Lack of transfer of lpr-type abnormalities (lymphoproliferation or lymphoid aplasia) in double congenic nude beige mice engrafted with lpr haematopoietic cells.

The aetiology of the autoimmune and lymphoproliferative syndrome caused by the murine lpr (lymphoproliferation) mutation was studied by the adoptive transfer methodology using non-irradiated athymic and natural killer (NK)-deficient C57BL/6 nude beige mice (B6 nubg) as recipients. The [lpr-->nubg] chimeras did not display the severe lymphoid organ aplasia shown by irradiated non-lpr recipients of lpr haematopoietic cells. However, nor did they either express the typical lpr phenotype features (hyperglobulinaemia, autoimmunity and lymphoid hyperplasia). Nevertheless, engraftment of lpr cells in the nubg recipients was shown by their much increased survival, the recovery of T-cell mitogen responsiveness in the spleen, and the presence of T-dependent immunoglobulin isotypes in their serum. The host of donor origin of serum immunoglobulin was studied by measuring IgG2a allotypes in the serum of [lpr-->nubg] chimeras made with different lgh-congenic mice. Interestingly, several months after grafting, the serum IgG2a was found to be mainly of lpr graft origin, suggesting that only lpr B cells could function in such chimeras. In conclusion, a lpr spleen cell graft reconstituted non-irradiated nubg recipients and induced neither a typical lpr syndrome nor a lpr-type graft-versus-host (GVH)-like disease. These features of the lpr syndrome are at variance with those of the phenotypically similar gld syndrome, since this mutation allows the transfer of a generalized lymphadenopathy disease by grafting gld spleen cells in nubg or irradiated recipients. Unlike the gld syndrome, the lpr gene might not only affect haematopoietic cells but also cells of the environment, which would interact in the same impaired process.     

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.     

Nocardia infections in congenitally athymic (nude) mice and in other inbred mouse strains.

The mortality rate and histopathological features of Nocardia asteroides and Nocardia brasiliensis infections in congenitally athymic (nude) mice of ICR and C3H/eB origins were quite different from what we found for Swiss white mice and other inbred mouse strains (namely, C57/BL/6J, New Zealand Black, BALB/c, CBA/LAC, and C3H/eB). The immunocompetent littermates of the congenitally athymic mice occupied an intermediate position between their athymic siblings and Swiss white mice in terms of their responses to both these organisms. Macrophage ingestion and destruction of N. brasiliensis, as demonstrated by electron microscopy, was found to occur. The T-lymphocyte appears to be an essential component in normal mouse resistance to infection by both N. asteroides and N. brasiliensis.     

Comparative patterns of serum immunoglobulin levels in specific-pathogen-free congenitally athymic (nude), hereditarily asplenic (Dh/+), congenitally athymic-asplenic (lasat) and splenectomized athymic mice.

Serial determinations of serum immunoglobulin levels were assessed in congenitally athymic (nude), hereditarily asplenic (Dh/+) and congenitally athymic-asplenic (lasat) mice and the results compared to normal intact littermate controls (nu/+), neonatally splenectomized nu/+ and neonatally splenectomized nude mice. Quantification of Ig levels was accomplished by radial immunodiffusion, for IgM, IgG1, IgG2a, IgG2b and IgA antibody isotypes. Intact spleen and/or thymus function was shown to have marked effects on the age-dependent development of serum IgM, IgG2b and IgA production. Furthermore, because of higher levels of IgA in congenitally athymic-asplenic mice and neonatally splenectomized nude mice v. sham splenectomized nude mice, it is suggested that an IgA-specific suppressor population resides in the spleen. Finally, because of frequent problems in the literature in interpretation of immunoglobulin values, the criteria for the statistical evaluation of such data in establishing normal serum Ig values and ascertaining real differences between treatment groups are emphasized.     

Structural and cell population changes in the lymph nodes of the athymic nude mouse

A recent tridimensional analysis of the lymph node demonstrated that its deep cortex is composed of grossly hemispherical "units," adjoining a portion of its peripheral cortex. Each deep cortex unit can be distinguished into a center and a periphery. The periphery was concluded to be a site for migration of circulating lymphocytes, the center, a site where T cells would participate in cellular immune responses. The aim of the present work was to determine the influence of the congenital athymic state on the development of the units and of other components in the lymph nodes of the nude mouse. For this, the lymph nodes at various anatomical locations in adult athymic nude mice were analyzed. The present study revealed that the athymic state did not inhibit the development of the units but severely depleted the lymphocyte population of their center only. However, it did inhibit the development of an area of peripheral cortex located over the middle part of a unit. Such an area of peripheral cortex is, thus, concluded to be thymus dependent, as is the center of a deep cortex unit. The athymic state also prevented the development of the cells of the nodules (germinal centers) and of much of the plasmocytes. On the other hand, it yielded to the enlargement of the follicles, the formation of new structures: medullary "lymphocyte clusters" and the transformation of the medullary venules into high endothelial venules. The various modifications of the nodal structures resulting from the congenital athymic state are discussed in relation to some functions of the organ.     

Hybridomas using athymic nude mouse injected with Crohn's disease (CD) tissue filtrate. Immunoreactivity of the hybridomas with CD sera.

Injections of Crohn's disease (CD) tissue filtrates produce lymphoma and hyperplastic lymph nodes from plasma cell hyperplasia (PCH) in athymic nude (nu/nu) mice; these lymphoid tissue contain an antigen(s) recognized by CD serum/gamma G immunoglobulin (IgG). To immortalize the "CD-reactive antigen(s)," the authors fused the lymphoid cells from a CD tissue filtrate primed nu/nu mouse with nonsecretory mouse myeloma cells. Hybrids were screened and selected based on their reactivity with CD serum IgG, but not with control serum IgG in an indirect immunofluorescence assay (IF). Two CD-positive hybridomas were examined by IF with sera from 47 CD, 38 ulcerative colitis (UC), 13 controls with other gastrointestinal diseases, 19 with autoimmune diseases, and 21 normal subjects. Sera from 16 CD patients (34%) reacted with the two hybridomas, but only one of 38 UC sera and none of the 53 other disease or normal control sera reacted. The immunoreactivity of CD sera was significantly higher than UC sera (P less than 0.01) and each of the other groups (P less than 0.007). Using immunoperoxidase techniques at light and electron microscopic levels, the authors localized CD-associated antigen(s) in the plasma membrane of the two hybridomas. Further characterization of these hybridomas and the immunoreactive protein(s) may provide an important probe(s) for the diagnosis and the understanding of the pathogenesis of CD.     

Thymic-dependent anti-hapten response in congenitally athymic (nude) mice immunized with DNP-thymosin.

Immunization of congenitally athymic (nu/nu) and adult thymectomized, irradiated bone marrow, reconstituted (TxBm) mice with DNP5-thymosin (dinitrophenylated-bovine thymosin fraction 5) was found to elcit IgM and IgG anti-DNP plaque-forming cells in these animals. Further studies indicated that this response was antigen specific and not due to polyclonal activation. Since the hormonal properties of the thymosin were retained following linkage with hapten and DNP-thymosin was immunogenic in CBA/N and CBA/N female X DBA/2 male)F1 male mice, animals previously shown to have an X-linked inability to respond to thymus-independent antigens, it was concluded that DNP-thymosin functions both as a hormone and as a T-dependent antigen in eliciting an immune response in nu/nu and TxBm mice. Additional support for this conclusion was provided by the demonstration that DNP-thymosin could specifically prime for and elicit an anamnestic response in nu/nu mice. These results indicate that further investigation of the immune activities of DNP-thymosin may provide valuable insight in characterizing the maturation of helper T cells and their subsequent interaction with B cells.     

Mycobacterium lepraemurium infection of nude athymic (nu/nu) mice.

Nude athymic (nu/nu) mice on a BALB/c background and their heterozygous euthymic litter mates (nu/+) were infected with either 10(8) or 10(6) Mycobacterium lepraemurium organisms intravenously or in the left hind footpad (LHF). After LHF infection with 10(8) M. lepraemurium organisms, nu/+ mice slowly developed a response that consisted of LHF swelling and local resistance to Listeria monocytogenes. The lower inoculum induced a proportionately lower response in nu/+ mice, but the nu/nu mice developed neither LHF swelling nor resistance to L. monocytogenes in response to either dose of M. lepraemurium. Counts of M. lepraemurium in the LHF revealed no difference between the nu/+ mice and nu/nu mice. After intravenous infection the nu/+ mice developed splenomegaly, but did not otherwise differ from nu/nu mice with respect to resistance to intravenous challenge with L. monocytogenes or growth of M. lepraemurium in the spleen. In light of the poor responsiveness of nu/+ mice in this experiment, they were then compared with CB6 and B6D2 mice, which are genetically susceptible and resistant to M. lepraemurium, respectively. These mice were infected with either 10(8) or 10(6) M. lepraemurium cells or 10(6) Mycobacterium bovis BCG cells in the LHF. Once again the nu/+ mice responded poorly to M. lepraemurium, the CB6 mice responded very strongly, and the B6D2 mice gave an intermediate response with respect to LHF swelling and resistance to L. monocytogenes. However, M. lepraemurium grew to higher numbers in the LHF of nu/+ and CB6 mice than in B6D2 mice, revealing, in CB6 mice, a dissociation between resistance to L. monocytogenes and M. lepraemurium. All three mouse strains responded strongly to M. bovis BCG, but there was a suggestion that nu/+ mice might be more susceptible to this agent than the other two strains. I concluded that the failure of nu/+ mice to restrict the growth of M. lepraemurium more than nu/nu mice was due to the intrinsic genetic susceptibility of both types of mice. In effect, the nu/+ mice behaved like nu/nu mice, as if they too were deficient in T lymphocytes that were responsive to M. lepraemurium.     

Reciprocal lymphoid cell homing studies using wild and LPR (lymphoproliferation) mutant C57BL mice

The injection of lymphoid cells from adult lpr mice into normal and athymic congenic mice does not transfer the lpr (lymphoproliferation) syndrome. We studied whether this phenomenon could be due to abnormal homing. The lymphoid cells from lpr donors do not show a marked deficiency of migration to lymphoid organs in comparison with cells from Wild donors and a T-cell lymphoma BL/VL3. The lymphoid organs of lpr recipients do not show an intrinsic abnormality as homing sites for lymphoid cells. The data reveal some minor migration preferences: the lpr cells migrate better than Wild cells into lpr lymph nodes (including athymic lpr hosts), whereas Wild cells migrate slightly better than lpr cells into Wild lymph nodes. In spite of such minor preferences, Wild cells can efficiently migrate into lpr lymphoid organs, as well as lpr cells into Wild lymphoid organs. Thus, the lack of transfer of lymphadenopathy in Wild recipients cannot be attributed to an alteration of lpr cell homing.     

Resistance to lymphoid engraftment in lpr recipients of normal bone marrow: characterization of chimeric stem cell, monocyte and peripheral lymphoid lineages

Lethally irradiated MRL/lpr mice reconstituted with T cell-depleted bone marrow stem cells from the non-autoimmune strain A. Thy had been shown to develop a state of long-term split chimerism; erythrocytes were derived from the A. Thy donor, while peripheral lymphocytes were derived from the lpr recipient. In contrast, recipients of the non-lpr-congenic strain, MRL/+, were fully repopulated in both lineages by donor-derived hematopoietic cells. In order to more fully understand the mechanisms responsible for this type of split chimerism, we have investigated additional genetic and developmental parameters. We found that histocompatible normal B cell precursors engrafted C3H/HeJ and C57BL/6/+ mice much better than they engrafted the corresponding lpr congenic strains, indicating that resistance to lymphoid engraftment was not unique to the MRL background. Bone marrow cells and peritoneal macrophages were found to express the donor H-2 phenotype in both non-lpr and lpr recipients, limiting resistance to the lymphoid lineage. Moreover, we showed that normal bone marrow stem cells passaged in an lpr host environment were subsequently able to repopulate the B cell lineage of non-lpr secondary recipients, proving that prelymphoid stem cells were intact. Although lymph node cells from A. Thy----MRL/lpr chimeras were lpr-derived, they did not show the abnormal surface marker expression associated with the lpr phenotype, nor did they develop lymphoid hyperplasia or elevated autoantibody levels. However, A. Thy----MRL/lpr chimeras differed from normal mice in that their spleens were markedly deficient in IgM+ B cells.     

Virus carrier state suppresses tumorigenicity of tumor cells in athymic (nude) mice.

Nude mice injected subcutaneously with normal uninfected BHK 21 cells or HeLa cells regularly develop large, rapidly-growing tumours at the subcutaneous site of inoculation. However, these same tumour cell lines when persistently infected with VSV or other enveloped RNA viruses are either rejected or form small nodules in nude mice. This rejection phenomenon probably involves some type of immunocyte since heavily-irradiated nude mice (500 rads) cannot reject persistently infected cells but develop large, rapidly-growing tumours which shed virus and defective interfering virus (DI) and which do not exhibit the lymphocytic infiltration observed in the nodules of unirradiated mice given persistently infected cells. Finally, it was possible to select a subline of BHK 21-VSV carrier cells which regularly produces large rapidly-growing tumours in normal unirradiated nude mice, although all these carrier cells express virus antigen and shed large amounts of mature infectious virus and DI both in vivo and in vitro.     

Follicular dendritic cell dysfunction and disorganization of lymphoid structures in MRL/lpr mice.

The follicular dendritic cell (FDC) in secondary lymphoid organs plays a key role in the function of the germinal center (GC) of the lymphoid follicle (LF) for regulation of the humoral immune response. MRL MpJ-lpr/lpr (MRL/lpr) mice, which have an abnormal humoral immune response, are a model for autoimmune disease. The present study was undertaken to investigate FDC dysfunction and LF disorganization in the spleen and lymph nodes of MRL/lpr mice. In 12-week-old MRL/lpr mice, antigen (Ag) trapping of FDC and half-life of trapped Ag on FDC was reduced. Although immunohistochemistry revealed well-developed FDC networks positive for complement receptors and having in vitro immune complex trapping in cryostat sections, Ag-trapping was not detected in 16-week-old MRL/lpr mice. Moreover a marked decrease in the number of GC and that of GC cell of residual GC in the MRL/lpr mice was observed by 12 weeks of age. Also reduced expression of intercellular adhesion molecule-1, FcgammaRII/III, and FDC-specific Ag (FDC-M1) on FDC was detected in 16-week-old MRL/lpr mice. The accumulation of double negative (CD4-CD8-) T cells, which is a characteristic feature of MRL/lpr mice, was observed around LF; however they did not infiltrate the LF and FDC network. In conclusion the loss of Ag-trapping on FDC and simultaneous disorganization of the lymphoid structure may be related closely to the immunologic abnormality observed in MRL/lpr mice.     

Use of nude (athymic) mice for the study of hypertrophic scars and keloids: vascular continuity between mouse and implants

Hypertrophic scars and keloids appear to be unique to humans since animals are not known to form these lesions. Therefore, in an effort to develop an experimental model for their study, implants of these human lesions were made in nude (athymic) mice (nu/nu) in suprascapular subcutaneous pockets. The implants were recovered from 2 to 246 days. By histological and fine structural parameters all implants remained viable and their morphological character was maintained. Selected mice were injected with barium to confirm by microangiography vascular flow between mouse and implant. Hoechst stain for DNA, used to distinguish mouse cells from human cells, confirmed vascular anastamosis between host and implant: barium-filled vessels in the interior of the implant demonstrated human endothelial cells. Peripheral vascularization of the implant with minimal ingrowth of mouse vessels occurs during the first 8 days. Anastamosis probably occurs sometime before 16 days postimplantation, or earlier, depending upon the availability of patent microvessels in the implanted tissue. The presence of the implant does not appear to prompt a continuing vascular growth into or throughout the implant. The time frame of 16 days postimplantation should be taken into account when developing schemata of experimental or therapeutic modalities.     

Natural killer cell activity in the rat. Analysis of effector cell morphology and effects of interferon on natural killer cell function in the athymic (nude) rat

Athymic (nude) rats were found to have increased levels of natural killer (NK) activity, 3? to 5?fold higher than in euthymic rats. Studies were performed to determine the nature of the NK cells in these animals and the basis for their increased cytotoxic reactivity. Large granular lymphocytes (LGL), which were previously shown to be the NK cells in euthymic rats, were increased 2- to 7-fold in the peripheral blood and spleen of nude rats. The LGL, enriched by centrifugation on discontinous Percoll density gradients, were shown to have augmented NK activity similar to that seen with LGL-enriched fractions from euthymic rats. These results indicate that the NK cells in euthymic and athymic rats are morphologically and functionally similar, and that the higher NK activity in nude rats appears to be mainly attributable to an increased proportion of effector cells. In a single-cell cytotoxicity assay, interferon pretreatment of LGL was shown to increase: (a) the percentage of LGL which form conjugates with target cells; (b) the percentage of conjugate-forming cells which kill; and (c) the kinetics of lysis. Different effects were seen depending on the target cell tested.     

Induction of early-phase endotoxin tolerance in athymic (nude) mice, B-cell-deficient (xid) mice, and splenectomized mice.

Early-phase endotoxin tolerance was inducible in mice which were T cell deficient (nude), B cell deficient (xid), or asplenic, which suggests that these lymphoid cell subsets and the spleen do not contribute significantly to the induction of acquired lipopolysaccharide hyporesponsiveness. C3H/HeJ mice did not exhibit the hematopoietic changes observed in mice made endotoxin tolerant, which suggests that multiple mechanisms may underlie lipopolysaccharide hyporesponsiveness.     

Trichinella spiralis infection in congenitally athymic (nude) mice. Parasitological, serological and haematological studies with observations on intestinal pathology.

In six experiments the course of a Trichinella spiralis infection in congenitally athymic (nu/nu) mice and their heterozygous thymus-bearing littermates (+/nu) was followed. In the +/nu mice worms were expelled at day 10 post infection. In nu/nu mice worms remained in the intestine until the end of the observation period (83 days post infection). In testing the yield of muscle larvae in +/nu and nu/nu mice 4--5 times more muscle larvae were isolated from nu/nu mice than from infected +/nu mice. The following phenomena were observed in +/nu mice only: anti-T. spiralis antibodies detected by immunofluorescence, intestinal plasma-cell production and intestinal eosinophilia. In nu/nu mice no blood eosinophilia was observed in contrast to the induction of eosinophilia both in infected +/nu and infected nu/nu mice reconstituted with thymuses from heterozygous littermates. Intra-epithelial lymphocytes, more numerous in +/nu than in nu/nu mice, were not attracted by Trichinella antigen. The data supported the hypothesis that worm expulsion is a T cell-dependent phenomenon. Plasma cell and antibody production as well as tissue and blood eosinophilia were shown to be thymus-dependent in a T. spiralis infection.