that the molecular structure of the mannose-containing glycosyl moiety of the two molecules may also be similar. Moreover, the intact or modified form of MAF and IFN activities of different LKF preparations showed a strong correlation, indicating that the production and denaturation of MAF activity were proportional to those of IFN antiviral activity. The results of this study provide strong evidence that MAF and IFN antiviral activities may reside in virtually the same molecular species.     

Stimulatory effect of Bestatin,a new specific inhibitor of aminopeptidases,on the blastogenesis of guinea pig lymphocytes

A potent protease-inhibitor of Actinomycetes origin, Bestatin. which is of dipeptide nature and inhibits aminopeptidase B and leucine-aminopeptidase competitively, strongly stimulates blastogenesis of small lymphocytes triggered with polyclonal mitogen. such as phytohemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM) and lipopolysaccharide of Escherichiae coli (LPS), whereas it inhibits DNA synthesis of normal resting lymphocytes. The stimulatory effect is non-selective with respect to the category of small lymphocytes, i.e. T- and B-lymphocytes, but strikingly selective with respect to the stage of blastogenesis: the stimulation is greatest at a relatively early stage, diminishes as mitogen-activation proceeds, and is not appreciable at a later stage of lymphocyte blastogenesis.The pattern of Bestatin stimulation on lymphocyte blastogenesis is specific for the mitogen used: in T-lymphocyte activation with PHA or Con A, the stimulation first increases and then decreases with increase in mitogen concentrations, whereas in B-lymphocyte activation with LPS, with increasing concentrations of the mitogen, the stimulation increases to a plateau at approximately 100 μg/ml of mitogen. The optimum concentration of Bestatin was found to be approximately 50 μg/ml (0.16 mM) for either PHA or Con A activation, and 50 to 75 μg/ml for B-cell activation with LPS. Bestatin must remain in cultures of T- and B-lymphocytes with polyclonal mitogens for at least about 24 and 16 hr, respectively, to exert its stimulatory effect on blastogenesis.Biochemical results, together with those from autoradiographic analyses, indicate that Bestatin increases the number of blastoid-transformed lymphocytes with polyclonal stimulants. It is suggested that aminopeptidases, possibly located at the cell surface, may play a role in the control of lymphocyte activation during immune responses.     

Biological and Biochemical Characterization of Macrophage Activating Factor (MAF) in Murine Lymphocytes: Role of Mannopyranosyl Residue of the MAF Molecule in Macrophage Activation

Activation of macrophages with macrophage activating factor (MAF) was evaluated by measuring the intracellular killing activity of murine macrophages against Salmonella typhimurium. Concanavalin A (Con A)-induced MAF-rich fraction was obtained by a Sephadex G-100 column, which contained molecules ranging from 25,000 to 67,000 daltons. The intracellular killing ability of mouse peritoneal macrophages against S. typhimurium was found to be increased by 0.1 m d-mannose as well as by Con A-induced MAF-rich fraction. Both 0.1 m d-mannose and MAF exhibited a similar timing pattern for macrophage activation. The same concentration of d-glucose or l-rhamnose did not change bacterial uptake and intracellular killing by macrophages. Moreover, when MAF-rich fraction was applied to a Con A-Sepharose column, a fraction that was adsorbed on Con A and eluted with 0.1 m α-methyl d-mannoside exhibited MAF activity. These results suggest the possibility that mannopyranosyl residues in the MAF molecules play an important role as a ligand in macrophage activation.     

Facilitation of adenylate cyclase stimulation in macrophages by lectins.

Preincubation of guinea pig peritoneal macrophages with concanavalin A (Con A) markedly enhanced the accumulation of 3′,5′-cyclic-adenosine monophosphate (cAMP) in response to the adenylate cyclase (AC) stimulators prostaglandin E₁ (PGE₁) and isoproterenol (IP). Basal cAMP levels were not altered. Maximal enhancement of cAMP accumulation was induced by preincubation with 50–100 μg/ml Con A for 10 min at 37 °C. Con A-induced facilitation of macrophage responsiveness was prevented by α-methyl-d-mannoside (αMM). No facilitation was induced by the divalent derivative, succinyl-Con A or by Con A immobilized on Sepharose beads. Con A-induced facilitation developed normally in macrophages treated with the microfilament blocking agent, cytochalasin B. The responsiveness of macrophages to PGE₁ and IP was also augmented by phytohemagglutinin (PHA) but wheat germ agglutinin (WGA), soy bean agglutinin (SBA), pokeweed mitogen (PWM), and Lotus tetragonolobus lectin (LL) showed no enhancing effect. The effect of Con A on cAMP levels was the result of augmented cAMP synthesis and not of reduced degradation or a block in cAMP egress from the cells. Lectin-induced facilitation of AC stimulation could be mediated via one of the following mechanisms: (i) induction of receptor clustering; (ii) causing a conformational change in the receptors; (iii) inhibition of negative cooperativity; (iv) causing an increase in membrane fluidity; (v) disruption of microtubules by acting as a Ca²⁺ ionophore; or (vi) inactivation of a sugar-containing inhibitor of AC.     

Characterization of macrophage activation factor, a lymphokine that causes macrophages to become cytotoxic for tumor cells.

Macrophage activation factor (MAF) was isolated from PPD-stimulated, BCG-immune mouse spleen cell culture fluids. In nine gel filtration runs on Sephadex G-100 or G-200, MAF was eluted in a single peak corresponding to a MW of 55,000 ± 1600. Recovery of activity was about 65%. Since the relative concentration curve of eluted MAF was wider than that of a single protein, MAF activity may be due to more than one protein with similar molecular weights. This possibility is strengthened by a broad elution range on DEAE cellulose chromatography, from a specific conductance of 3.5 to 8.5 mmho/cm, at pH 7.9. MAF was labile at both pH 4 and 10, and was destroyed by proteolytic enzymes. Eighty percent was destroyed by heating at 56 °C for 30 min. In affinity chromatography experiments, MAF did not bind to Con-A Sepharose; but it was bound to insolubilized Cibacron-blue and was eluted by an increase in ionic strength.     

Suppression of lymphokine production: II. Macrophage-dependent inhibition of production of macrophage activating factor

The ability of different populations of macrophages to affect the production of macrophage activating factor (MAF) by stimulated T lymphocytes was investigated. We found that activated macrophages, infiltrating MSV-induced regressing tumors or macrophages recovered from the peritoneum of mice injected with Corynebacterium parvum, were able to actively suppress the production of MAF. MAF production by antigen-stimulated MSV-immune or -alloimmune spleen cells and by normal spleen cells stimulated by Con A was susceptible to macrophage-dependent suppression to a similar extent. In contrast, resident macrophages or those elicited by light mineral oil or proteose-peptone did not affect MAF production. While suppressor macrophages added at the time of the lymphocyte stimulation inhibited MAF production, the same cells added 4–6 hr after stimulation were ineffective. Therefore, it seems that the macrophages suppressed the early events of lymphocyte activation leading to MAF production. Suppressor macrophages, by inhibiting MAF production, may limit the expansion of the cytotoxic activity. This regulation of macrophage functions, mediated by the effects of suppressor macrophages on T lymphocytes, could be responsible for an insufficient antitumor cytotoxic response by macrophages.     

Thymocyte-stimulating factor from peripheral blood cells.

Human peripheral blood leukocytes (HPBL) produce a thymocyte-stimulating factor (TSF-HPBL) that enhances the phytohemagglutinin (PHA) and concanavalin A (Con A) responsiveness of murine thymocytes. This activity is considerably specific for thymocytes. TSF-HPBL is not mitogenic by itself. Experiments with cell cultures pretreated with carbonyl iron particles showed that phagocytic cells are not involved in the production of mouse and rat TSF but are involved in the production of TSF-HPBL. The dose-response profile to PHA of murine thymocytes cultured in the presence of TSF-containing supernatants is similar to that of mature, immunocompetent spleen cells. TSF-HPBL, however, does not enhance the PHA responsiveness of murine thymocytes at low (<0.25 μg/microwell) concentrations of mitogen. TSF enhances the PHA and Con A responsiveness of the high-density subpopulations of thymocytes isolated on a Ficoll-Hypaque gradient. In general, the enhancing effect of TSF-HPBL on these subpopulations of thymocytes is smaller than that exerted by TSF. While supernatants containing TSF confer to thymocytes the ability to participate in a mixed lymphocyte reaction, this effect is not exerted by supernatants containing TSF-HPBL. A factor enhancing the PHA and Con A responsiveness of murine thymocytes is also produced by murine peripheral blood leukocytes (TSF-MPBL). This factor, similarly to TSF-HPBL, is produced by phagocytic cells and does not confer to murine thymocytes the ability to participate in a mixed lymphocyte reaction. Human T-cell lines do not enhance the PHA or Con A responsiveness of murine thymocytes. TSF-HPBL has a molecular weight of about 30,000 daltons, as measured by Sephadex filtration. Its half-time of inactivation as 56 °C is 162 ± 8 min.     

d-Mannose as a component of the macrophage surface receptor for macrophage-activating factor (MAF) in mice

Mouse peritoneal exudate macrophages were rendered cytostatic and cytolytic for various mouse tumor cells in vitro by exposure to partially purified lymphokines containing macrophage-activating factor (MAF) at 37 °C for 2 hr. The macrophage activation disappeared completely when either 0.1 Md-mannose or 0.1 M methyl-d-mannoside was present with MAF. On the other hand, neither d-galactose nor d-glucose inhibited the activation, and l-fucose, l-rhamnose, and N-acetyl-d-glucosamine inhibited it only partially. Incubation of either macrophages or MAF with 0.1 Md-mannose for 2 hr had no effect on activation of the macrophages by the MAF. Treatment of the macrophages by α-d-mannosidase rendered them no longer responsive to MAF. Macrophages treated by either neuraminidase or proteolytic enzymes, but not with β-d-galactosidase lost their ability to respond to MAF. Treatment of MAF with α-d-mannosidase did not affect MAF activity. In addition, MAF activity was not reduced by passage through a column of immobilized concanavalin A. In an absorption experiment, the presence of d-mannose was shown to prevent the adsorption of MAF to macrophages, while d-galactose did not. Treatment of macrophages with plant lectins having affinity for d-mannose, sialic acid or l-fucose prevented the adsorption of MAF, but the other lectins did not. Mouse MAF failed to adsorb to guinea pig peritoneal exudate macrophage, which were suggested as having a fucose-containing glycolipid as a lymphokine receptor. Taken together, these results strongly suggest that the receptor for MAF on mouse macrophages may be a glycoprotein containing d-mannose and sialic acid as essential components.     

Biochemical and functional characterisation of macrophage stimulating factors secreted by mitogen-induced goldfish kidney leucocytes

Mitogen-stimulated goldfish kidney leucocytes secrete a number of different macrophage activation factors (MAF) that induce profound physiological changes in macrophages. MAF produced by goldfish kidney leucocytes was characterised using fast performance liquid chromatography (FPLC) and bioassays that measured MAF-induced respiratory burst (RB) and nitric oxide (NO) responses of activated macrophages. Mitogen-induced fish kidney leucocyte supernatants were fractionated using gel permeation FPLC (GP-FPLC) and the ability of different fractions to induce NO or RB measured. A MAF of M(r) 50 kD, that induced a potent nitric oxide response in both a long-term goldfish macrophage cell line (GMCL) and in in vitro-derived fish kidney macrophages (IVDKM) was identified. The GP-FPLC partially purified 50 kD MAF activity occasionally induced significantly higher nitric oxide production than that of the crude MAF preparations. This increase in the NO-inducing activity was due to segregation of the 50 kD MAF from a novel macrophage deactivating molecule of M(r) 10-12 kD present in crude MAF preparations. This 10-12 kD molecule was shown to inhibit nitric oxide production in cytokine-activated goldfish macrophages. Mitogen-induced fish kidney leucocyte supernatants contained two distinct MAFs that induced the respiratory burst in GMCL and IVDKM: the 50 kD and 30 kD proteins. The partially purified 30 kD MAF primed goldfish macrophage for increased RB activity after only 6 h of treatment, and continued to augment the RB activity after 24 h of stimulation. In contrast, the GP-FPLC partially purified 50 kD molecule also primed the RB after only 6 h of stimulation, but subsequently deprimed the RB after 24 h of stimulation, an effect similar to that observed for crude MAF preparations. The 50 kD MAF activity was further purified using chromatofocusing FPLC (C-FPLC) using basic pH gradients and was shown to consist of two distinct NO-inducing molecules (> pI 9.3). Mitogen-stimulated fish kidney leucocytes secrete several factors that profoundly affect the anti-microbial responses of teleost macrophages and which undoubtedly are responsible for regulating teleost macrophage function in vivo.     

Lymphokines inhibit macrophage RNA synthesis

The effects of lymphokine (LK) preparations on the incorporation of [3H]uridine into macrophage RNA were investigated. Supernatants from murine spleen cells activated in vitro by alloantigens or Con A, and shown to contain macrophage-activating factor (MAF), were used as the source of LK. It was observed that such LK preparations contain factor(s) causing a profound inhibition of [3H]uridine incorporation into the RNA of proteose-peptone-elicited peritoneal macrophages. Such RNA-labeling inhibitory factor (RIF) was absent in control supernatants from nonstimulated cultures, and showed activation curves similar to that of MAF. RIF activity was not due to altered permeability of macrophages to [3H]uridine nor to the changes in the specific activity of the pool of RNA precursors, but rather reflected an altered metabolism of RNA. The inhibition of RNA synthesis was dependent upon the presence of nanogram amounts of LPS as a costimulator. Moreover, the response to RIF appeared to be genetically controlled since macrophages from C3H/HeJ mice were not affected by RIF, while C3H/HeN mice were fully responsive. In parallel cultures of macrophages, LK were also tested for their MAF activity, and a strong similarity between the biological conditions in which MAF and RIF activities were expressed could be demonstrated. The assay for RIF provides a new and convenient parameter for measuring macrophage-sensitive LK activity that might be very useful for monitoring purification or for screening of T-cell hybridoma supernatants.     

Passive transfer of experimental allergic encephalomyelitis in the Lewis rat with activated spleen cells: differential activation with mitogens

It has previously been shown that spleen cell transfer of clinical EAE requires donor cells to be cultured in vitro prior to transfer. Donor cells must be stimulated when cultured, and either Con A or the encephalitogen, guinea pig myelin basic protein (BP), satisfies this stimulation requirement. Following recovery from passive disease, recipients of these in vitro cultured cells will subsequently develop clinical symptoms of EAE sooner than controls when challenged with BP in complete Freund's adjuvant (BP-CFA). In the present study, three T-cell mitogens were evaluated as donor cell stimulants in the required in vitro culture period. Pokeweed mitogen (PWM) as well as Con A stimulated the donor cell population to the degree that clinical EAE could be transferred with 5 × 10⁶ cultured viable cells. Con A at culture levels below 0.25 μg/ml did not yield transfer active cells even though proliferation levels were similar to those found at concentrations of Con A that did yield transfer active cells. Phytohemagglutinin (PHA)-stimulated cultures did not transfer clinical disease even though the degree of lectin induced proliferation ([³H]thymidine uptake as well as recovered cells from culture) was equivalent to the PWM- or Con A-stimulated, transfer positive, cultures. Mixing experiments suggested that the inability of PHA or low doses of Con A to induce transfer active cells was not due to the induction of suppressor cells. Although cells cultured with PHA do not transfer clinical EAE, recipients of these cells as well as recipients of either PWM- or Con A-stimulated donor cells develop an early appearance of disease upon subsequent challenge with BP-CFA. This included cells incubated with a concentration of Con A (0.1 μg/ml) which did not induce cells capable of transferring clinical EAE. These results suggest that PHA and perhaps the low dose of Con A may stimulate the proliferation of the EAE effector cell precursor population without causing the additional differentiation of this precursor population into the effector cell population which is capable of transferring clinical disease. Alternatively, PHA may expand only the helper cell population while effective doses of Con A and PWM would expand both helper and effector cell populations.     

Properties of some defective strains of tobacco mosaic virus and their behaviour as affected by inhibitors during storage in sap

From the type strain of tobacco mosaic virus, defective strains were isolated that produced chlorotic or ringspot type symptoms in tobacco and were difficult to transmit without carborundum in the inoculum. Their concentration was less than 0–1 μg/ml of sap instead of the usual 2 mg/ml with the type strain. Phenol extracts of infected leaves were a little more infective than extracts in buffer, whereas phenol extracts of leaves infected with type strain were very much less infective than extracts in buffer. Electron microscopy of infective sap rarely showed any virus particles, but preparations concentrated by ultracentrifugation contained virus particles, many of which were broken or seemed inadequately assembled. Changing the ambient temperature at which infected plants were kept from 20 to 35°C did not increase the amount or improve the appearance of the virus. Some of the strains were inactivated during heating for 10 min between 70 and 80 °C. Undiluted sap lost its infectivity in 3 days at 20 °C, as did the type strain when diluted to 0–1 μg/ml in sap from healthy leaves. This is because substances that inhibit infection were produced by microbes in the sap. The ability of sap from healthy leaves to inhibit infection increased by more than twenty-five times when left 3 days at 20 °C. Infectivity of appropriate mixtures of type strain and aged sap was restored by diluting them in buffer. Sodium azide at 0·02% in sap prevented formation of the inhibitor. The infectivity of the defective strains increased when inoculated together with the type strain.     

Serum of rabbits infected with Treponema pallidum (Nichols) inhibits in vitro transformation of normal rabbit lymphocytes.

Serum collected from outbred male New Zealand white rabbits infected intratesticularly with Treponema pallidum (Nichols) was assayed for ability to alter transformation of normal rabbit peripheral blood lymphocytes (PBL) in vitro. Sera collected from 25 infected rabbits inhibited [³H]thymidine incorporation by normal rabbit PBL stimulated with concanavalin A (Con A, 16μg/ml), relative to PBL cultured in normal rabbit serum (NRS). Maximal inhibitory activity was detected in serum collected at the time of peak orchitis. The degree of inhibition was related to the concentration of syphilitic serum in PBL cultures. Inhibition of Con A stimulation was reversed by increased mitogen concentration. Sera which depressed Con A stimulation also depressed lymphocyte transformation induced by oxidation with sodium m-periodate (NaIO₄). Cytotoxic activity was detected in occasional sera. All sera were heat inactivated at 56 °C for 30 min prior to testing. Both freshly collected sera and sera stored at ?70 °C significantly inhibited PBL transformation. These results suggested that serum of syphilitic rabbits contains one or more inhibitors of in vitro lymphocyte transformation.     

Effect of T- and B-lymphocyte mitogens on interactions between lymphocytes and macrophages.

The mitogenic response of murine T cells ² to Con A, S-Con A and PHA was found to be macrophage-dependent. Optimal mitogenic responses were obtained when macrophage-depleted T-cell populations were reconstituted with 5% normal peritoneal macro-phages. Studies were carried out to investigate the effect of T- and B-cell mitogens on in vitro physical interactions between murine lymphocytes and macrophages. This was done by determining the number of T- or B cells binding to macrophages in the absence and in the presence of T- and B cell mitogens, and comparing the results of these experiments with the induction of lymphocyte proliferation. Con A increased the binding of T cells to macrophages when used in mitogenic doses (1–5 μg/ml). Dose response experiments showed that the same dose of Con A which produced maximal mitogenic stimulation also induced the greatest number of T cells to bind to macrophages. Nonmitogenic doses of Con A (20–50 μg/ml) did not enhance the binding of T cells, while identical doses of S-Con A both induced T cell mitogenesis and increased the number of T cells bound to macrophages. Similar results were obtained with PHA. None of the B-cell mitogens tested (LPS, EPO 127 and LAgl) increased the binding of either T or B cells to macrophages. PWM, which is mitogenic for both T and B cells, increased the binding of T cells to macrophages, but not that of B cells. In brief, the four T-cell mitogens tested (Con A, S-Con A, PHA, and PWM) induced specific physical interactions between T cells and macrophages, while none of the B-cell mitogens had any effect on the physical interactions between either B or T cells and macrophages when used in mitogenic doses.     

Lymphokine-mediated induction of cytolytic activity in a T cell hybridoma

Functionally inducible CTL hybridomas were constructed by fusing alloantigen-specific T cells (C57BL/6 alpha-DBA/2) with cells from the rat thymoma line W/FU (C58NT)D. A cloned hybridoma line (KSH.4.13.6) that was specifically cytolytic in the presence of activated rat spleen cell supernatant fluid (rat Con A SN) lost activity when transferred to normal medium. However, a cytolytic activity could be reinduced by culturing KSH.4.13.6 cells in medium containing rat Con A SN or secondary mixed leukocyte culture SN. By using various sources of SN, it was found that cytolytic induction required two different factors. PMA-induced EL-4 SN and SN from antigen-activated cloned T cells, neither of which were capable of inducing cytolytic activity alone, were able to synergize in the cytolytic induction of KSH.4.13.6 IFN-gamma and IL 1 failed to induce cytolytic activity even in the presence of EL-4 SN. Furthermore, this hybridoma produced macrophage activating factor (MAF) upon culture in rat Con A SN, although MAF production could not be induced by either specific antigen or lectins. The kinetics of induction and loss of cytolytic activity mediated by rat Con A SN were similar to those of the induction of MAF production. However, EL-4 SN, which by itself was incapable of inducing cytolytic activity, was able to induce MAF production in the KSH.4.13.6 hybrid to an extent similar to that induced by rat Con A SN. These results suggest that the induction of cytolytic activity and of MAF production in this cloned hybridoma cell line are regulated by different mechanisms. Such a functionally inducible T cell hybrid may provide a tool for biochemical and molecular analysis of T cell function and regulation, and of the characterization of cytokines required for CTL differentiation.     

Studies on obtaining meiotic chromosomes for analysis in the bull. II. Direct and culture methods

Testicular tissue was obtained by biopsy or by aspiration following castration or slaughter from 32 healthy bulls, 12 to 16 mos of age, using a 20G 2″, 19G 1 1/2″, 18G 1 1/2″ or 16G 1 1/2″ needle attached to a 20 ml syringe. Meiotic preparations were made by processing the tissue directly and/or after 24 hrs in culture. The tissue culture medium consisted of minimum essential medium (Eagle) supplemented with 10% heat inactivated fetal bovine serum, 4 mM L-glutamine, 20 μg/ml follicular stimulating hormone, 40 i.u./ml human chorionic gonadotrophin, 5 μg/ml testosterone and 25 mM HEPES buffer. The pH was adjusted to 7.0. Culture conditions were: cell concentration approximately 100 cells/ml; incubation temperature 31°C, and atmosphere 5% CO₂, 95% air. Satisfactory preparations were obtained from only 40% of biopsies processed directly, but from 57% of biopsies cultured for 24 hrs. With the procedure that was finally developed using a 16G 1 1/2″ needle enveloped in a sterile plastic bag, 87% of biopsies cultured for 24 hrs gave preparations suitable for meiotic analysis.     

Characterization of a human mitogenic factor (MF) released by phytohemagglutinin-activated lymphocytes.

Phytohemagglutinin (PHA)-stimulated human lymphocytes release soluble products upon subsequent incubation in fresh medium, which are strongly mitogenic for other lymphocytes. In the present investigation, some of the biochemical properties of such a factor (MF) were investigated. It was found that serum is not required in the production of MF. The mitogenic factor was stable at 56 °C for 30 min and at 80 °C for 10 min but was destroyed by treatment at 100 °C for 1 min. By gel chromatography on Sephadex the mitogenic activity was found in fractions corresponding to a molecular weight of 40,000–55,000. Moreover, isoelectric focusing indicated an isoelectric point at pH 8.0–8.5. By subjecting MF to CM-32 cellulose ion-exchange chromatography, all activity was detected in the adsorbed fractions. PHA was studied in parallel in some of the experiments. The results clearly showed that MF is distinct from PHA which induces the release of MF. MF was not adsorbed to concanavalin A-Sepharose.     

Correlation of the fluorescent banding pattern and ultrastructure of a human chromosome

After incubation with Con A, cultured melanoma cells B16-C2W stuck firmly to the dish wall and could be detached neither with trypsin and pronase treatment nor with EDTA treatment, whereas the control cells were easily released from the dish wall by the same treatments. This effect became evident within 5 min of incubation at 37 °C with Con A in a greater concentration than 5 μg/ml. Resistance to trypsinization arose more rapidly as the temperature increased up to 22 °C and changed substantially around 15 °C. By addition of α-methyl-d-mannoside which combines specifically with Con A, the resistant cells became de novo susceptible to trypsinization within 5 min of incubation at 37 °C. The reversing effect of the inhibitor was also temperature dependent. The appearance of resistance to trypsinization was observed without divalent cations (Ca²⁺ and Mg²⁺), but was inhibited by pretreatment with 10^?4 M 2,4-dinitrophenol. The temperature-dependence and the concentration of Con A required for agglutination of freed cells was the same as for induction of resistance to trypsinization.