Th1 response in Salmonella typhimurium-infected mice with a high or low rate of bacterial clearance.

Previous studies have shown that the capacity to clear an attenuated strain of Salmonella typhimurium after the second week of infection varies widely among mouse strains. Bacterial clearance is mediated by CD4+ T cells and is regulated in part by the H-2 complex. The aim of the present study was to compare the patterns of cytokine mRNA expression in the spleens of C57BL/6 (H-2b) and CBA (H-2k) mice, which exhibit a low and a high rate of bacterial clearance, respectively. A transient increase in interleukin-12 (IL-12) mRNA levels was found in both mouse strains. Gamma interferon (IFN-gamma) gene expression was higher and more sustained in C57BL/6 than in CBA mice. No increase in IL-4 mRNA was detected. A transient increase in IL-10 mRNA was found in C57BL/6 mice. Separation of spleen cells into CD4+ and CD4- fractions showed that CD4+ T cells produced the bulk of IFN-gamma in both mouse strains and of IL-10 in C57BL/6 mice. Infection of H-2 congenic mice induced a higher level of IFN-gamma mRNA expression by CD4+ T cells in mice with a low rate of clearance (H-2b) than in mice with a high rate of clearance (H-2q). Treatment of infected C57BL/6 mice with anti-IFN-gamma or anti-CD4 monoclonal antibodies indicated that IFN-gamma participates in resistance in the early phase of infection, but not in bacterial clearance, and that CD4+ T cells mediate bacterial clearance during the 3rd week of infection. Taken together, these results suggest that defective bacterial clearance in H-2b mice is not linked to defective IFN-gamma production and that CD4+ T cells mediate bacterial clearance by an IFN-gamma-independent mechanism.     

Polyclonal activation of human peripheral blood B lymphocytes: III. Cellular interaction and immunoregulation of immunoglobulin-secreting cells induced by formaldehyde-fixed Salmonella paratyphi B

We studied the interactions between regulatory cells and B cells in cultures of peripheral blood leukocytes (PBL) stimulated with various polyclonal B-cell activators, i.e., pokeweed mitogen (PWM), Staphylococuss aureus Cowan I (SAC), and Salmonella paratyphi B (SPB). Polyclonal B-cell activation in cultures was evaluated by enumeration of cells secreting immunoglobulin (Ig) and/or by quantitation of Ig released into culture supernates. Cord blood T cells and PBL after activation with concanavalin A or by prolonged incubation were used as sources of suppressor T cells. In coculture experiments, SPB-induced generation of Ig-secreting cells (ISC) was not affected, whereas PWM-induced generation of ISC was inhibited by suppressor T cells. The effects of suppressor T cells on SAC-induced generation of ISC was variable. Monocytes were required for PWM-induced generation of ISC; in contrast, SPB- and SAC-induced B-cell activation were not dependent on monocytes. Moreover, SPB-induced B-cell activation appeared to be susceptible to monocyte-mediated suppression. These results indicate that monocyte-mediated suppression is exerted directly on B cells rather than indirectly via suppressor T cells.     

Role of H-2 and non-H-2 genes in control of bacterial clearance from the spleen in Salmonella typhimurium-infected mice.

The ability of mice to clear Salmonella typhimurium from their spleens in the late phase of infection was studied after inoculation with a temperature-sensitive mutant. Clearance of bacteria was delayed in C57BL/6 mice compared with BALB/c, C3H/HeJ, DBA/2, A/J, and CBA mice. The responses of F1 hybrids, backcrosses, and recombinant inbred strains derived from C57BL/6 and BALB/c (both Itys) and of H-2 congenic mice were analyzed. The results showed that the low rate of bacterial clearance was recessive, that the rate of clearance was under polygenic control, and that an H-2-linked gene(s) plays a major role. Among H-2 congenic mice with a C57BL/10 background, three phenotypes of bacterial clearance could be distinguished: high (H-2j, H-2q, and H-2u), intermediate (H-2d, H-2f, H-2k, H-2p, H-2r, H-2s, and H-2v), and low (H-2b) rates. The effect of the H-2 complex was apparent with different genetic backgrounds (Itys and Ityr). In recombinant inbred strains derived from C57BL/6 (Itys) and A/J (Ityr) mice, the effect of the H-2b haplotype on bacterial clearance appeared to be fully expressed only in strains carrying the Itys allele.     

Fc receptors on human blood B lymphocytes.

The frequency of Fc-receptor positive B lymphocytes in human blood was investigated. Under the conditions used heat-aggregated gammaglobulin binding and EA(ox)-rosette formation labelled the same lymphocyte populations. Using various techniques, double marking and cell separations the proportion of Fc-receptor positive cells within the surface Ig carrying population was estimated to be between 11-8 and 36-2%. The proportion of SIg carrying cells within the population forming EA-rosettes was between 11 and 26-4%. This represents extreme values due to known technical circumstances.     

Complement receptors and B lymphocytes

Most of the biological processes depend on cell-to-cell and protein-to-cell interactions, which take place through receptors present on the cell surface. Various physiological systems are linked by such interactions, as is the case for innate and adaptative immune response. There is increasing evidence that two of the main actors involved in host defense, namely, the proteins of the complement system (nonspecific response) and the B lymphocytes (specific response), are strongly connected through the complement receptors displayed on the B-cell surface. Many parameters account for the importance of these molecules: (1) their diversity in terms of binding specificity allows them to interact with different fragments resulting from complement activation and C3 component proteolysis; (2) the structures of their extra- and intracytoplasmic domains differ from one receptor to another, controlling their interactions with other nall surface molecules as well as pathogens and regulating cell signaling; (3) their expression on the majority of the cells involved in immune response, especially B lymphocytes, make them an essential link between specific and nonspecific immune responses. This review deals with these different aspects, taking into account the most recent data.     

Localization of dysfunctional tight junctions in Salmonella enterica serovar typhimurium-infected epithelial layers

Infection of polarized MDCK epithelial layers by Salmonella enterica serovar Typhimurium is accompanied by increased tight junction permeability and by contraction of perijunctional actinomyosin. We localized dysfunctional tight junctions in serovar Typhimurium-infected MDCK layers by imaging apical-basolateral intramembrane diffusion of fluorescent lipid and found that loss of the apical-basolateral diffusion barrier (tight junction fence function) was most marked in areas of prominent perijunctional contraction. The protein kinase inhibitor staurosporine prevented perijunctional contraction but did not reverse the effects of serovar Typhimurium on tight junction barrier function. Hence, perijunctional contraction is not required for Salmonella-induced tight junction dysfunction and this epithelial response to infection may be multifactorial.     

Isolated hapten-binding receptors of sensitized lymphocytes. V. Cellular origin of receptor molecules

Chimeric mice were constructed by injection of thymus cells into nu/nu mice. The thymus cells carried a different immunoglobulin (Ig) heavy chain linkage group than the cells of the recipients. The chimeric animals were then immunized with the hapten (4-hydroxy-3-nitro-phenyl)acetyl (NP) on an appropriate carrier, and NP-binding receptor molecules were isolated from the spleen cells of the animals. Receptor molecules and antibodies were analyzed for the presence of two Ig heavy chain variable region (V_H) markers which are specific for the anti-NP response of mice carrying the Ig^b allogroup, namely the NP^b idiotpye and the heteroclitic fine specificity of hapten binding. Two types of receptor molecules are obtained by our method, those carrying determinants of Ig constant domains (anti-Ig⁺ receptors) and others lacking such determinants (anti-Ig^? receptors). The two V_H region markers were found on anti-Ig^? receptors but not on anti-Ig⁺ receptors and humoral antibodies when the T cells in the chimeric mice carried the Ig^b allogroup, whereas the B cells were Ig^a/a. Conversely, anti-Ig⁺ receptors and humoral antibodies but not anti-Ig^?receptors, expressed the two markers when the B cells of the chimeras carried the Ig^band the T cells the Ig^a allogroup. From these results and previous data showing that anti-Ig^? receptors are enriched together with T lymphocytes and anti-Ig⁺ receptors together with B cells, we conclude that the V_H-bearing anti-Ig^? receptors are not only present on T cells, but are indeed synthesized by these cells.     

The radiosensitivity of T and B lymphocytes in mice.

The radiosensitivity of T and B lymphocytes in spleens of specific pathogen-free C3Hf/HeMs male mice was studied by the direct and indirect immunofluorescence technique. It was found that the radiobiological parameters characterizing the survival curve of Bpsi lymphocytes were DO = 200 R and n = 1-00. The T lymphocytes, on the other hand, were shown to consist of two distinct subpopulations with respect to their radiosensitivity. The radiobiological parameters of the radiosensitive fraction of T lymphocytes were Dq = 185 R, DO =195 R and n = 2-50. The DO value of the radioresistant T lymphocyte subpopulation was practically unmeasurable. It was estimated that approximately 8 per cent of the T lymphocytes present in the spleen of normal C3Hf mice belonged to this radioresistant subpopulation.     

T and B lymphocytes in pituitary dwarf Snell-Bagg mice.

The lymphocyte composition of the thymus and spleen from weaned (4 month old) hypopituitary dwarf Snell-Bagg mice were compared to those of their phenotypically normal littermates and of hormone (somatotropic hormone plus thyroxine)-treated individuals. Detection of cells bearing receptors for peanut agglutinin, physical analysis and measurement of in vitro reactivities to phytohaemagglutinin and concanavae intra-thymic lymphocyte population of dwarf mice. Examination of spleen-cell suspensions demonstrated a slightly higher frequency of T lymphocytes (Thy 1-2+ alpha-Naphthyl esterase+, high electrophoretic mobility) and lower frequency of B lymphocytes (surface immunoglobulin+, low electrophoretic mobility) in dwarf mice than in control mice. The degree of splenocyte responsiveness to T- and B-cell mitogens, however was similar in the two mouse types. High mobility (T) splenic cells were found to exhibit a smaller modal volume in dwarf mice (110 micron3) than in control mice (122 micron3) but this difference was not corrected by hormone administration. More pronounced were the quantitative differences between the spleens of hormone-deficient and normal mice. Thus, when expressed as a function of body weight, the numbers of splenic T and B lymphocytes in untreated dwarf mice were about half the corresponding values in hormone-reconstituted or normal littermates. These data suggested that in adult life, developmental hormones exert little direct effect on the thymus lymphocytes but influence the size of the pool of both peripheral T and B lymphocytes.     

Demonstration of Fc receptors on the surface of B lymphocytes.

Human blood lymphocytes were tested by an immunofluorescence technique for surface immunoglobulin and by a rosette test with IgG-sensitised red cells for Fc receptors. From combined tests and experiments involving fractionation of lymphocyte populations it is concluded that Fc receptors are demonstrable on most B lymphocytes by the rosette test, but only if the red cells are optimally sensitised. These observations are advanced as an explanation of the discrepant results of surface marker tests on B cells.     

Antigen activation of human B lymphocytes bearing artificial antigen receptors.

When highly purified human and murine B cells are challenged in vitro with certain so called "T cell-independent" activators such as the polyclonal B cell activator lipopolysaccharide (LPS) or the clonally specific B cell activator dinitrophenyl-conjugated polymerized flagellin (DNP-POL), mouse, but not human, cells differentiate into immunoglobulin-secreting cells. However, results from this study show that DNP-POL can cause human B cell differentiation in a T cell-independent manner when the antigen is concentrated onto the cells via artificially incorporated palmitate-modified anti-DNP mouse IgA molecules. This response is comparable in magnitude to that induced by a T cell-dependent polyclonal B cell activator, pokeweed mitogen, in unfractionated mononuclear cell cultures, suggesting that DNP-POL induced polyclonal B cell differentiation. DNP-POL binding to the artificial receptor molecules on B cells did not cause cellular proliferation, even in unfractionated mononuclear cell populations. These results are similar to those obtained in previous studies using mouse B cells in which the artificial receptor was unable to act as a transmembrane signaling element. From these studies, we conclude that B cells express clonally unrestricted, presumably low-avidity, endogenous receptor for POL, and that signaling through this receptor activates B cell differentiation but not cell proliferation.     

Cellular cooperation in lymphocyte activation. II. Cooperative response of human peripheral T and B lymphocytes to rabbit anti-human beta 2-microglobulin.

In the present study we attempted to clarify the effects of anti-beta 2-microglobulin (a-beta 2m) on lymphocyte activation. Neither a-beta 2m IgG fraction nor F(ab')2 had a mitogenic effect on either highly purified T or B lymphocytes alone, while their mitogenic effect was observed when T and B lymphocytes were appropriately reconstituted. When T lymphocytes were reconstituted with mitomycin C (MMC) treated B lymphocytes, a negligible decrease in the response to a-beta 2m was observed compared to the response of an untreated mixture to a-beta 2m. On the other hand, when B lymphocytes were reconstituted with MMC-treated T lymphocytes, the response was markedly diminished. It was found, moreover, that the response of T lymphocytes separated by a semipermeable membrane from MMC-treated B lymphocytes was not enhanced, while a mixture of T and MMC-treated B lymphocytes in the same chamber showed a marked response. These results lead to the conclusion that the cells responding to a-beta 2m are mainly T lymphocytes whose response is strongly enhanced by B lymphocytes, and that for the mitogenic effect of a-beta 2m direct cell-to-cell interaction between T and B lymphocytes is necessary.     

Subpopulations of mouse spleen lymphocytes. III. Cellular interactions in the response to concanavalin A.

The profile of response to concanavalin A (con A) of purified mouse T cells was found to differ appreciably from that of non-fractionated spleen cells, in agreement with results previously published by other investigators. Experiments designed to elucidate the reasons underlying these differences have revealed that the response of the spleen cells to con A is determined by a complex interplay between several cell types. (a) B cells contribute to the overall incorporation of thymidine in the presence of con A-stimulated T cells. However, the B cells participate in the response only if the T cells are dividing. (b) A population of 'adherent cells' is present in the spleen, which enhances the stimulation of the spleen cells by low doses of con A but suppresses the response to high doses of mitogen. These adherent cells include most likely the conventional macrophages, but probably also a population of 'suppressor T cells'. (c) Such 'suppressor T' cells can be readily detected among the peritoneal exudate cells. Addition of the exudate cells to cultures of purified T cells enhances the response to low doses of con A. This effect can be further increased by treating the peritoneal cells with a cell T-specific antiserum and complement, i.e. by eliminating the T cells.     

Cellular immunity in pregnancy: subpopulations of T lymphocytes bearing Fc receptors for IgG and IgM in pregnant women.

Studies on the change of peripheral T and B lymphocytes and T cells bearing Fc receptors for IgG and IgM in pregnant women were performed by using rosette-formation tests. There was no significant difference in the proportion of T and B lymphocytes between pregnant and non-pregnant women. The percentage of T cells bearing Fc receptors for IgG in the T lymphocytes which are considered to have suppressive activity increased in the various stages of pregnancy and post-partum as compared with that in non-pregnant women. On the contrary, the percentage of T cells bearing Fc receptors for IgM in the T lymphocytes which have a helper function decreased in pregnant and post-partum women. The results of this investigation suggest that the depression of cell-mediated immunity during pregnancy depends on the qualitative change of T lymphocytes, i.e. increased suppressor and decreased helper T lymphocyte subpopulations.     

Cellular DNA content of T helper, T suppressor and B lymphocytes in SLE.

The ratio of in vivo activated T helper, T suppressor and B cells in the blood of patients with SLE has been studied through simultaneous subset specific immunofluorescence labelling and the analysis of cellular DNA content with the help of flow fluorimetry. In the active stage of the disease an increase was observed in the proportion of proliferating cells either among the T helper cells or the B cells. Occasionally, however, the rate of proliferation in both subsets grew at the same time. Cases with a high number of activated B cells have proved to be more serious. The percentage of the T suppressor cells in the S-G2-M phase of the cell cycle increased moderately in both the active and the inactive stages of the disease. Our results are also discussed in relation to the pathogenetic mechanism of SLE.     

Cellular mechanisms in the immune response to malaria in Plasmodium vinckei-infected mice.

Infection of mice with the malaria parasite Plasmodium vinckei vinckei is 100% lethal. However, after two infections followed by drug cure, BALB/c mice develop a solid immunity which is antibody independent but mediated by CD4+ T cells. To elucidate the mechanisms of this immunity, spleen cells from immune mice were challenged in vitro with lysates of P. vinckei-infected or uninfected erythrocytes. The parasite antigen induced proliferation of T cells from immune mice but not from nonimmune mice. When gamma interferon production by cells from immune mice was assayed at the single-cell level, 1 to 3 cells per 1,000 cells were found to release this cytokine when exposed to antigen. In contrast, the numbers of interleukin 4 (IL-4)-producing cells from both immune and control mice were < or = 4 per 10(6) cells, regardless of antigen exposure. Investigation in a bioassay showed that P. vinckei antigen induced the release of IL-4 from spleen cells of immune mice but not from those of control mice. Nevertheless, that IL-4 is of minor significance in this system is also suggested by the absence of elevation of immunoglobulin E levels in blood samples from these mice, in contrast to what is seen with P. chabaudi infection, in which IL-4-producing Th2 cells are of major importance for immunity during later phases of infection. Taken together, the present results indicate that immunity to P. vinckei is a Th1 response, with gamma interferon being an important protective factor. Whether or not the Th1 response, through overproduction of tumor necrosis factor alpha, is also responsible for pathology and death in this infection remains to be clarified.