Differential ultraviolet-B-induced immunomodulation in XPA, XPC, and CSB DNA repair-deficient mice.

Ultraviolet B irradiation has serious consequences for cellular immunity and can suppress the rejection of skin tumors and the resistance to infectious diseases. DNA damage plays a crucial role in these immunomodulatory effects of ultraviolet B, as impaired repair of ultraviolet-B-induced DNA damage has been shown to cause suppression of cellular immunity. Ultraviolet-B-induced DNA damage is repaired by the nucleotide excision repair mechanism very efficiently. Nucleotide excision repair comprises two subpathways: transcription-coupled and global genome repair. In this study the immunologic consequences of specific nucleotide excision repair defects in three mouse models, XPA, XPC, and CSB mutant mice, were investigated. XPA mice carry a total nucleotide excision repair defect, whereas XPC and CSB mice only lack global genome and transcription-coupled nucleotide excision repair, respectively. Our data demonstrate that cellular immune parameters in XPA, XPC, and CSB mice are normal compared with their wild-type (control) littermates. This may indicate that the reported altered cellular responses in xeroderma pigmentosum patients are not constitutive but could be due to external factors, such as ultraviolet B. Upon exposure to ultraviolet B, only XPA mice are very sensitive to ultraviolet-B-induced inhibition of Th1-mediated contact hypersensitivity responses and interferon-gamma production in skin draining lymph nodes. Lipopolysaccharide-stimulated tumor necrosis factor alpha and interleukin-10 production are significantly augmented in both XPA and CSB mice after ultraviolet B exposure. Lymph node cell numbers were increased very significantly in XPA, mildly increased in CSB, and not in XPC mice. In general XPC mice do not exhibit any indication of enhanced ultraviolet B susceptibility with regard to the immune parameters analyzed. These data suggest that both global genome repair and transcription-coupled repair are needed to prevent immunomodulation by ultraviolet B, whereas transcription-coupled repair is the major DNA repair subpathway of nucleotide excision repair that prevents the acute ultraviolet-B-induced effects such as erythema.

UVB irradiation modulates systemic immune responses by affecting cytokine production of antigen-presenting cells.

The immunosuppressive effects of UVB irradiation have been well documented. The production of cytokines by keratinocytes is considered to play a major role in the induction of local as well as systemic immunosuppression. It is thought that partly due to the interaction of locally produced cytokines with antigen-presenting cells (APC) systemic effects, like antigen-specific tolerance, can be induced. In this study we examined the effect of UVB irradiation on cytokine profiles of peripheral APC as well as the functional consequences. Our results indicate that UVB irradiation impairs T(h)1-mediated immune responses in vivo by suppression of the systemic IL-12p70 production. Splenic APC from UVB-exposed mice showed an enhanced production of prostaglandin E(2), IL-1, IL-6 and tumor necrosis factor-alpha after in vitro stimulation. Also, spleen cells from UVB irradiated IL-4(-/-) mice showed increased IL-6 levels. These APC were less efficient in inducing IFN-gamma production by CD4(+) T cells and suppressed IgM production by B cells. We conclude that the altered cytokine profile of peripheral APC can be responsible for the systemic effects of UVB irradiation on the T(h)1/T(h)2 balance as well as on B cell responses.

Activation and re-activation potential of T cells responding to staphylococcal enterotoxin B.

To elucidate the parameters that lead to superantigen induced non-responsiveness, an in vitro model for studying primary and secondary responses to the bacterial superantigen staphylococcal enterotoxin B (SEB) was established. Upon re-activation with SEB, in vitro SEB primed T cells show an early proliferative response that 'quenches' in time and is severely impaired 3 days after re-stimulation. Despite their overall impaired proliferative capacity and IL-2 production, these T cells are able to produce IFN-gamma and to up-regulate activation markers CD69 and IL-2R alpha upon re-stimulation with SEB, demonstrating that SEB non-responsiveness is not absolute. Rather, it reflects the inability to mount an ongoing proliferative response upon re-stimulation with SEB. Our results also demonstrate that SEB-induced non-responsiveness is not simply the result of presentation in the absence of co-stimulation, since presentation of SEB on highly purified dendritic cells during the primary response did not prevent the induction of non-responsiveness. As previously shown, SEB induces a Th1 phenotype in responding CD4+ T cells. Skewing towards a Th2 phenotype by adding IL-4 and antibodies to IFN-gamma did not prevent the induction of non-responsiveness by SEB. Interestingly, T cells pretreated with plate-bound anti-CD3 epsilon and anti-V beta 8 were also non-responsive to SEB re-stimulation. Thus, non-responsiveness to SEB (defined here as inability to produce IL-2 and proliferate) seems to reflect an intrinsic inability of previously activated T cells to respond to SEB, probably reflecting differences in signal transduction pathways used by naive versus previously activated T cells.

Modulation of systemic cytokine levels by implantation of alginate encapsulated cells.

The availability of cell lines that are transfected with IL-4, IL-5 and IFN-gamma cytokine genes permits the prolonged in vivo delivery of functional cytokines in relatively large doses for the modulation of specific immune responses. Often the transfected cells are xenogeneic or allogeneic to the experimental animal and have to be encapsulated in such a way that no cellular response by the host will be induced. Alginate has proven to be a simple matrix for encapsulating cells under mild conditions suitable for in vivo implantation. Encapsulated cells express the transfected IL-4 gene for at least 14 days after in vivo implantation and were shown to be functional during that period by modulating ongoing IgE responses. The application of adherent growing transfected cells permits dose-response titrations and provides an easy method for local and systemic cytokine delivery. Alternatively, hybridoma cells can be encapsulated and the secreted antibody monitored in the serum. It was found that no host immune response was triggered by alginate encapsulated cells. The efficiency of treatment by encapsulated hybridoma cells was shown to be equivalent to that of injecting purified antibodies.

Cytokine Detection and Modulation in Acute Graft vs. Host Disease in Mice.

A murine model for acute lethal graft vs. host disease (GVHD) was used to study the role that a number of cytokines play in the development of lethal GVHD. In this study we focused on the role of IL-1, IL-2, IL-4, IL-6, IFN-gamma and TNF-alpha. Lethally irradiated (C57BL x CBA)F1 mice were reconstituted either with 10(7) allogeneic BALB/c spleen cells or with a similar number of syngeneic cells, as a control. A significant rise in serum levels of IL-6, TNF-alpha and IFN-gamma levels was found in allogeneically reconstituted mice. This is in contrast to the syngeneic control group in which no rise was seen. Serum IL-2 and IL-4 levels were below the detection limit. In the supernatant of Con A stimulated spleen cells from allogeneically reconstituted mice IL-6, IFN-gamma and TNF-alpha concentrations were increased. The expression of mRNA for cytokines as detected by reverse transcription PCR was studied in spleen cells. In the allogeneic reconstituted mice the mRNA expression of IL-1alpha, IL-2, IL-6, IFN-gamma and TNF-alpha displayed faster kinetics compared with that in syngeneic reconstituted mice. The effect of treatment with recombinant cytokines, antibodies to cytokines and to cytokine receptors on the development of GVHD was investigated. Administration of recombinant IL-2 to allogeneically reconstituted mice strongly increased the morbidity and mortality whereas injection of IL-1alpha and TNF-alpha did not influence survival. Administration of antibodies against IL-2 or the IL-2 receptor decreased the morbidity and mortality. Anti-IL-6, anti-IFN-gamma, and anti-TNF-alpha mAB, on the other hand, did not affect the morbidity and mortality of GVHD. The results of this study suggest successive waves of cytokine-secreting cell populations consistent with the induction of an inflammatory response in the development of acute GVH disease.

The Effect of IFN-gamma, Alum and Complete Freund Adjuvant on TNP-KLH Induced Ig.G(1), IgE and IgG(2a) Responses in Mice.

Adjuvants are considered to play an important role in directing the isotype and amount of antibodies produced upon immunization by conducting the development of either Th-1 or Th-2 cells upon T-cell stimulation. This is based on the different cytokine production patterns that were observed after in vitro resttmulation of T cells isolated from mice immunized with antigen either adsorbed on alum or emulsified in complete Freund adjuvant (CFA). However, other studies suggest that primarily the type of antigen determines which isotypes are produced and to what extent. In these studies, however, IgE was not determined. Therefore, this study examined whether alum and CFA influenced the amount and/or ratio of IgG(1), IgE and IgG(2a) produced after TNP-KLH immunization. Similar levels of IgG(1), IgE and IgG(2a) antibodies were found upon immunization with TNP-KLH either adsorbed on alum or emulsified in CFA. Moreover, administration of IFN-gamma in combination with TNP-KLH adsorbed on alum did not increase the amount of IgG(2a) produced. IFN-gamma treatment resulted in an increased IL-6 and decreased IFN-gamma production by spleen cells upon Con A stimulation, whereas it did not change the IL-4 production in similar conditions. The presented results suggest that upon immunization with TNP-KLH high IL-4 levels are produced, resulting in an antibody response that is dominated by IgG(1), independent of the adjuvant employed. The IL-4 inducing property of TNP-KLH is substantiated by the finding that repeated immunization of mice with TNP-KI, without adjuvant, increases the serum total IgE level. The presented data suggest that the carrier part of TNP-KLH preferentially results in Th-2 cell activity after which the adjuvant merely enhances the antibody responses generated.

Cytokines in lethal graft-versus-host disease.

Graft-versus-host disease (GVHD) is caused by donor T lymphocytes that recognize foreign antigens on host tissues. This leads to T cell activation, which involves a cascade of events including the transcription of genes for cytokines and their receptors and the production of cytokines. One of the first cytokines to appear is interleukin 2 (IL-2). IL-2 production enhances the IL-2 receptor expression and leads to T cell proliferation. As a further step, differentation of T cells occurs, which results in the production of a certain pattern of cytokines. These cytokines influence the expression of cell surface antigens and adhesion molecules, and are able to activate other cell types such as cytotoxic T cells, macrophages and natural killer cells, which might act as effector cells in tissue destruction. Insight into the sequential expression of the various cytokines involved might enable a more effective treatment of GVHD. Therefore, we investigated the occurrence of cytokines in a murine model for acute GVHD. We addressed in particular the period early after allogeneic reconstitution.

In vivo kinetics and characterization of IFN-gamma-producing cells during a thymus-independent immune response.

Using immunohistochemical techniques, we studied IFN-gamma-producing cells (IFN-gamma-PC) in vivo during immune responses to thymus-independent type-2 (TI-2) Ag. Detection of IFN-gamma-PC in cryostat sections of spleen-tissue was performed with an enzyme labeled mAb directed against IFN-gamma. After TNP-Ficoll immunization, IFN-gamma-PC and TNP-specific antibody-forming cells (TNP-AFC) displayed similar kinetics reaching a maximum number at day 5 to 7. The IFN-gamma-PC were localized in the same compartment as TNP-AFC and a part of them in juxtaposition to TNP-AFC. Immunization with other TI-2 Ag resulted also in a significant increase of the number of IFN-gamma-PC. In a parallel experiment we found both in vivo and in an ELISA-spot assay a significant increase of the number of IFN-gamma-PC and IFN-gamma-spot-forming-cells, respectively, in spleens of mice 6 to 7 days after TNP-Ficoll immunization. Double staining of spleen sections for IFN-gamma and surface Ag revealed that 5 to 7 days after TNP-Ficoll immunization, +/- 40% of the IFN-gamma-PC expressed the MT4 Ag (CD4), +/- 50% the Lyt-2+ Ag (CD8) and +/- 10% the asialo-GM1 Ag (NK cell). This study represents the first description of the in vivo activity and characterization of IFN-gamma-PC during a TI-2 immune response. Moreover, the presented data confirm suggestions from in vitro investigations that IFN-gamma and T cells may play a direct role in the in vivo regulation of a primary immune response against a TI-2 Ag.

Modulation of total IgE levels in serum of normal and athymic nude BALB/c mice by T cells and exogenous antigenic stimulation.

Several different grades of T-system impairment were studied for their effects on the total serum IgE concentration in BALB/c mice. Homozygous athymic nu/nu mice and their heterozygous nu/+ littermates were compared for serum IgE levels while kept under either barrier-maintained or conventional conditions. The results show a paradox between the T-cell dependency of the IgE immune response and the increased levels of serum IgE in the absence of T cells. Both barrier-maintained and conventionalized nu/nu mice have at least twofold increased serum IgE levels as compared to nu/+ mice. With age, IgE levels increased faster and reached higher plateau values in nu/nu than nu/+ mice. Moreover, after adult thymectomy of BALB/c mice the serum IgE levels increased up to 15-fold at 4 months of age, while infusion of immunocompetent T cells in nude mice resulted in a 2- to 5-fold decrease of the IgE level.

Lipopolysaccharide-induced suppression of graft-versus-host reactivity in mice.

Reconstitution of lethally irradiated mice with spleen cells from donors that had been treated with lipopolysaccharide (LPS) intravenously and allogeneic spleen cells subcutaneously leads to a suppressed anti-host delayed-type hypersensitivity (DTH). Either donor injection alone proved to be ineffective. The state of suppression appeared to be antigen-specific, but, depending on the experimental conditions, also anti-host DTH to third-party alloantigens could be suppressed. The suppression was mediated by a population of Thy-1- suppressor cells that could also be induced in athymic nude mice. The suppressor cells specifically adhered to anti-kappa-coated plastic plates, but were not adsorbed by passage through a Sephadex G-10 column. Thus, it appears that the combined donor treatment with LPS and allogeneic spleen cells induces a population of B cells that can suppress anti-host immune reactivity.

The effect of cyclophosphamide on B cells and 'background' immunoglobulin-secreting cells in mice.

The influence of cyclophosphamide (CY) was studied on the B-cell compartment of mice. This was done at five different levels: (a) the serum immunoglobulin (Ig) levels; (b) the numbers of 'background' Ig-secreting cells; (c) the incidence of surface Ig+ B cells; (d) the capacity of lipopolysaccharide-reactive B cells to give rise to a polyclonal IgM- and IgG-response in vitro; and (e) the capacity of long-lived memory B cells to give rise to an adoptive anti-sheep red blood cell plaque-forming cell response in vivo. A single injection of 300 mg CY/kg body weight (BW) decreased the numbers of background IgM-, IgG- and IgA-secreting cells in spleen, bone marrow and lymph nodes to minimum values of about 25% of normal at day 7. The incidence of surface Ig+ B cells also gradually decreased after CY treatment. The functional capacity of the B cells, however, was completely abolished one day after a single injection of 300 mg CY/kg BW. This was found for the lipopolysaccharide-reactive B cells, which largely represent newly formed, short-lived B cells as well as for long-lived memory B cells. The decrease of background Ig-secreting cells following a single injection of 300 mg CY/kg BW was followed by a gradual recovery with a substantial overshoot peaking about 40 days after CY injection. After multiple injections of 100 mg CY/kg BW, the minimum values of background Ig-secreting cells in the various lymphoid organs were lower than after a single injection of 300 mg CY/kg BW, but in this case the recovery was not associated with an overshoot reaction. Remarkably, the serum Ig levels were less profoundly affected than the numbers of Ig-secreting cells in the various lymphoid organs.

The effect of corticosteroids upon murine B cells in vivo and in vitro as determined in the LPS-culture system.

The influence of the synthetic corticosteroid dexamethasone sodium phosphate (DEXA) upon mouse B cells was studied. This was done by in vivo treatment of mice with a single or multiple injection of DEXA, and by culturing murine spleen cells and bone marrow cells in vitro in the presence of different concentrations of DEXA. The effect of DEXA on the B-cell compartment was assayed by polyclonal stimulation of the B cells by Escherichia coli lipopolysaccharide (LPS) in vitro and subsequent measurement of the Ig-secreting cell response in the protein A plaque assay. DEXA treatment could greatly reduce the number of B cells in the spleen, but the bone marrow B-cell compartment was quite resistant to DEXA. The in vitro LPS-induced IgM response of the residual B cells from both spleen and bone marrow and their capacity to switch from IgM to IgG and IgA secretion were not affected. These data indicate that DEXA can decrease the total number of B cells but not the functional capacity of the residual LPS-reactive B cells. This was confirmed at the clonal level by limiting dilution culture experiments. The contrasting effects of DEXA on splenic and bone marrow B cells was also found when the cells were exposed to the drug in vitro. It was found that 10(-8) M DEXA in vitro reduced the response of splenic B cells to LPS by more than 80%, while a similar reduction of the response by bone marrow B cells required a 1000-fold higher concentration.

Cellular and humoral immunity in patients with hyperthyroid Graves' disease before, during and after antithyroid drug treatment.

Many reports of thyroid stimulating immunoglobulins (TSI) in relation to treatment of Graves' disease have been published and with variable results concerning prediction of permanent remission or relapse after therapy. A range of methods has been used and little has been published measuring TSI by using their ability to stimulate cyclic AMP production in human thyroid cells in monolayer culture. We therefore conducted a prospective study of the predictive value of such an assay in patients with hyperthyroid Graves' disease before, during and after treatment of one year with methimazole and thyroid hormone substitution. Furthermore, the possible relationship between activated suppressor T lymphocytes and TSI in patients followed before, during and after medical therapy has been studied. Patients were divided into two groups; group I, 15 patients, who stayed in remission and group II, 14, who relapsed during the first year after discontinuation of therapy. Mean TSI activity did not differ between the two groups before and during the first half year of medication. In the second half year of treatment, however, mean TSI activity was significantly lower in group I. TSI activity at the end of treatment appeared to have no value in predicting final outcome. Increased TSI activity in group II during treatment was reflected in an increased pertechnetate thyroidal uptake as compared to that in group I. There was no relationship between changes in TSI activity and T cell subsets (Leu 1, 2a, 3a). We found no difference in T lymphocytes between the two groups at any time during observation. Subsets of T lymphocytes in both patient groups did not differ from normal.(ABSTRACT TRUNCATED AT 250 WORDS)

Construction of human-mouse T cell hybrids that express human T cell-associated surface antigens and allow the chromosomal localization of these antigens.

We have constructed somatic cell hybrids between the murine T cell line BW5147 and cells from patients suffering from T cell acute lymphoblastic leukemia. The obtained hybrid clones were analyzed for expression of human T cell antigens and presence of human chromosomes. T cell hybrids derived from fusion between the BW5147 cell line and bone marrow cells from a patient with pre-T acute lymphoblastic leukemia (TdT+/HLA-DR+/Tp41+/T11+/T1-/T6-/T4-/T8-/T3-) appeared to express the human T cell antigen Tp41, which can be recognized by the monoclonal antibodies 3A1 and WT1. Although this panel of hybrid cells contained all human chromosomes, no other T cell antigens were expressed. Fusion of the BW5147 cell line with peripheral blood cells from a patient with a more mature T cell acute lymphoblastic leukemia (TdT+/HLA-DR+/Tp41+/T11+/T1+/T6-/T4+/T8+/T3-) resulted in a panel of hybrid clones that expressed not only the Tp41 antigen, but also the human T cell antigens T1 and T4; two hybrids even expressed the T3 antigen. This panel of hybrids also contained the whole human genome. The two panels of human-mouse T cell hybrids allowed us to assign the genes coding for the human T cell antigens Tp41, T1, and T4 to human chromosomes 17, 11, and 12, respectively. Furthermore, these data support our previous suggestion that the expression of human lymphoid differentiation antigens in human-mouse lymphoid hybrids is influenced by the differentiation stage of the fusion partners.

Complement split product C5a mediates the lipopolysaccharide-induced mobilization of CFU-s and haemopoietic progenitor cells, but not the mobilization induced by proteolytic enzymes.

Intravenous (i.v.) injection of mice with lipopolysaccharide (LPS), and the proteolytic enzymes trypsin and proteinase, mobilizes pluripotent haemopoietic stem cells (CFU-s) as well as granulocyte-macrophage progenitor cells (GM-CFU) and the early progenitors of the erythroid lineage (E-BFU) from the haemopoietic tissues into the peripheral blood. We investigated the involvement of the complement (C) system in this process. It appeared that the early mobilization induced by LPS and other activators of the alternative complement pathway, such as Listeria monocytogenes (Lm) and zymosan, but not that induced by the proteolytic enzymes, was absent in C5-deficient mice. The mobilization by C activators in these mice could be restored by injection of C5-sufficient serum, suggesting a critical role for C5. The manner in which C5 was involved in the C activation-mediated stem cell mobilization was studied using a serum transfer system. C5-sufficient serum, activated in vitro by incubation with Lm and subsequently liberated from the bacteria, caused mobilization in both C5-sufficient and C5-deficient mice. C5-deficient serum was not able to do so. The resistance of the mobilizing principle to heat treatment (56 degrees C, 30 min) strongly suggests that it is identical with the C5 split product C5a, or an in vivo derivative of C5a. This conclusion was reinforced by the observation that a single injection of purified rat C5a into C5-deficient mice also induced mobilization of CFU-s.

Regulation of delayed type hypersensitivity to host histocompatibility antigens during graft-versus-host reactions.

During GvH reactions in irradiated mice a variety of specific anti-host immune responses may occur. One of these is the occurrence of DTH to the host histocompatibility antigens, which may account for the inflammatory aspects of GvH. During acute as well as delayed GvH reactions the occurrence of anti-host DTH precedes the clinical symptoms of GvH disease. The anti-host DTH is mediated by long-lived, recirculating Lyt-1 + 2- T cells that need to proliferate in the irradiated recipients in order to display maximum activity. Not all host histocompatibility antigens can elicit anti-host DTH. H-2I and Mls-locus coded alloantigens do, but H-2K/D coded alloantigens and non-H-2 alloantigens other than Mls-locus coded alloantigens do not. This correlates with the ability to elicit proliferative mixed lymphocyte reactions in vitro but is in contrast to the response of nonirradiated mice to sc administered alloantigens. Under the latter conditions, all histocompatibility antigens induce a state of antigen-specific DTH. While the anti-host DTH is mediated by long-lived, recirculating Lyt-1 + 2- T cells, their response can be amplified by short-lived, sessile Lyt-1 + 2 + T cells. The latter T cell subset reacts to the host H-2K/D alloantigens and/or to non-H-2 alloantigens other than Mls-locus coded products. These cells alone cannot mount an anti-host DTH response. The anti-host DTH can be mitigated by Ts cells and non-T suppressor cells. Appropriate Ts cells can be readily induced by iv preimmunization of the donors with 5 X 10(7) irradiated, recipient-type spleen cells. Non-T suppressor cells can be induced by iv injection of bacterial LPS and simultaneous sc injection of 1 X 10(7) recipient type spleen cells. The suppression induced by these protocols shares several characteristics. In both cases the suppression is long-lasting, i.e., lasts at least 50 d, is transferable to syngeneic mice by spleen and lymph node cells, and both suppressive systems affect the induction of anti-host DTH as well as already activated anti-host DTH reactive T cells. Furthermore, while Ts cells and non-T suppressor cells are specific with regard to their antigen recognition, they are both able to suppress the DTH to a completely different set of host alloantigens. This, however, only occurs if the latter are inherited by the irradiated recipients as bystanders to the type of alloantigens that had activated the suppressor cells in the lymphoid cell donors.

T-lymphocyte subpopulations in patients with various courses after hepatitis B virus infection.

The course of a hepatitis B virus infection is probably determined by the cellular immune response of the host, which is partly regulated by the T helper and T suppressor cells. We therefore counted immunoregulatory T-cell subsets in the peripheral blood of 97 patients with various courses of hepatitis B virus infection: 23 of these patients were asymptomatic HBsAg carriers without detectable liver disease, 13 had chronic persistent hepatitis B, 19 had chronic active hepatitis B (11 HBeAg, 8 anti-HBe), 7 had chronic active hepatitis with anti-HBs or anti-HBc, and 35 were healthy controls with anti-HBs after recovery from acute hepatitis B. Peripheral blood mononuclear cells were specifically labelled with monoclonal Leu-1 (T cells), Leu-2a (T suppressor/cytotoxic cells), and Leu-3a (T helper cells) antisera and analyzed by flow cytometry. Leu-3a/Leu-2a ratios for patients with persistent virus infection did not differ from those found for patients who cleared the hepatitis virus antigens. There was, however, evidence that the number of T suppressor cells in the subgroup of patients with ongoing chronic active hepatitis and anti-HBe had decreased. These findings suggest that elimination of hepatitis B virus is unlikely to be related to the relative number of peripheral T-cell subsets. The few patients who develop chronic active hepatitis after partial clearance of the virus probably have an enhanced immunoreactivity compared with those with the commoner courses of this disease.