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.

Comparison of the sensitivity of the protein A plaque assay and the immunofluorescence assay for detection of immunoglobulin producing cells.

This paper reports a comparative study on the sensitivity of the protein A plaque assay and the cytoplasmic immunofluorescence assay for detection of immunoglobulin (Ig) producing cells in cell suspensions of murine lymphoid organs. The protein A plaque assay was found to detect as many or several times more Ig producing cells than the immunofluorescence assay, depending on the age and antigenic load of the mice, and upon the Ig class and organ studied.

Improvement of the protein A plaque assay for immunoglobulin secreting cells by using immunoglobulin-depleted guinea pig serum as a source of complement.

This paper describes a modification of the protein A hemolytic plaque assay for the enumeration of immunoglobulin (Ig)-secreting cells independent of antibody specificity of the Ig. This assay was originally developed by Gronowicz et al. (1976), and is based upon binding of the Fc portion of IgG to protein A. Ig-secreting cells are mixed with protein A-coated sheep erythrocytes, developing rabbit anti-Ig antiserum and guinea pig serum as a source of complement. This mixture is either pipetted between two microscope slides, or added to agarose and plated on a petri dish or microscope slide. The hemolytic plaques are enumerated after incubation at 37 degrees C. Here we show that purification of the guinea pig complement over a Sepharose-protein A column in order to eliminate the IgG fraction facilitates plaque formation. This modification reduces the incubation period required for plaque formation, and yields a higher number of, and more discrete plaques, than the original method.

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.

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.

Quantitative aspects of the nonspecific humoral immune response to sheep erythrocytes.

The kinetics and magnitude of the nonspecific humoral immune response was studied at the cellular level in mice immunized with sheep erythrocytes (SRBC). Intravenous injection of the antigen evoked, in addition to a specific anti-SRBC response, a nonspecific response of all immunoglobulin (Ig) classes and subclasses. This nonspecific response peaked on day 4 or 5 after immunization, irrespective of the Ig class. The absolute number of nonspecific Ig-secreting cells induced by immunization varied with the different Ig-classes, and it was not proportional to the background level of Ig-secreting cells of the various classes. The nonspecific IgM-IgG1- and IgG2-response was 3 to 4 times as large as the antigen-specific responses of these classes. The nonspecific IgA-response, however, was many times greater.

The effect of corticosteroids upon the number and organ distribution of "background" immunoglobulin-secreting cells in mice.

The influence of the synthetic corticosteroid dexamethasone sodium phosphate (DEXA) upon the immunoglobulin (Ig)-secreting cells was studied in not intentionally immunized BALB/c mice. This was done for IgM-, IgG-, and IgA-secreting cells in spleen, mesenteric lymph nodes (MLN), and bone marrow (BM). A single injection of DEXA (16 to 144 mg/kg body wt) markedly reduced the number of Ig-secreting cells in spleen and MLN within 1 day, but hardly affected their number in the BM. The decrease was immediately followed by a recovery and, at the highest doses and especially in MLN, by an overshoot. Two weeks after the initial decrease a second decrease was found. When mice were subjected to daily treatment with DEXA during 1 week, initially a recovery pattern was found in spleen and MLN similar to that found after a single injection of a high dose. In this case, however, the effects were less dose dependent, and the overshoot reaction was followed by a period of subnormal numbers of Ig-secreting cells which lasted at least 1 week. This late effect of DEXA not only occurred in spleen and MLN, but also in the BM. The most prominent effect of daily treatment with DEXA was the long-lasting decrease of the number of IgG-secreting cells starting 1 week after withdrawal of treatment. This decrease was associated with a severely decreased serum IgG level.

B memory cells in the thymus: part of the pool of potentially circulating memory cells.

Immunization of mice with sheep red blood cells (SRBC) or Escherichia coli lipopolysaccharide (LPS) induces the appearance of B memory cells in the thymus. In this paper the origin of these B memory cells was investigated. Therefore, mice primed with either SRBC or LPS 6 months previously and nonprimed mice were joined for parabiosis. Four weeks later the parabiotic mice were separated from each other. Another 3 weeks later thymus cells from the primed and nonprimed mice were transferred separately into lethally irradiated mice in order to determine the adoptive PFC response. It was found that the 4-week period of parabiosis could account for the appearance of a distinct population of B memory cells in the thymus of the nonprimed mice. This result suggest that the B memory cells which appear in the thymus belong to the pool of potentially circulating memory cells.

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.

Frequencies of background cytoplasmic Ig-containing cells in various lymphoid organs of athymic and euthymic mice as a function of age and immune status.

The distribution of background Ig-secreting cells, measured as cells containing cytoplasmic immunoglobulin (C-Ig cells), over spleen, bone marrow, lymph nodes and Peyer's patches was studied in congenitally athymic (nude) mice and heterozygous euthymic mice as a function of age and immune status (germ-free (GF) vs specific pathogen-free (SPF]. In young athymic as well as in young euthymic mice, the spleen was found to contain the great part of all C-Ig cells, irrespective of whether the mice were GF or SPF. The number of C-Ig cells in the spleen was found to be rather constant over the life span, while the number of C-Ig cells in the bone marrow of all groups of mice greatly increased with age. This indicates that the relative shift of C-Ig cells to the bone marrow is neither dependent on the presence of the thymus, nor on the microbiological status of the mice. However, at young and intermediate age the microbiological status of the mice did affect the total number of C-Ig cells per mouse. This was mainly due to the effect upon the bone marrow, mesenteric lymph nodes and Peyer's patches. At these ages the background Ig synthesis in these organs appeared to be mainly dependent on external antigenic stimulation, in contrast to the spleen, where the Ig synthesis appeared to be mainly due to endogenous stimulation. The Ig (sub)class distribution of the C-Ig cells was different for all different organs tested. Hardly or no difference in percentage distribution was found between the GF nude and GF heterozygous mice. Most C-Ig cells in spleen, bone marrow and lymph nodes of the GF mice were of the IgM isotype. C-IgG and C-IgA cells occurred in substantial percentages only in bone marrow and lymph nodes. In the lymph nodes of GF nude mice a remarkably high percentage of C-IgA cells was found.

Kinetics of recovery of serum Ig levels and of cytoplasmic Ig positive cells in various lymphoid organs of nude mice after thymus transplantation.

The long-term effects of thymus transplantation in nude mice were studied with regard to the number of cytoplasmic immunoglobulin positive plasmablasts and plasma cells (C-Ig cells) in various lymphoid organ and their immunoglobulin (Ig) class distribution profile. These data were correlated with the serum Ig levels of the same mice. Four weeks after thymus transplantation, the number of C-Ig cells in the spleen of nude mice had increased two- to three-fold over that found in normal nude mice and normal heterozygous littermates of the same age. This overshoot subsided at 8 weeks after thymus transplantation. The increase of the C-Ig cell number in the other lymphoid organs tested (bone marrow, mesenteric lymph nodes and Peyer's patches) started later than in spleen, and did not show a clear overshoot. Almost complete recovery of the C-Ig cell pattern to that of normal littermates was found 32 weeks post-transplantation. Analysis of the Ig class distribution of the C-Ig cells showed that the increase of the C-Ig cell numbers after thymus transplantation in nude mice was almost exclusively confined to IgG1, IgG2 and IgA. The increase of C-IgG1 and C-IgG2 cells in spleen and bone marrow correlated with a simultaneous increase of the serum IgG1 and IgG2 levels, suggesting that these organs are the major source of serum IgG in young adult mice.

Leukocyte mobilization in mice by polyanions. Decline of leukocyte mobilizing capacity after transplantation of lymphoma.

The leukocyte mobilizing polyanions dextran sulphate (DS) and polymethacrylic acid (PMAA) were administered to AKR and (C57BL x CBA) F1 mice at various times after transplantation of syngeneic lymphoma cells. In nonleukaemic mice DS and PMAA increased the number of circulating leukocytes 3--4-fold. The extent of leukocyte mobilization in leukaemic mice depended on the interval between transplantation of the lymphoma cells and injection of the polyanion. During the development of leukaemia in AKR as well as in (C57BL x CBA) F1 mice the capacity to react upon injection of polyanions with leukocyte mobilization gradually decreased. For DS, this decrease started before the number of leukocytes increased in the peripheral blood. On the other hand, the capacity for PMAA-induced leukocyte mobilization was fully preserved for several more days. In heavily leukaemic mice neither DS nor PMAA could further increase the number of peripheral blood leukocytes. In such mice the distribution pattern of leukaemic blast cells, small lymphocytes, granulocytes and monocytes was also hardly or not affected by injection of the polyanion.

The distribution of cytoplasmic immunoglobulin containing cells over various lymphoid organs of congenitally athymic (nude) mice as a function of age.

The distribution of cells containing cytoplasmic immunoglobulin (C-Ig cells) over various lymphoid organs was studied in congenitally athymic (nude) mice as a function of age. The C-IgM, C-IgG and C-IgA cells were enumerated in spleen, bone marrow, mesenteric lymph node and Peyer's patches of nude mice and their heterozygous littermates of 6, 40 and 100 weeks of age. In the nude as well as in the heterozygous mice an age-related shift was observed in the localization of the C-Ig cells. In young mice of both groups the majority of these cells resided in the spleen, whereas in adult and old mice the bone marrow was found to be the major C-Ig cell organ, indicating that this shift is not dependent on the presence of the thymus. In young and adult nude and heterozygous mice C-Ig cell numbers in the spleen were comparable, whereas C-Ig cell numbers in the other lymphoid organs were higher in the heterozygous mice than in the nude mice. The total C-Ig cell number in young and adult nude mice was lower than in heterozygous mice of the same age, whereas in old nude mice they were as high as in heterozygous mice of the same age, indicating a retarded development of the immunological activity in nude mice. C-Ig cells in nude mice were almost exclusively of the IgM class, although in the bone marrow of the oldest animals also a substantial number of C-IgG and C-IgA cells was observed. Our finding that nude mice can live up to at least two years of age indicates that the age-related deterioration of the thymus-dependent limb of the immune system is not the cause of ageing, but rather a consequence of it.

Immunoglobulin isotype expression. II. Frequency analysis in mitogen-reactive B cells.

The frequency of lipopolysaccharide (LPS)-reactive B cells developing into clones that secrete the various immunoglobulin (Ig) classes has been determined in vitro, in cells from BALB/c mice, under culture conditions which detect all growth-inducible cells. Secretion of the different Ig classes was assessed in the protein A plaque assay for Ig-secreting, plaque-forming cells by using developing antisera specific for either IgM, IgG1, IgG2a, IgG2b, IgG3 or IgA. In all lymphoid organs tested (spleen, bone marrow, mesenteric lymph nodes and thoracic duct), a considerable proportion of all B cells (5-20%) was induced by LPS to yield a clone of IgM-secreting cells. Frequency determinations of LPS-reactive cells giving rise to descendants secreting other Ig isotypes revealed that, on an average, and irrespective of the origin of the cells, 7% of all IgM-secreting clones switched to the synthesis of IgG1, 39% to IgG2, 41% to IgG3 and 1% to IgA. Roughly the same frequencies of B cells switching CH gene expression were found among spleen cells of athymic nude mice. No correlation was found between the clonal frequencies of CH gene expression in polyclonally activated B cells and the in vivo "background" Ig-secreting cells suggesting that the CH gene expression in B cells is influenced by the quality of stimulation and other regulating influences.

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.