Many Bacterial Species are Mitogenic for Human Blood B Lymphocytes

Thirty bacterial species were tested for their ability to stimulate to increased DNA synthesis in human blood lymphocytes. A definite stimulation was obtained with eighteen bacterial species. For three of these species ten different strains of each were tested, and all increased DNA synthesis. The maximum response was after 3--4 days of culture, suggesting a mitogenic effect. This was confirmed by the induction of polyclonal antibody production shown by a plague assay, which was positive for nine of eleven species tested. Most bacterial species increased the DNA synthesis in B-lymphocyte-enriched and unseparated lymphocytes but had negligible activity on T-lymphocyte-enriched cultures. Among bacteria with a mitogenic effect and ability to induce polyclonal antibody production are Staphylococcus aureus strain Cowan I with a high content of protein A and many common human pathogens such as Haemophilus influenzae, Mycobacterium tuberculosis, Neisseria gonorrhoeae, Streptococcus group A and Streptococcus pneumoniae.     

DNA-Synthetic Responses of Fractionated Human Lymphocytes Exposed to Lymphocyte-Derived Mitogenic Factor

Human peripheral lymphocytes that have been exposed to phytohemagglutinin (PHA) release mitogenic factors (MF) on subsequent incubation in fresh medium. In this investigation, MF-rich supernatants were analyzed for their ability to induce DNA synthesis in various fractions of peripheral human lymphocytes. MF was found to induce higher DNA synthesis in T-cell preparations that in unfractionated lymphoid preparations or B-cell-rich fractions. Evidence is presented showing that some types of non-T-cell can inhibit the MF response of T cells. These cells are nonadherent and nonphagocytic and require DNA synthesis to express their inhibitory activity. These results suggest that the regulation of the action of lymphocyte-derived MF is cell-mediated.     

Evidence for the Release of Mitogenic Factors by Both T and B Lymphocytes in the Human

Lymphocytes incubated with antigens or nonspecific stimulatory agents may release factors that induce DNA synthesis in other lymphocytes. The aim of this investigation was to determine whether such mitogenic factors (MF) are produced by both T and B cells in the human. Both T- or B-cell-enriched cell preparations from peripheral blood were found to release MF during or after incubation with phytohemagglutinin or pokeweed mitogen. Concanavalin A covalently bound to Sepharose triggered B-cell preparations to higher MF release than either T cells or unfractionated lymphocyte suspensions. These results strongly indicate that both T and B cells in the human are able to produce MF.     

Lactate Dehydrogenase Isoenzyme Pattern: Marker for Differentiation of Lymphocytes

The lactate dehydrogenase isoenzyme pattern has been determined in different murine thymocyte cell populations. Enrichment of thymocytes with more mature cells through total irradiation results in a smaller percentage of LDH-1, LDH-2, LDH-3 and a greater percentage of LDH-4 and LDH-5. Fractionation of normal thymocytes by velocity sedimentation at unit gravity yields a fraction of cells with a high sedimentation rate. LDH-1 and LDH-2 formed a smaller percentage of the total enzyme activity in these cells. These findings indicate that the LDH isoenzyme distribution is a marker for differentiation of thymocytes.     

Corticosteroid-Induced Modulation of Immunoglobulin Secretion by Human B Lymphocytes: Potentiation of Background Mitogenic Signals

Abstract

The modulation of immunoglobulin (Ig) secretion of human peripheral blood mononuclear cells by in vitro hydrocortisone (HC) was evaluated. A marked enhancement of Ig secretion was observed in unstimulated cultures in the presence of HC as compared to cultures without HC. The augmented response was not due to a direct induction of Ig secretion by HC, but resulted from an enhancement or unveiling of the background mitogenic signal provided by the supplemental serum used in culture. HC-induced augmentation of Ig secretion was only seen in unstimulated cultures performed in fetal calf serum (FCS) which is known to possess mitogenic properties. In contrast, HC had no significant effect on Ig secretion of cultures performed in human serum, which provides little or no background mitogenic signal. On the other hand, no enhancement of Ig Secretion by HC was seen in cultures maximally stimulated with pokeweed mitogen regardless of whether FCS or human A serum was used. The mechanisms of this modulation of Ig secretion are unclear at present and may include a synergy between corticasteroids and background mitogenic signals (such as FCS) indirectly triggering B cells and/or the dampening of a negative immunoregulatory effect of an accessory cell.     

Corticosteroid-Induced Modulation of Immunoglobulin Secretion by Human B Lymphocytes: Potentiation of Background Mitogenic Signals

The modulation of immunoglobulin (Ig) secretion of human peripheral blood mononuclear cells by in vitro hydrocortisone (HC) was evaluated. A marked enhancement of Ig secretion was observed in unstimulated cultures in the presence of HC as compared to cultures without HC. The augmented response was not due to a direct induction of Ig secretion by HC, but resulted from an enhancement or unveiling of the background mitogenic signal provided by the supplemental serum used in culture. HC-induced augmentation of Ig secretion was only seen in unstimulated cultures performed in fetal calf serum (FCS) which is known to possess mitogenic properties. In contrast, HC had no significant effect on Ig secretion of cultures performed in human serum, which provides little or no background mitogenic signal. On the other hand, no enhancement of Ig Secretion by HC was seen in cultures maximally stimulated with pokeweed mitogen regardless of whether FCS or human A serum was used. The mechanisms of this modulation of Ig secretion are unclear at present and may include a synergy between corticasteroids and background mitogenic signals (such as FCS) indirectly triggering B cells and/or the dampening of a negative immunoregulatory effect of an accessory cell.     

Enhancement of Human B Lymphocyte Differentiation in Vitro by Thyroid Hormone

A study was made of the regulatory influence of a thyroid hormone, triiodothyronine (T₃), on human blood T- and B-lymphocyte responses in vitro. Blood lymphocytes were stimulated with the T-cell mitogens concanavalin A (Con A) and phytohaemagglutinin (PHA), with the T-dependent B-cell activator pokeweed mitogen (PWM), and with the T-independ-ent B-cell activator Staphylococcus aureus Cowan I bacteria in the presence of various hormone concentrations. T₃ did not stimulate T-cell proliferation. The number of immuno-globulin-containing and -secreting cells (plasmablasts) was increased in PWM and Staph. aureus cultures treated with T₃. The maximal enhancement was reached at a concentration of 10⁻⁹-10⁻⁷ M T₃. Cell fractionation techniques revealed that T₃ apparently had a direct stimulatory effect on B-cell differentiation.     

Characterization of Human Lymphocyte Surface Receptors for Mitogenic and Non-mitogenic Substances

To compare the receptor patterns for mitogenic and non-mitogenic substances, surface glycoproteins of human lymphocytes were labelled with the lactoperioxidase-catalysed iodination technique and with a galactose oxidase-tritiated sodium borohydride technique. Labelled cells were detergent-solubilized, and the lysates were allowed to react with insolubilized purified mitogenic lectins, phytohaemagglutinin, leucoagglutinin and an insolubilized non-mitogenic lectin, oxidized leucoagglutinin. Lectin-reactive protein were eluted with sodium dodecyl sulphate (SDS) buffer. Cell membrane components reactive with anti-lymphocyte globulin (ALG) were retrieved by indirect immunoprecipitation with protein-A-bearing staphylococcus Cowan I strain (SaCI). Lectin-and ALG-reactive proteins were analysed by SDS polyacrylamide gel electrophoresis. Iodinated glycoproteins regularly showed four major components with molecular weight of 120,000, 70,000 60,000 and 43,000 daltons, respectively, on 7% gels. An additional broad peak in the molecular weight range 20,000–35,000 daltons found on 10% gels. Tritiated glycoproteins also showed four major components with MW 120,000, 70,000, 60,000 and 42,000, respectively, which reacted with lectin and ALG. In addition, ALG reacted with some glycoproteins with MW between 150,000 and 230,000 daltons. On 10% gels additional lectin and ALG-binding glycoproteins with MW around 30,000 daltons were found. The similarity in structures bound by mitogenic and non-mitogenic substances indicated that lymphocyte activation may depend on some properly conferred by the mitogen.     

Immunochemical Characterization of a Human B Lymphocyte Differentiation Antigen (p65)

A human B-cell differentiation antigen (BDA-1) with a molecular weight of 65,000 was Identified by use of an anti-B-cell xenoantiserum. BDA-1 was isolated by immunoprecipitation of cell lysates from a B-lymphoblastoid cell line (SB) and from blood B lymphocytes (sIg⁺ER⁻) of four normal individuals. It was not detectable in cell membrane lysates from two T-lymphoblastoid cell lines (HSB-2 and MOLT-3) or from enriched normal T cells (sIg⁻ER⁺). BDA is a singlechain molecule since its migration in SDS-PAGE gels was not altered by heating the protein to 100 C or by treatment with 2-mercaptoetbanol. Immunodepletion experiments demonstrated that the antigen recognized by anti-BDA xenoantiserum has neither structural nor antigenic relationships with la-like antigen or β-microglobulin.     

Enumeration of Absolute Numbers of T and B Lymphocytes in Human Blood

A new immunofluorescence test permits the direct enumeration of T and B lymphocytes in unseparated blood. B cells were identified with antiimmunoglobulin sera and T cells by a rabbit anti-human brain serum rendered unreactive with B cells by sequential absorption with erythrocytes, liver, and either chronic lymphocytic leukaemia cells or a cell line, BRI-8, with B-cell surface characteristics. T and B cells were also enumerated with lymphocyte suspension purified by either Ficoll-lsopaque density sedimentation or by a two-step gelatin-polystyrene bead column method. T cells in Ficoll-purified suspension were also enumerated by rosette formation with sheep erythrocytes. Comparison of the T-B proportions evaluated with the different cell preparation showed that the cell separation procedures can give rise to a deviation in the T:B ratio in favour of B cells. The extent of this induced bias related inversely to lymphocyte yield. Staining of whole blood preparations or high-yield purified cells enables absolute numbers of T and B lymphocytes to be calculated. We consider this measurement considerably more informative than relative proportions of T and B cells.     

Polyclonal Activation of Human Peripheral Blood B Lymphocytes by Formaldehyde-Fixed Salmonella paratyphi B

B-cell 'activation' in cultures stimulated with pokeweed mitogen (PWM), Staphylococcus aureus strain Cowan I, or formaldehyde-fixed Salmonella paratyphi B (SPB) was evaluated by enumeration of cells secreting immunoglobulin (Ig) and by quantitation of Ig released into culture supernatants. A dissociation between these two values was found after day 6 in cultures activated with PWM or SPB, suggesting that Ig-secreting cells (ISC) are heterogeneous in terms of Ig secretion rate. Generation of ISC in cultures activated with PWM or SPB was partially inhibited by hydroxyurea, but [g levels in culture supernatants were not affected. These results indicate that there are at least two subpopulations of ISC in stimulated peripheral blood lymphocyte cultures, one sensitive to, and the other resistant to, hydroxyurea. The hydroxyurea-resistant subpopulation appeared to be more mature and to release almost all of the Ig detected in culture supernatants. Furthermore, time-course studies of ISC numbers and Ig levels showed that each ISC in SPB-stimulted cultures (but not in PWM-stimulated cultures) was more active in Ig synthesis and secretion after day 8 than before day 6, indicating that after day 8 most of the ISC in cultures activated with SPB were hydroxyurea-resistant. These studies suggest that SPB is another useful polyclonal B-cell 'activator' for studies of human B-cell differentiation and function, and that SPB defines two distinct subsets of B cells.     

Recombinant Protein A of Non-Staphylococcal Origin is Not Mitogenic for Human Peripheral Lymphocytes

Recombinant protein A(SpA) produced in Escherichia coli or Bacillus subtilis bacteria did not induce activation of human peripheral mononuclear cells, whereas SpA preparations obtained from naturally occurring Staphylococcus aureus bacteria as well as recombinant SpA from Staphylococcus xylosus were potent mitogens. Further purification of SpA from S. aureus showed that the mitogenic material was concentrated in the side fractions containing more basic molecules. Some staphylococcal enterotoxins are mitogenic for human cells and in order to test whether contaminating enterotoxins would be responsible for the mitogenic effect of SpA preparations, rabbit antibodies were produced against enterotoxin A and B. These antibodies inhibited activation of human cells induced by the enterotoxins used for immunization but did not affect the activation induced by SpA preparation. The addition of selected human sera to in vitro cultures resulted in an inhibition of the response induced by low doses of SpA. There was no clear relationship between these effects and the content of IgG antibodies against staphylococcal enterotoxins A, B, and Cl in the sera. Thus, we conclude that the mitogenicity of SpA preparations is caused by contaminating molecules, probably not enterotoxins A, B, or Cl.     

Mitogenic Activation of Human Lymphocytes: a Protein A Plaque Assay Evaluation of Polyclonal B-Cell Activators

Using the protein A plaque assay, the capacity of various polyclonal B cell activators to induce differentiation in human B lymphocytes was investigated. Dextran sulphate and native Dextran were both virtually devoid of mitogenic properties, Lipopolysaccharide, however, was round to be a potent mitogen in human cells that, although giving rise to low DNA synthetic response, induced high numbers of immunoglobulin-synthesizing cells. Mean plaque-forming cell (PFC) numbers in healthy blood donors assayed on the optimal day (days 5–7) were 23, 493 IgM/10⁴ cells, 11, 288 IgG/10⁶ cells, and 2643 IgA/10⁶ cells. Values obtained in spleen cells, peaking at days 4–6, were slightly higher. Purified protein derivative (PPD) was equally or oven more-effective than lipopolysaccharide (LPS) in generating PFC of different subclasses in peripheral blood with mean of 29, 241 IgM/10⁶, 21, 269 IgG/10⁶, and 3681 IgA/10⁶. PPD furthermore induced a marked DNA synthetic response in human lymphocytes. These data suggest that LPS and PPD may both be used as functional markers in human cells when analysing patients with a suspected immunodeficiency state. It is suggested that cultures should be assayed using the protein A plaque assay, thereby being able not only to investigate the individual immunoglobulin classes but also to avoid the possible hazards involved in measuring antigen-specific responses in patients whose prior immunization to the antigen tested can never be totally excluded     

Induction of Natural Killer Cell Activity and Allocytotoxicity in Human Peripheral Blood Lymphocytes after Mixed Lymphocyte Culture

The K-562 tumour cell is a highly susceptible target for natural killer (NK) cell lysis by lymphocytes of human peripheral blood. We have studied the antigenic relationship between the recognition sites for lysis of lymphoid and various tumour target cells by cytolytic T lymphocytes (CTL) and NK cells induced in mixed lymphocyte cultures (MLC). The characteristics of these two effector cell types have also been investigated. It was demonstrated that fresh NK cells lose their NK lytic activity when cultured alone. Cell lines not susceptible to lysis by fresh NK cells are lysed by MLC-induced NK cells. There is no antigenic relationship between the recognition sites for the alloreactive T lymphocytes and MLC-generated NK cells expressed on the lymphoid target cells and the tumour target cells, respectively. The MLC-generated alloreactive T cells and NK cells are not identical. The MLC-generated NK cells are different from the fresh NK cells present in the peripheral blood.     

Induction of sIg-Negative B Lymphocytes for Differentiation by PWM

It has been reported in the literature that PWM-responsive E lymphocytes are sIgM,⁺ sIgD^-, lack receptors for mouse erythrocytes, and are larger than PWM-negative lymphocytes. This paper describes the observation of B cells among the thymocytes from a patient with myasthenia gravis. In the thymocytes, actually no slg⁺cell wan found, and Staphylococous aureus Cowen strain I could not induoe the mononuolear cells in the thy-mus to differentiate. It was surprising that 6.9 per cent of the cells were BA-1 positive cells. At the same time, in the presence of PWM, 9680 ± 553 cells out of 1 × 10^--⁶ thymocytes could be induced to differentiate to immunoglobulin-secreting cells (ISC) in culture. This result suggests that PWM might be able to induce sIg^-B lymphocytes to differentiate as well.     

Stimulation of Human B Lymphocyte Differentiation by the Neuropeptides Substance P and Neurokinin A

Substance P (SP) at a concentration of 10(-7) M significantly increased the number of IgG-producing cells induced by the polyclonal activator Staphylococcus aureus protein A (SpA) in 11 out of 22 cultures of enriched human blood B lymphocytes, in nine cultures SP did not significantly affect the SpA response and in three cultures IgG secretion was decreased in the presence of SP. Stimulation by SP was observed in cultures at days 6 and/or 8. In 3 out of 4 cell cultures depleted of monocytes SP did not affect the cell response to SpA stimulation. SP antagonists inhibited the enhancing effect of SP on B-cell antibody secretion induced by SpA. SP alone did not stimulate B lymphocytes. Neurokinin A (NKA) had similar effects as SP and enhanced the IgG secretion induced by SpA in 5 out of 9 experiments, in two experiments was inactive, and in one decreased the IgG secretion. The effect of SP and NKA on B lymphocytes suggest that the neuropeptides interact with the regulation of the immune response.