A new type of mitogenic factor produced by Streptococcus pyogenes.

A new type of mitogenic factor (protein) was purified from the culture supernatant of a strain of Streptococcus pyogenes by SP-Sephadex C-25 column chromatography, preparative isoelectric focusing and reversed-phase high-performance liquid chromatography. The purified factor, showing marked mitogenic activity in rabbit peripheral blood lymphocytes, gave a single-band staining for protein on SDS-PAGE. The molecular weight of the purified mitogenic factor was determined to be 25,370, which was different from those calculated from reported amino acid sequences deduced from 4 different nucleotide sequences of 3 kinds of streptococcal pyrogenic exotoxins (two SPEAs, SPEB and SPEC). The amino acid sequence of the N-terminal region of the purified mitogenic factor was determined to be Gln-Thr-Gln-Val-Ser-Asn-Asp-Val-Val-Leu-Asn-Asp-Gly-Ala-Ser-Lys-Tyr-Leu- Asn-Glu - Ala-, which was also different from the reported N-terminal sequences deduced from the 4 different nucleotide sequences. These data indicate that this mitogenic factor is distinct from the already described streptococcal pyrogenic exotoxins.     

Stimulation of human B lymphocytes by Nocardia-delipidated cell mitogen and derived fractions from N. opaca. Structure-activity relationship

Nocardia-delipidated cell mitogen (NDCM), a particulate fraction prepared from Nocardia opaca, is able to stimulate the proliferation of small resting human B lymphocytes and their differentiation into Ig-secreting cells. This fraction contains two active structures: the cell wall peptidoglycan (PG) and a fraction (Cy I) derived from the cytoplasmic compartment. Treatment of insoluble PG with various bacteriolytic enzymes showed that the minimal structure required for mitogenic activity is more complex than that required for the differentiation of human lymphocytes. The mitogenic activity of cell wall fractions varies in different bacterial species; that prepared from N. opaca is the more potent. Both mitogenic structures of N. opaca induce higher responses in infant and adult PBL as compared to cord lymphocytes. The differentiation of B lymphocytes into Ig-secreting cells induced by PG fractions is T-dependent.     

Proliferation of human B lymphocytes mediated by a soluble factor

Recent studies have established the ability of a proportion of activated human B lymphocytes to undergo G1 phase cell cycle progression and subsequent S phase entry on exposure to factor(s) present in lectin-stimulated mononuclear cell-conditioned media. One factor capable of stimulating activated human B lymphocyte proliferation may be separated from peripheral blood lymphocyte-conditioned media by successive ammonium sulfate precipitation, ion exchange, and gel filtration chromatography. The isolated factor is distinct from the other well-described cytokines, possesses a molecular weight of 12,000-13,000, has a mildly acidic isoelectric point (at pH 6.3-6.6), is protease sensitive, and is relatively heat sensitive. The human B cell mitogenic factor possesses functional and cellular specificity in that its action is restricted to B lymphocytes and its function is proliferative. The production of the B cell mitogenic factor by T lymphocytes is augmented by the presence of a macrophage and further stimulated by syngeneic B cells.     

Concanavalin A as an Inducer of Human Lymphocyte Mitogenic Factor

IT is likely that pharmacological products of antigen : lymphocyte interaction (“lymphokines”) act as mediators and regulators of a variety of cellular immune responses^1,2. This view is strengthened by demonstrations that phytomitogen lectins induce lymphocytes to generate products with similar biological activities^3,4 and physicochemical characteristics⁵, to the lymphokines. Increasing evidence suggests that mitogenic lymphokines may mediate lymphocyte transformation responses in vitro and facilitate lymphoid cell cooperation in vivo (refs. 1,2,6–9). The study of mitogenic factor production by phytomitogens which may predominantly activate thymus-dependent lymphocytes (Concanavalin A (ConA))^8,9 provides a model approach to the investigation of lymphokine function in man. Powles et al.⁴ have described a ConA-induced mitogenic factor which stimulated autologous human lymphocytes only, whereas antigen-induced lymphocyte factors generally stimulate both allogeneic and syngeneic lymphocytes^11–13. Interest in ConA as an inducer of human lymphocyte mitogenic factors would be widened if conditions were found in which ConA stimulated human lymphocytes to generate products which were mitogenic for both allogeneic and autologous lymphocytes. As a lymphokine stimulant, ConA has the advantage that it is largely removed from culture fluids by absorption to cross-linked dextrans (‘Sephadex G-50’) or serum glycoproteins¹⁴. Here we demonstrate that a ‘Sephadex’-binding fraction of ConA (ConA- V) induces human lymphocytes to generate a mitogenic factor which activates both allogeneic and autologous lymphocytes.     

Cellular regulation of human lymphocyte mitogenic factor release in vitro]

Production of the mitogenic facotr by human lymphocytes stimulated with phytohemagglutinin (PHA) was investigated. Anti-PHA antibodies were used for studying the culture medium mitogenic activity. The mitogenic factor production markedly increased after lymphocyte irradiation. When macrophages were eliminated, using iron powder, mitogenic factor generation was also increased. It was demonstrated that lymphocyte irradiation and macrophage elimination stimulated the mitogenic factor production throgh different mechanisms.     

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.     

Isolation,X-Ray Structure and Biological Activity of Deacetyl-dihydrobotrydial Produced by Botrytis squamosa

Mitogenic substances on human peripheral blood mononuclear leukocytes were screened from culture filtrates of microorganisms newly isolated from soil and sea water by measuring [³H]- thymidine incorporation into the cells. Strong mitogenic activity was found in marine bacteria, particularly in marine vibrios. These mitogen samples exhibited neither hemagglutinating activity nor leukoagglutinating activity. They could scarcely stimulate murine lymphocytes.

Cell-cell interaction among leukocyte subsets in response to a bacterial mitogen was investigated using the most powerfully mitogenic sample (culture filtrate of strain H 52–2). A slight decrease in the mitogen response was observed on depletion of plastic surface adherent cells. Separation of T and non-T cells from each other by erythrocyte-rosette sedimentation resulted in a markedly diminished mitogen response. Considerable restoration of the mitogen response was obtained when T cells were mixed with mitomycin C-treated adherent cells or mitomycin C-treated non-T lymphocytes, or when non-T lymphocytes were mixed with mitomycin C-treated T cells.     

The role of monocytes in human lymphocyte activation by mitogens.

Studies were performed to determine the role of monocytes in human lymphocyte activation by mitogens. Velocity sedimentation at 1 x G in a new apparatus was utilized to obtain highly purified lymphocyte fractions (LF) nearly free of monocytes (0.02 to 0.4%) and a fraction (MF) enriched for monocytes (64 to 92%). The average peak responses of the lymphocyte fractions to phytohemagglutinin, concanavalin A, and pokeweed mitogen were 19, 10, and 9% of the responses achieved with unfractionated lymphocyte cultures containing approximately 20% monocytes. These changes were not attributable to altered dose requirements. When mitomycin-C-treated MF cells were used to reconstitute LF cultures, it was found that 4% monocytes fully restored the response to phytohemagglutinin whereas 8 to 16% monocytes were required for a normal response to the other mitogens. Higher numbers of MF cells produced supranormal responses, with 35 to 50% monocytes resulting in the optimal stimulation. Allogeneic monocytes were able to fully reconstitute the response of LF, and 2-mercaptoethanol (50 microM) was only slightly effective. In exploring possible mechanisms by which monocytes potentiate the mitogenic activity of lymphocytes, it was found that the supernatants of MF cultures could partially, but not completely, reconstitute LF responses, suggesting that contact with MF may be required for optimal effectiveness. Addition of graded numbers of monocytes to LF altered both the kinetics of the response and the peak level of proliferation. Monocyte depletion also resulted in markedly decreased survival of cultured unstimulated LF. These observations suggest a variety of possible effects of monocytes in potentiating mitogenic responses, including contact-mediated interactions with lymphocytes (possibly to present the mitogen optimally); enhancement of proliferation kinetics and the size of the responding subpopulation, and maintenance of a requisite growth factor(s) in the culture. Small differences in the monocyte content of cultured lymphocyte preparations may thus account for many of the often observed variations in mitogen responsiveness.     

Production of interleukin 1 activity by normal human peripheral blood B lymphocytes

Interleukin 1 (IL 1) production by normal human B lymphocytes was investigated. Normal human peripheral blood B lymphocytes were purified by sequential separation with the use of Ficoll-Hypaque gradient centrifugation, sheep red blood cell rosette formation, Percoll gradients, and treatment with monoclonal antibodies (anti-Leu-M1, B73.1, and T101) and complement. Both purified large B lymphocytes (BL) and small B lymphocytes (BS) produced IL 1-like (thymocyte co-mitogenic and fibroblast mitogenic) activities in response to lipopolysaccharide. Maximal production of IL 1 activity by both BL and BS occurred at 48 hr. The m.w. of IL 1 activities from both BL and BS were about 20,000 with high pressure liquid chromatography, and the major isoelectric point of BL- and BS-derived IL 1 activity was 7.0. A rabbit anti-human monocyte IL 1 antiserum inhibited the activity of B cell-derived IL 1, suggesting antigenic similarities of monocyte- and B lymphocyte-derived IL 1 moieties. These data suggest that normal B lymphocyte-derived IL 1 activity is biochemically and immunologically similar to monocyte-derived IL 1.     

Human lymphocyte mitogenic factor: synthesis by sensitized thymus-derived lymphocytes, dependence of expression on the presence of antigen

Lymphocyte mitogenic factor (LMF) was obtained from human lymphocytes stimulated with tetanus toxoid. LMF proved to be a soluble lymphokine produced by a sensitized T lymphocyte in response to stimulation with antigen. LMF induces proliferation in cells which normally do not proliferate in response to antigen (thymocytes, B lymphocytes). This function is expressed only in the presence of antigen. LMF produced in response to stimulation with a specific antigen is able to cooperate with more than one antigen in recruiting cells into division.LMF is a nondialyzable protein different from other lymphokines as judged by the kinetics of its release. It elutes in the postalbumin fraction of Sephadex G200.     

Biological Activities of Guinea Pig Mitogenic Factor from Immune Lymphocytes Stimulated with Antigen

Mitogenic factor was produced by sensitized guinea pig lymph node cells stimulated with a specific antigen. Both T lymphocytes and macrophages were required for the production of this factor. The culture supernatant of lymphocytes containing the mitogenic factor exhibited a strong helping effect on the proliferative response of T lymphocytes to phytohemagglutinin (PHA). Mitogenic factor and the factor with the helping activity coeluted in the molecular weight range of 25,000-35,000 daltons in gel filtration. Furthermore the fraction containing mitogenic factor was found to support the proliferation of lymphoblasts induced by PHA or antigen, suggesting that the mitogenic factor may be the guinea pig equivalent of T cell growth factor (TCGF) reported in the mouse, rat, and human. On the other hand, the T cell-activating monokine of the guinea pig, possessing the helping activity for the proliferative response of T lymphocytes to PHA, never exhibited TCGF-like activity.     

Plasminogen activator is an apparent lymphocyte mitogen

Culture fluids of avian sarcoma virus (ASV)-transformed but not normal chicken embryo cells frequently elicited a mitogenic response in normal avian and murine lymphocytes. We examined the possibility that plasminogen activator (PA) might be responsible for the observed mitogenic effect. PA activity, present in culture medium, was correlated positively with lymphocyte mitogenic capacity. Treatment of cells with phorbol myristate acetate, which elevates PA levels, increased mitogenesis. Similar treatment with dexamethasone, which inhibits PA biosynthesis and/or secretion, reduced lymphocyte mitogenic activity. Addition to culture fluids of either benzamidine or diisopropylfluorophosphate, both specific PA inhibitors, blocked lymphocyte proliferative responsiveness to culture fluids. In contrast, neither epsilon-amino-caproic acid nor trasylol, which inhibits plasmin esterase activity but not PA, abrogated lymphocyte responsiveness. Furthermore, purified urokinase, an enzyme of similar substrate specificity to PA, had lymphocyte stimulatory activity. These results strongly suggest that PA can function as a lymphocyte mitogen.     

Partial purification and characterization of porcine platelet-derived growth factor (PDGF)

Platelet derived growth factor (PDGF) has been partially purified from porcine platelets. Purification steps included heparin-agarose chromatography of the material released by thrombin-stimulated washed porcine platelets and Blue-Sepharose chromatography. Preparative isoelectric focusing showed that isoelectric point of porcine PDGF is at pH 10.0–11.0 and elution experiments from sodium dodecyl sulfate (SDS) polyacrymlam de gels indicated that its molecular weight is close to 30 kD. The immunoglobulin fraction prepared from anti-human PDGF serum inhibited the mitogenic activity of porcine PDGF. These experiments suggest a homology of porcine and human PDGF. Porcine platelet factor 4 and porcine platelet basic protein were devoid of significant mitogenic activity.     

Regulatory Factors of Lymphocyte-Lymphocyte Interaction: II. Characterization of Human Mitogenic Factor Derived from the Culture Supernatant of a Mouse-Human Hybridoma

Cell hybridization of phytohemagglutinin (PHA)-stimulated human peripheral blood lymphocytes (PBL) with murine lymphoma (EL-4) provided three hybridomas (MHH-16, MHH-20, and MHH-22) which spontaneously produced human mitogenic factor (MF). MHH-16 was serially subcloned by limiting dilution procedures, which resulted in maintaining two subclones producing human MF spontaneously for more than one year (PQL-3 and PQL-5 subcloned lines). Human MF (MHH-MF) derived from supernatants of PQL-5 line cultures had a molecular weight (m.w.) of about 26,000–30,000 daltons (the major peak) with a minor peak with an m.w. of 15,000 daltons on Sephadex G-100 chromatography, and at a high concentration of NaCl (1 m), the activity of the 26,000–30,000-m.w. fraction became weak and that of the 15,000-m.w. fraction became predominant. MHH-MF had an isoelectric point of pH 5.0–6.5. On DEAE-cellulose chromatography, MHH-MF was eluted at a fairly low salt concentration (sodium phosphate buffer 0.02 M, pH 8.0, NaCl 10 mm). After periodate treatment of this MHH-MF, the mitogenic activity almost disappeared. MHH-MF was relatively unstable to heating at 56 C for 20 min. In the presence of tunicamycin (0.3μg/ml), an inhibitor of N-linked glycosylation, the synthesized MHH-MF showed a decrease in m.w. as follows: the major peak shifted from 26,000–30,000 to 23,000 daltons and the minor peak from 15,000 to 10,000 daltons on Sephadex G-100 chromatography. In internal labeling experiments with [³H]leucine, the ³H-labeled MF was partially purified, with mitogenic activity as a guide. This ³H-labeled MHH-MF fraction could be absorbed by PHA blasts but not by normal PBL. On SDS-PAGE under reducing conditions, only the radioactive peak of the 15,000-dalton fraction was recovered. MHH-MF obtained from the hybridoma culture supernatants may be a dimer of the 15,000-dalton fraction and a glycoprotein.     

Inhibition of interleukin 1 activity by a factor in submandibular glands of rats

With the sequential use of ammonium sulfate precipitation, gel filtration and chromatofocusing, we have partially purified from extracts of the submandibular glands of rats a factor (referred to as submandibular gland's immunosuppressive factor or SMG-ISF) capable of inhibiting the in vitro proliferation of mitogen- and antigen-stimulated murine lymphocytes. The semi-purified suppressor fractions had an isoelectric point of 4.4 to 4.5 and consisted of at least three molecular species. These active fractions suppressed the mitogenic effects of Concanavalin A phytohemagglutinin, and lipopolysaccharide. In vitro immune reactions such as the mixed lymphocyte culture MLC reaction and the production of cytotoxic T lymphocytes (CTL) across major histocompatibility barriers in mice were also suppressed. These in vitro immunosuppressive effects required the addition of the suppressor fractions early after the initiation of the cultures and were reversed if the factor was removed from the cultures at least 48 to 72 hr before the completion of the assays. The active fractions did not affect the proliferation of CTLL 2 cells induced by interleukin 2 (IL 2), but inhibited the mitogenic and co-stimulatory effects of IL 1 on mouse thymocytes, and in this effect showed a dose-response relation suggestive of a competitive mechanism. These characteristics of SMG-ISF indicate a specific inhibition of the activity of IL 1.     

Purification and biochemical characterization of human lymphocyte mitogenic factor (LMF).

Supernatants of human T lymphocytes stimulated by TT antigen release two factors that induce mitogenesis in autologous and allogeneic B lymphocytes. These factors are precipitated by 60% ammonium sulfate and 50% ethanol, and are both destroyed by heating to 70 degrees C for 5 min. By equilibrium ultracentrifugation there was a peak of mitogenic activity in the fraction with a specific gravity of 1.3147 corresponding to a partial specific volume of 0.761. After ultrafiltration through an Amicon XM50 membrane, the concentrate was chromatographed on a Sephadex G-200 column. Mitogenic activity was found only in the post-albumin fraction. When the post-albumin fraction was run on an isoelectrofocusing column, two distinct mitogenic factors were identified. The major peak of mitogenic activity (LMF) had a pI of 6.68 +/- 0.05 and the minor peak (MMF) had a pI OF 7.27 +/- 0.05. Amino acid analysis of LMF identified it as a protein and PAGE showed that LMF probably was a tetramer with a m.w. of 80,000.     

Mitogenic activity of Cratylia mollis lectin on human lymphocytes.

The mitogenic effect of Cratylia mollis seed lectin preparations containing two (Cramoll 1,4) or one molecular form (Cramoll 1) showed activity similar to the well known T-cell mitogen, concanavalin A (Con A). The effect on human lymphocytes was analyzed through a colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). Inhibition of lymphocyte proliferation with methyl-alpha-d-mannoside (both preparations) indicated that the mitogenic effect involved carbohydrate lectin binding sites.     

The superantigenic activity of streptococcal pyrogenic exotoxin B is independent of the protease activity

The nature of the mitogenic activity of pyrogenic streptococcal exotoxin B, also known as streptococcal cysteine protease, has been debated in the literature. Streptococcal exotoxin B has been shown to cleave interleukin-1beta precursor and create biologically active interleukin-1beta, a major cytokine mediating inflammation and shock. This activity could mimic the mitogenicity and cytokine release induced by superantigens in lymphocyte stimulating experiments. In this study, the protease activity of streptococcal exotoxin B was irreversibly inhibited by covalent binding of a tripeptide and the superantigenic properties of streptococcal exotoxin B were found not to be influenced by this inactivation. Native as well as protease-inactivated streptococcal exotoxin B was shown to stimulate T-cell proliferation without a need of metabolically active antigen presenting cells. Furthermore, streptococcal exotoxin B-induced T-cell proliferation was shown to require HLA-DQ since addition of HLA-DQ monoclonal antibodies totally inhibited the mitogenic activity of streptococcal exotoxin B, indicating that streptococcal exotoxin B, as other superantigens, makes direct contact with the T-cell receptor via HLA class II. The aim of this study was to characterize the relationship between the proteolytic and superantigenic properties of streptococcal exotoxin B.     

Role of interleukin 2 on enhancement of concanavalin A-induced human peripheral blood lymphocyte proliferation by murine B cell mitogens

Murine B cell mitogens such as bacterial lipopolysaccharide (LPS), butanol-extracted water soluble adjuvant (Bu-WSA), dextran sulfate (DS), synthetic muramyl dipeptide (MDP), and its analog MDP-Lys (L18) do not show any mitogenic ability in vitro on human peripheral blood lymphocytes or mixed cell populations of purified T and B cells obtained from the lymphocytes in an ordinary culture system. However, these mitogens are capable of enhancing the mitogenic effect of concanavalin A (Con A) in the cultures. In the presence of one of these mitogens, the activity of interleukin 2 (IL 2), but not interleukin 1, in the supernatants obtained from cultures containing Con A-stimulated T cell and B cell populations was higher than that of control cultures. The role of the newly produced IL 2 in the synergistic effect of the mitogens in human lymphocyte cell cultures was discussed.     

Functional and immunological characterization of the stimulation of human lymphocytes with a mitogen from group A streptococci (SM)

Streptococcal mitogen (SM), an extracellular product of group A streptococci, is non specifically mitogenic for both B and T lymphocytes. The mitogenic activity of SM is resistant to digestion with trypsin, to heating at 100 °C for 5 min, and to treatment with dithiothreitol. The proliferative response of lymphocytes from patients with a history of rheumatic fever is similar to that of lymphocytes from healthy donors when stimulated with optimal concentrations of SM, but is significantly reduced when low doses of SM are used. T lymphocytes stimulated with SM acquire Ia antigens and the ability to stimulate allogeneic and autologous lymphocytes in mixed lymphocyte reactions. An involvement of Ia antigens in these reactions is indicated by the specific block by monoclonal antibodies to human Ia antigens.