Lymphocyte function in experimental african trypanosomiasis: mitogenic effects of trypanosome extracts in vitro.

Extracts of Trypanosoma brucei and Trypanosoma congolense were incubated in vitro with nonimmune lymphocytes of mice, rats, guinea pigs, and rabbits in order to test for mitogenic effects or for other characteristics of polyclonal B lymphocyte activators. Trypanosome extracts (TE) were not mitogenic for spleen cells of mice, rats, and guinea pigs in vitro, nor did the parasite extracts alter the mitogenic responses of lymphocytes from these animals to known B- and T-cell mitogens. TE also failed to induce polyclonal antibody synthesis in mouse spleen cell cultures in an in vitro antibody response system, in contrast to the effects of bacterial lipopolysaccharide, a known polyclonal B cell activator. Rabbit spleen cell and peripheral blood lymphocyte cultures, however, were stimulated by TE to undergo blastogenesis in vitro. Incubation of rabbit lymphocytes with phytohemagglutinin (PHA) and TE or anti-rabbit immunoglobulin serum and TE revealed an additive effect only in terms of the TE-plus-PHA culture responses; these findings suggest that a non-PHA responsive lymphocyte population, possibly B lymphocytes, is stimulated by TE in rabbits. The relationship of trypanosome-induced lymphocyte mitogenic stimulation to other immunological dysfunctions occurring in chronic African trypanosomiasis is discussed.     

Mycoplasma neurolyticum: a potent mitogen for rat B lymphocytes.

A mitogen prepared from Mycoplasma neurolyticum has been demonstrated to induce extensive transformation of in vitro cultured rat B lymphocytes. The data summarized in this report show that rat thymus cells as well as hydrocortisone-resistant thymocytes were not activated by this mitogenic agent. On the other hand, spleen cells obtained from thymectomized, lethally irradiated and bone marrow-reconstituted rats were extensively activated by M. neurolyticum. Furthermore, M. neurolyticum was shown to induce the development of antibody-producing cells, as attested by the appearance of direct plaque-forming cells against sheep red blood cells and trinitrophenylated sheep red blood cells in spleen cell cultures exposed to this mitogen. It was also demonstrated that stimulation of rat lymphocytes by this mitogen was inhibited by anti-rat immunoglobulin antibodies. In view of these data, it was suggested that M. neurolyticum, which activates mouse B lymphocytes, is a potent mitogen for rat B lymphocytes as well. This mitogen is a significantly more powerful mitogen for rat B lymphocytes then any other known mitogens. The availability of such mitogenic material in the rat system will enable studies on control mechanisms of action and differentiation of rat B lymphocytes.     

Mitogenic Properties of Rabbit Anti-Human β2-Microglobulin for Murine B Cells

Rabbit antisera against human β₂-microgluhulin were found to be mitogenic for mouse spleen cells, giving rise to a peak DNA synthetic response on day 2 in cultures containing serum-free medium. The mitogenic effect was shown on cells from spleens of nude 'athymic' mice and on spleen B cells, but no effect was found on cortisone resistant mouse thymocytes or on spleen T cells. Thus, it was concluded that the serum was mitogenic for mouse B cells but not for T cells. This conclusion was confirmed by experiments showing that the antiserum was able to induce polyclonal antibody synthesis in mouse spleen cells in culture. The activity of the scrum was absorbed by pure human β₂-microglobulin as well as by mouse thymocytes and bone marrow cells     

Mitogenicity of Naegleria fowleri extract for murine T lymphocytes

The whole-killed pathogenic free-living amoeba, Naegleria fowleri, contained mitogenic activity (NFM) for mouse spleen cells. Similar preparations from the non-pathogenic amoeba N. gruberi and the pathogenic Acanthamoeba culbertsoni lacked mitogenic activity. Fluids from N. fowleri cultures, containing amoeba antigens, also failed to cause proliferation of mouse spleen lymphocytes. Spleen cells from athymic nude mice failed to respond to NFM. In addition, nylon wool non-adherent, but not the adherent, spleen cell subpopulation proliferated in the presence of NFM. These results show that the factor(s) is mitogenic for T lymphocytes. The spleen cells from mice treated with cyclophosphamide doses known to deplete T suppressor cell activity from this organ failed to respond to NFM, indicating that NFM may be mitogenic for T suppressor cells.     

Specific and Non-specific Activation of Human and Mouse Lymphocytes in Vitro by Pathogenic and Apathogenic Neisseria Strains

In vitro lymphocyte responses to gonococcal antigen (T2), apathogenic Neisseria pharyngis (APN), PPD and PHA were studied in patients with uroarthritis and in healthy controls. Only the T2 antigen induced a significantly higher DNA synthetic response in cells from patients than in controls, whereas no difference was observed with the other substances. Peak responses occurred on day 6 of culture with a concentration of 4 × 10⁷ bacteria/ml. In order to test whether lymphocyte responses were the result of specific sensitization, experiments were carried out with cord blood cells. These cells reponded to both T2 and APN with maximum activity on day 6 of culture. Since specific sensitization was ruled out in these experiments, we concluded that T2 and APN were capable of inducing a non-specific mitogenic response in cord blood cells. The mitogenic activity of T2 and APN was further elucidated in experiments with mouse lymphocytes. Both substances induced increased DNA synthesis in mouse spleen cells, with peak activity on days 2–3 of culture. Stimulation occurred in spleen cells depleted of macrophages, in anti-Thy 1.2 treated spleen cells and in Nude spleen cells, but not in thymus cell cultures. It was concluded that T2 and APN contained polyclonal B cell activating (PBA) substances, which was subsequently proved by their ability to induce a polyclonal antibody response in cell culture. The B cell subpopulation that responds to the mitogenic properties of these bacteria is normally absent from blood, present as a minor proportion of cord blood lymphocytes and present as a major cell population in spleen. Therefore, in spite of the mitogenic properties of these bacterial strains, they are useful tools for the elucidation of a specific reactivity in patients with uroarthritis.     

Mitogenic effect of beryllium sulfate on mouse B lymphocytes but not T lymphocytes in vitro

Beryllium metal and its salts can produce disease in man and in animal models. Beryllium disease is thought to involve cell-mediated immunity and an antigen-dependent response by beryllium-specific T cells. Beryllium salts have been shown to stimulate lymphocyte proliferation and release of lymphokines, and to induce granuloma formation and delayed-type hypersensitivity reactions in mice, guinea pigs and man. The studies described here were designed to test the hypothesis that a second lymphocyte population, B cells, may be responding nonspecifically to beryllium. Different populations of BDF1 mouse lymphocytes were cultured in the presence of varying concentrations of beryllium sulfate (BeSO4), and the increase in 125-iodouracildeoxyriboside uptake after 72 h in culture was determined. The data show that BeSO4 is weakly mitogenic for normal mouse spleen cells. Furthermore, BeSO4 is mitogenic for normal and nude mouse spleen B cells and not for spleen T cells or thymocytes in vitro. These findings suggest that BeSO4 can stimulate B cells nonspecifically, and support the hypothesis that polyclonal activation of B cells by beryllium may occur.     

Mitogenic activity of Mycoplasma pulmonis. I. Stimulation of rat B and T lymphocytes.

The mitogenic activity of Mycoplasma pulmonis towards both rat B and T lymphocytes has been demonstrated. The data summarized in this report show that spleen cells obtained from T X BM rats were extensively activated by M. pulmonis. Furthermore, M. pulmonis has been demonstrated to induce the development of antibody producing cells, as attested by the appearance of direct plaque forming cells against SRBC and TNP-SRBC in spleen cultures exposed to this mitogen. It was also demonstrated that rat thymus cells and a part of the lymph node T-cell population responding to either Con A or PWM, were stimulated by M. pulmonis, the response being weaker than that of rat B-cell populations. It was thus concluded that M. pulmonis activates both rat B and T lymphocytes. This mitogenic stimulation, however, is not equally exerted on both these populations, being strongly effective upon B cells and less so on T cells.     

Sendai virus glycoproteins are T cell-dependent B cell mitogens

UV-inactivated Sendai virus is mitogenic for murine splenocytes, whereas infectious Sendai virus kills spleen cells in vitro. The isolated hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins of Sendai virus are also mitogenic for cultured mouse spleen cells. A mixture of these glycoproteins (1 microgram/well) gives maximum stimulation 96 h after culture initiation. Viral proteins remaining insoluble after Triton X-100 extraction are also mitogenic for mouse spleen cells, with maximum stimulation occurring at 72 h after culture initiation with 1 to 5 microgram/well. On the basis of protein concentration, the HN and F glycoproteins are approximately three times more mitogenic than the Triton X-100-insoluble material. The mitogenic response of the HN and F glycoproteins has two components, a T cell-independent B cell proliferation, which is less than one-half of the total stimulation observed, and a T cell-dependent B cell proliferation. In contrast, the Triton X-100-insoluble material is a T cell-dependent B cell mitogen. Purified T lymphocytes do not respond to the mitogenic signal of either HN-F or Triton X-100-insoluble material.     

中药芦笋促T细胞有丝分裂活性的观察

低浓度芦笋原汁(0.1～1.0％)可明显促进正常小鼠胸腺细胞的增殖,也可刺激正常小鼠脾脏细胞的增殖,但对裸鼠脾脏细胞没有任何作用。由此提示,芦笋具有促T淋巴细胞有丝分裂的活性,而对B淋巴细胞没有作用。与常用的T细胞促有丝分裂素植物血凝素(PHA)及刀豆蛋白A(ConA)不同,芦笋原汁对人、绵羊、豚鼠和鸡的红细胞均无凝集作用。     

B-lymphocyte activation with an extract of Nocardia brasiliensis.

An extract from the pathogenic actinomycete Nocardia brasiliensis was mitogenic for murine lymphocytes. This deoxyribonucleic acid-synthetic response of whole spleen cells peaked after 48 h in culture at concentrations of Nocardia extract ranging from 10 to 200 micrograms/ml. The extract appeared to be a mitogen for B lymphocytes since cultures of spleen cells from congenitally athymic nude (nu/nu) mice and of antithymocyte serum plus complement-treated spleen cells from conventional (+/+) mice responded as well as untreated spleen cells from normal +/+ mice. Furthermore, thymocytes did not respond mitogenically to the extract. Mitogenic responses were stimulated in spleen cells from H-2(a), H-2(b), H-2(d), and H-2(k) mice, including lipopolysaccharide-nonresponder C3H/HeJ mice. This Nocardia extract also stimulated polyclonal B-cell activation to the hapten trinitrophenyl, serum protein human gamma globulin, and several mammalian erythrocytes in cultures of cells from both euthymic and nude mice. Additionally, the requirement for helper T cells in the primary in vitro immune response to sheep erythrocytes could be circumvented by the addition of this Nocardia extract. These results indicate that an extract from the pathogen N. brasiliensis can nonspecifically activate murine B lymphocytes and raise the possibility that polyclonal activation of B lymphocytes may contribute to the pathogenesis of nocardiosis.     

The 39-kilodalton outer membrane protein of Proteus mirabilis is an OmpA protein and mitogen for murine B lymphocytes.

Partial amino acid sequence analysis of a major outer membrane protein of Proteus mirabilis (39-kDa protein) indicates that it is an OmpA protein. The mitogenic activities of the 39-kDa protein for murine lymphocytes were also investigated with T lymphocytes isolated by passing spleen cells over columns of nylon wool fiber and B lymphocytes obtained by treating spleen cells with monoclonal antibodies to Thy1 plus complement. The 39-kDa protein showed little activity in stimulating T cells to proliferate but was strongly mitogenic for B cells.     

Immunocompetent Cells in Man. IV. The Inhibition of Pokeweed Mitogen-Induced Blastogenesis of Normal Human Peripheral Leukocytes by Trypsin: A B-Cell Mitogen of Mouse, but not of Human, Lymphocytes

Normal human peripheral blood lymphocytes were tested for their blastogenic response in vitro to trypsin. Although it has been shown by other investigators that mouse B lymphocytes are stimulated by trypsin to undergo blastogenesis, we have shown that human lymphocytes are not stimulated by trypsin. Paradoxically, trypsin inhibits the response of human lymphocytes to pokeweed mitogen, a B-cell stimulant, without significantly affecting the response of these cells to phytohemagglutinin, a T-cell stimulant. These results serve to underline the functional dissimilarity of mouse spleen cells and human circulating lymphocytes, at least with respect to their blastogenic responses to mitogenic agents.     

Circulating soluble immume complexes in recurrent oral ulceration and Beh?et's syndrome.

Cell wall fraction of Listeria monocytogenes (LCWF), a B cell mitogen for mouse spleen cells, is also mitogenic for human adult and cord peripheral blood lymphocytes. Purified B-cell suspensions responded to LCWF in vitro proliferation, to a similar extent as the unfractionated suspensions. Furthermore, LCWF-induced B cell differentiation into IgM-containing cells and their percentage correlated significantly with the extent of lymphocyte proliferation.     

Immunocompetent Cells in Man. IV. The Inhibition of Pokeweed Mitogen-Induced Blastogenesis of Normal Human Peripheral Leukocytes by Trypsin: A B-Cell Mitogen of Mouse,but not of Human,Lymphocytes

Normal human peripheral blood lymphocytes were tested for their blastogenic response in vitro to trypsin. Although it has been shown by other investigators that mouse B lymphocytes are stimulated by trypsin to undergo blastogenesis, we have shown that human lymphocytes are not stimulated by trypsin. Paradoxically, trypsin inhibits the response of human lymphocytes to pokeweed mitogen, a B-cell stimulant, without significantly affecting the response of these cells to phytohemagglutinin, a T-cell stimulant. These results serve to underline the functional dissimilarity of mouse spleen cells and human circulating lymphocytes, at least with respect to their blastogenic responses to mitogenic agents.     

Activation of murine B lymphocytes by Neisseria meningitidis and isolated meningococcal surface antigens.

Heat-killed Neisseria meningitidis was found to be a potent mitogen for mouse splenic lymphocytes. Results obtained with different cell separation techniques indicated that the bacteria acted to selectively induce proliferation of B lymphocytes. First, partial or total depletion of T lymphocytes by treatment with various anti-T-cell antisera plus complement did not affect the ability of the remaining spleen cells to proliferate in response to N. meningitidis. Second, T lymphocytes purified by affinity chromatography through an immunoglobulin-antiimmunoglobulin-coated glass bead column were unresponsive to meningococcal stimulation, even when provided with a source of macrophages (irradiated or mitomycin C-treated spleen cells). Finally, treatment of spleen cells with soy bean agglutinin showed that, whereas the soy bean agglutinin-positive population (B-enriched lymphocytes) was highly responsive to stimulation by N. meningitidis, the soy bean agglutinin-negative population (T-enriched lymphocytes) displayed only a background level of proliferation when exposed to the bacteria. Isolated meningococcal surface antigens such as lipopolysaccharide (LPS) and outer membranes also possessed mitogenic activity and induced proliferation of B lymphocytes in a dose-dependent manner. Both LPS and non-LPS components contributed to the mitogenicity of outer membranes since the addition of outer membrane preparations to spleen cells from the low LPS responder C3H/HeJ mouse strain gave rise to a high level of proliferative activity.     

Immunocompetent cells in man. IV. The inhibition of pokeweed mitogen-induced blastogenesis of normal human peripheral leukocytes by trypsin: a B-cell mitogen of mouse, but not of human, lymphocytes.

Normal human peripheral blood lymphocytes were tested for their blastogenic response in vitro to trypsin. Although it has been shown by other investigators that mouse B lymphocytes are stimulated trypsin to undergo blastogenesis, we have shown that human lymphocytes are not stimulated by trypsin. Paradoxically, trypsin inhibits the response of human lymphocytes to pokeweed mitogen, a B-cell stimulat, without significantly affecting the response of these cells to phytohemagglutinin, a T-cell stimulant. These results serve to underline the functional dissimilarity of mouse spleen cells and human circulating lymphocytes, at least with respect to their blastogenic responses to mitogenic agents.     

In Vitro and in Vivo Stimulation of Murine Lymphocytes by Human Respiratory Syncytial Virus Strains

Respiratory syncytial virus (RSV) strains of subtype A (A2, WV9894, and WV12138) and of subtype B (WV1293, WV4843, and WV6873) are mitogenic in vitro for unprimed BALB/c spleen cells. The virus also triggered splenocytes in vitro to secrete immunoglobulins. Plaque-purified and UV-irradiated materials of both RSV subtypes produced comparable levels of DNA synthesis. Infectious materials of both subtypes also induced pronounced responses. Lymphocyte activation with UV-inactivated RSV strain A2 was dose-dependent and maximal responses occurred after 4-5 days of incubation. The virus preparations were mitogenic for spleen cells depleted of T lymphocytes by treatment with anti-Thy 1.2 and complement and for lymphocytes of congenitally athymic mice (nu-nu). They were also mitogenic for highly purified T lymphocytes separated by panning of spleen cells on anti-mouse Ig-coated Petri dishes, suggesting that both B and T lymphocytes respond to the mitogenic activity of RSV. Moreover, mice infected intranasally with RSV strain A2 generated local as well as peripheral cellular and humoral responses.     

The mechanism of interaction between T and B lymphocytes in the in vitro response to sheep erythrocytes. Non-specific collaboration across a dialysis membrane.

Purified T lymphocytes from normal mouse spleen restored the antibody-forming cell response to sheep erythrocytes of various B lymphocyte populations when separated from them by a cell-impermeable dialysis or nucleopore membrane. A 2-5-fold increase occurred when spleen cells from neonatally thymectomized mice or congenitally athymic mice, and an adherent spleen cell population were used as sources of B cells. Antigen was required in both T- and B-cell compartments, but a nonspecific reconstitution occurred when the antigens present in the two compartments were different. It is concluded that during the first 20 hours of culture, T lymphocytes produce a non-specific factor in response to antigen. Although the factor acts at a distance, it does not have a non-specific mitogenic effect upon all B lymphocytes. Some of its properties are similar to those of other reported T-cell factors and of the 'sheep erythrocyte reconstitution factor', found naturally in some batches of foetal calf serum.     

Ontogeny of mitogenic responsiveness to PHA and sheep erythrocytes and lymphokine production in foetal guinea-pigs.

Pre-culture of spleen cells for 24 h before addition of activator greatly improved the mitogenic response to phytohaemagglutinin (PHA) and sheep erythrocytes (SRBC). Spleen cells were separated into plastic-adherent and non-adherent populations and in some experiments purified further to 'macrophages' and 'lymphocytes' respectively. A critical mixture of lymphocytes and macrophages (10:1) gave twice the mitogenic response of lymphocytes alone to PHA and SRBC in cultures of foetal, juvenile and adult guinea-pig spleen cells. Vigorous mitogenic responses to PHA and SRBC were found at 36 days of gestation. The mitogenic response increased from 46-56 days of gestation to above adult level. Lipopolysaccharide (LPS) obtained by the Westphal method was a superior mitogen to LPS extracted by the Boivin technique. Plaque-forming cells (PFC) could not be induced in vitro at any age even with the addition of LPS. Evidence was obtained for mitogenic and blastogenic lymphokines produced by lymphocytes activated with PHA or produced by lymphocytes activated with PHA or SRBC (but not LPS). These lymphokines were produced by activated splenic lymphocytes of a 40-day-old foetus; older animals showed no evidence for a quantitative increase in lymphokine production. The onset and maturation of mitogenic responsiveness in the guinea-pig and human foetus is compared by age-equivalence.     

Density gradient electrophoresis of mouse spleen lymphocytes. Differential mobilities of macrophages, responding and stimulating cells in mixed lymphocyte culture

Preparative density gradient electrophoresis has been employed for the separation of BALB/c mouse spleen T and B lymphocytes, on the basis of their surface charge. The high mobility cells were found to be predominantly T lymphocytes, whereas the low mobility cells were B lymphocytes. One way mixed lymphocyte cultures were prepared using electrophoretically separated BALB/c lymphocytes, either as responding or stimulating cells and unfractionated CBA/H/T6j mouse spleen lymphocytes, in the appropriate combination. The responding lymphocytes were found only among the high mobility cells which are primarily T lymphocytes. In contrast, the stimulating cells were found in the intermediate and low mobility fractions which contain the B lymphocytes and macrophages. Minimal stimulation in mixed lymphocyte culture was observed by T cells in this allogeneic system. Ia-positive cells were found only in the intermediate and low mobility fractions, which exhibited the capacity to stimulate allogeneic cells in MLC. Macrophages, identified by nonspecific esterase staining, exhibited intermediate electrophoretic mobility, their peak height coinciding with the maximum observed stimulation.