Further characterization of the autologous mixed lymphocyte reaction: induction of double negative gamma delta T lymphocytes.

To investigate the specific nature of the autologous mixed lymphocyte reaction (AMLR), we applied a method in which mixtures of NY-nonadherent responder cells and NY-adherent stimulator cells were treated with neuraminidase before culture and then cultured to assay the AMLR. This method produced a marked enhancement of DNA replication in the responder cells and the results were reproducible, regardless of the individuals tested. Using this method, we were able to make the following observations regarding the specific nature of the AMLR. (i) The AMLR is an IL-2-independent reaction, as revealed by bioassay to detect the presence of IL-2 by a blocking test using anti-IL-2R sera and as shown by the absence of mRNA for IL-2 in Northern hybridization. (ii) It is also HLA-DR dependent as proven by the fact that anti-DR sera almost completely inhibited the reaction. (iii) The AMLR was also found to induce the generation of activated CD4+ helper T cells in direct response to stimulation by NY-adherent cells, in which HLA-DR antigens were involved. (iv) Also, it induced the generation of CD4-CD8- double-negative (DN) lymphocytes, including gamma delta T cells with a cytotoxic activity against NK-resistant target cells and with a variety of lymphocyte activation markers (CD56, HLA-DR, CD25, transferrin receptors, CD38, and LFA-1). However, the AMLR did not induce the generation of NK cell markers CD16 and CD57. (v) The DN lymphocytes and gamma delta T cells appeared to be generated from the precursors of CD4-CD8- DN cells, in direct response to the stimulator cells. These results strongly suggest that the AMLR may be a phenomenon which induces the proliferative response of gamma delta T cells and their precursors, in addition to that of alpha beta T cells, particularly of CD4+ helper T cells.     

A critical role for Fc gamma RIIB in the induction of rheumatoid factors

Rheumatoid factors (RF) are autoantibodies with specificity for the Fc portion of IgG, and IgG-containing immune complexes are likely to be the major source of RF autoantigens. Therefore, the activation of RF-producing B cells could be controlled specifically through recognition of IgG immune complexes by the low-affinity IgG FcR, FcgammaRIIB, a potent negative regulator of the BCR. To test this possibility, we determined the development of RF in C57BL/6 (B6) mice lacking FcgammaRIIB, in relation to the H2 haplotype, complement C3, and the Y-linked autoimmune acceleration (Yaa) mutation. FcgammaRIIB-null B6 mice displayed substantial anti-IgG2a RF activities in their sera, in addition to anti-DNA autoantibodies. Their RF and anti-DNA responses were linked to the H2(b) haplotype, but were suppressed almost completely by the H2(d) haplotype. Strikingly, the absence of C3 failed to modulate RF production, but strongly inhibited anti-DNA production. Furthermore, we observed that partial FcgammaRIIB deficiency (i.e., heterozygous level of FcgammaRIIB expression) was sufficient to induce the production of RF and anti-DNA autoantibodies in the presence of the Yaa mutation. In contrast to FcgammaRIIB, the deficiency in another BCR negative regulator, CD22, was unable to promote RF and anti-DNA autoimmune responses in B6 mice. Our results indicate that RF autoimmune responses are critically controlled by FcgammaRIIB, together with the H2(b) and Yaa gene, while C3 regulates positively and specifically anti-DNA, but not RF autoimmune responses.     

Fate of surface immunoglobulin during induction of lymphocyte proliferation

The modulation of immunoglobulin on the surface of rabbit B lymphocytes by goat antibodies with specificity for rabbit surface membrane immunoglobulin or by such goat antibodies covalently linked to Sepharose was studied in relation to the proliferative response to these agents. Although the induction of DNA synthesis was greater in the presence of Sepharose-linked antibody than in the presence of free antibody, modulation of surface membrane immunoglobulin was induced with free but not with Sepharose-linked antibody. Thus, in the presence of free antibody the surface membrane immunoglobulin content of cells was rapidly decreased and remained at a low level throughout the culture period, whereas the surface immunoglobulin content of cells incubated with Sepharose antibody was essentially unaltered. The surface immunoglobulin lost from cells incubated with free goat antibodies reappeared slowly upon further incubation in culture medium devoid of antibody, and such reappearance of rabbit surface membrane immunoglobulin was inhibited by puromycin. Upon culture with Sepharose-linked antibody the surface membrane immunoglobulin content of B cells was unaffected by puromycin. This result was interpreted as indicating that surface membrane immunoglobulin loss followed by reappearance does not occur. Lastly, the linkage of surface membrane immunoglobulin to cytoskeletal elements induced by free antibody was not induced by Sepharose-linked antibody as judged from differences in detergent solubilization characteristics. Possible mechanisms to account for these differences in surface membrane immunoglobulin modulation as they relate to the proliferative response are considered.     

Continuous induction of unscheduled DNA synthesis by gamma irradiation

The induction of DNA-synthesis in non-S-phase cells is a very sensitive measure of a preceding damage of DNA. Usually, in an in vivo-in vitro test (treatment of an animal, incorporation of H3-thymidine in a cell suspension) the damaging of DNA takes place hours to days before the evaluation. In this case, the time course of the UDS-induction after a single dose of 1 Gy gamma irradiation was observed over a long period of time (21 months). C57 black mice served as test animals. In an age of about 80 days they were irradiated and the induction of unscheduled DNA synthesis was measured at ten time intervals during the whole life-span of the animals. Although the repair in this gamma radiation damage in DNA is a very quick process--with centrifugation in alkaline sucrose a half-life of some minutes is found--an induction of unscheduled DNA synthesis could be seen at the irradiated animals until the end of their life (640 days). The reason for this could be permanent disorders in cellular regulation caused by the gamma irradiation.     

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

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

Intestinal intraepithelial lymphocyte gamma delta-T cell-derived keratinocyte growth factor modulates epithelial growth in the mouse

Keratinocyte growth factor (KGF) promotes intestinal epithelial growth. To understand the relevance of intraepithelial lymphocyte (IEL)-derived KGF expression on epithelial growth, we used a mouse model of villus atrophy by the administration of total parenteral nutrition, and a model of villus hypertrophy by the creation of a short bowel syndrome. KGF expression was confined to gammadelta-TCR(+) IELs. IEL-derived KGF expression was highest in the crypts, somewhat less in the lower portion of villi, and markedly lower in the upper portion of villi. Total parenteral nutrition administration was associated with a down-regulation of IEL-derived KGF expression, and short bowel syndrome was associated with an up-regulation of IEL-derived KGF expression. In the absence of gammadelta-TCR(+) IEL, using gammadelta(-/-) mice, intestinal epithelial cell proliferation decreased in control, and in both mucosal atrophy (22% decline) and mucosal hypertrophy (14%) models. These results show that KGF from IELs is an important factor for maintenance of intestinal epithelial cell proliferation and villus growth.     

The effect of low exposure-rate gamma irradiation on T and B lymphocyte function in the mouse

The effect of chronic irradiation on T and B cell numbers and function was studied in mice. Cobalt 60 gamma radiation at 6 R/hour reduced the numbers of anti-SRBC PFC in the spleen, with minimal levels recorded after total exposures of 1000-2000 R. Recovery was incomplete after 1000 R, reaching only 40-50 per cent of normal in four months and remaining at that level for the animal's lifetime. The long-term deficiency in PFC formation was not due to a quantitative lack of T or B cells since normal cell numbers were observed in the spleen 60-144 days after 1000 R. Adoptive transfer studies with combinations of bone marrow and thymus cells, or of splenic T and B cells, from normal and irradiated mice, revealed functional defects in both cell compartments during the first two months. Normal and near normal function of T and B cells occurred 100 days postirradiation, a time when the splenic in vivo response was still only 50 per cent of the controls. The latter observation suggests that the microenvironment of the chronically irradiated spleen alters factors regulating T and B cell interactions in response to a T-dependent antigen.     

Aryl hydrocarbon hydroxylase induction in human lymphocyte cultures by 2,3,7,8-tetrachlorodibenzo-p-dioxin

Aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity is induced in cultured human lymphocytes by 2,3,7,8-tetrachlorodibenzo-$\text{p}$-dioxin (TCDD) at a concentration in the growth medium 40 to 60 times less than the concentration of 3-methylcholanthrene (MC) necessary for maximal hydroxylase induction. In cultured lymphocytes from 19 individuals, the extent of hydroxylase induction by TCDD or MC ranged between 1.7- and 2.9-fold. Those individuals having (presumably under genetic control) lower basal and MC-inducible hydroxylase activities in their lymphocytes also have lower TCDD-inducible hydroxylase activity. Because of the day-to-day experimental variability, the variations within each assay, and for several other reasons discussed, we suggest that the observed variance of expression of hydroxylase induction more closely fits a unimodal, polygenic ($\text{i.e.}$ 2 or more genes) pattern rather than the trimodal (single gene) form of inheritance proposed recently by Kellermann and coworkers.     

ROS induction and structural modification in human lymphocyte membrane under the influence of carbon nanotubes

To assess the potential risks of using the artificial nanostructures the structural state of the human lymphocyte membrane and lipid peroxidation under the influence of multi-walled carbon nanotubes with metal impurities was studied. The ability of carbon nanotubes to induce the formation of reactive oxygen species in cells was examined. A dose-dependent increase in reactive oxygen species formation in lymphocytes, which was not registered in cells pre-incubated with N-acetylcystein, after exposure to carbon nanotubes was shown. The addition of iron chelator deferoxamine to carbon nanotubes has also resulted in a decrease of reactive oxygen species. The mechanism of the activation of lipid peroxidation under the influence of carbon nanotubes and a structural modification of human lymphocyte membranes were discussed.     

IL-4 (B cell stimulatory factor 1) overcomes Fc gamma receptor-mediated inhibition of mouse B lymphocyte proliferation without affecting inhibition of c-myc mRNA induction

Mouse B cells are stimulated to proliferate by Fab'2 fragments of rabbit anti-mouse Ig antibodies. Proliferation is inhibited, however, in the presence of IgG anti-mouse Ig. We have previously shown that this inhibition is mediated by binding of the IgG anti-Ig to receptors for Fc gamma R on B cells. This report describes conditions under which IgG anti-mu or anti-delta will induce proliferation despite Fc gamma R engagement. Culture supernatants of Con A-stimulated, Il-4-secreting Th cell lines, but not of Il-2-secreting Th cell lines, will co-stimulate with IgG anti-Ig to induce small B cells to incorporate [3H]TdR. This co-mitogenic activity is inhibitable by anti-IL-4 antibodies and can also be induced by Il-4 affinity purified from the T cell supernatants or by supernatants containing rIl-4. B cells precultured with Il-4 for 18 h, while still expressing normal levels of Fc gamma R, also proliferate to IgG anti-Ig. We have previously shown that Fc gamma R-mIg cross-linking will inhibit mIg-dependent increases in c-myc mRNA levels. We investigated whether Il-4 allows B cells to respond to IgG anti-Ig by elevating c-myc. The data show that Il-4 has little effect on c-myc mRNA levels in either IgG or Fab'2 anti-Ig-containing cultures.     

ROS induction and structural modification in human lymphocyte membrane under the influence of carbon nanotubes

To assess the potential risks of using artificial nanostructures, the structural state of the human lymphocyte membrane and lipid peroxidation under the influence of multiwalled carbon nanotubes with metal impurities was studied. The ability of carbon nanotubes to induce the formation of reactive oxygen species in cells was examined. A dose-dependent increase in reactive oxygen species formation in lymphocytes, which was not registered in cells preincubated with N-acetylcysteine, after exposure to carbon nanotubes was shown. The addition of iron chelator deferoxamine to carbon nanotubes has also resulted in a decrease of reactive oxygen species. The mechanism of the activation of lipid peroxidation under the influence of carbon nanotubes and a structural modification of human lymphocyte membranes were discussed.     

Early changes in the lymphocyte surface membrane of rats after whole-body irradiation with neutrons and gamma rays

Biochemical changes in lymphocyte plasma membranes were studied 3 and 18 h after whole-body exposure of rats to neutrons and gamma-rays at doses from 2 to 6 Gy. It was shown that fast neutrons, with an average energy of 1.5-2.0 MeV, increased the rate of lipid peroxidation more markedly than gamma-rays did. In addition, there was an increase in the number of free aminogroups on the thymocyte surface. Dose- and time-dependent parameters of changes in the aminogroup content on the cellular surface were quantitatively different after the effect of radiation with different LET.     

Role of calcium in gamma interferon induction: inhibition by calcium entry blockers

Mitogen-induced gamma interferon production by human lymphoid cell cultures was studied in the presence of calcium entry blockers. A dose-dependent inhibition was found in the presence of drug concentrations down to 10(-5) M. This finding shows that calcium flow through lymphocyte membranes after oxidation of membrane-bound galactose residues is also critical for triggering interferon production.     

In vitro induction of lymphocyte responsiveness by a Strongylus vulgaris-derived mitogen

Proliferation in vitro of peripheral blood lymphocytes both from horses infected with Strongylus vulgaris and from helminth-free ponies was observed in the presence of extracts of the fourth and fifth stage larvae and adults of S. vulgaris. In addition, S. vulgaris extracts induced transformation in cultures of peripheral blood lymphocytes from sheep and dogs and in mouse spleen cell cultures. Nylon wool non-adherent, T cell enriched fractions of lymphocytes from both mice and horses were stimulated by the S. vulgaris larval mitogen while no proliferation was observed in cultures containing nylon wool adherent, B cell enriched fractions. Macrophage co-operation appeared not to be necessary for S. vulgaris mitogen-induced transformation of spleen cells. The S. vulgaris mitogen stimulated a subpopulation of mouse spleen cells different from those responsive to PHA, Con A and LPS. These cells might be T helper cells since B cells were stimulated to proliferate in the presence of both T cells and S. vulgaris larval mitogen. In addition, the supernatant of in vitro cultured larvae of S. vulgaris induced slight, but significant transformation of equine peripheral blood lymphocytes. Therefore, it is possible that the S. vulgaris mitogen released by both viable parasites and degenerating larvae might induce T cell dependent production of immunoglobulin in vivo and account for the beta-globulinaemia, of which IgG(T) is a major component, in S vulgaris infected horses.     

Immunological induction of T lymphocytes: role of antigen and the lymphocyte costimulator.

In vitro T cell activation requires both antigen presentation and a second stimulus provided by the lymphocyte costimulator. Neither alone is sufficient to induce specific T cell activation. The S+ phenotype of stimulating cells is dependent on the metabolic activity of these cells. This finding is consistent with the notion that production and/or release of the costimulator is a function of metabolically active cells. The costimulator acts at an early stage of the interaction between lymphocyte and antigen, and the costimulator, or a separate maintenance factor, is required throughout the culture period for the expression of full cytotoxic activity. The lymphocyte costimulator is not strain specific but is phylogenetically specific. The activation of cytotoxic T cells by S+ cells is also phylogenetically specific, and this specificity of cellular activation can be accounted for by the species specificity of the lymphocyte costimulator.