Pathological changes of thymic epithelial cells and autoimmune disease in NZB, NZW and (NZB × NZW)F1 mice

An extensive histological study was carried out of NZB, NZW and (NZB × NZW)F₁, (BWF₁), mice of all ages between birth and 18 months. The thymuses of these mice were compared to those of three normal mouse strains. The study of the NZW mice showed that these mice, although they only occasionally have weakly positive Coombs' tests, may develop a renal disease probably of an autoimmune nature, similar to that of the NZB and the BWF₁ mice. Mice of all the three NZ strains developed lesions of the skin, liver, intestines, lymphatic tissues and kidneys much resembling those occurring in human systemic lupus erythematosus (SLE), neonatally thymectomized mice and, with the exception of the renal changes, the lesions of graft versus host disease.

The comparative study of the thymus in autoimmune and normal strains, revealed that important changes of the large medullary epithelial cells, involved in the formation of Hassall's corpuscles, occur very early in the three autoimmune strains. In the NZB mice the large epithelial cells are severely decreased in number in the first weeks following birth. The depletion of epithelial cells could be ascribed to a secondary degeneration of these cells soon after birth.

In contrast with the NZB mice, an extensive hyperplasia of the large epithelial cells and Hassall's corpuscles was observed in the NZW and BWF₁ mice, and was already apparent in the newborn animal. Many of the epithelial aggregates seemed to have been invaded by lymphoid cells. Both epithelial cells and the lymphoid cells engaged in this process showed a variety of degenerative changes. As in the NZB, a depletion of epithelial cells occurred in a later phase, at the age of 8 months in the BWF₁ and at 1 year in the NZW. In the majority of young mice of the normal strains invasion of islands of epithelial cells by lymphoid cells may also be observed, although this process is far less extensive than in the autoimmune strains and does not result in either epithelial hyperplasia or depletion of epithelial cells.

The described phenomenon of invasion of epithelial structures in the thymus by subsequently disintegrating lymphoid cells seems to support Burnet's concept, that so-called `forbidden clones' of lymphoid cells are eliminated in the thymus.

     

Natural cytotoxic autoantibody against thymocytes in NZB mice

NZB mice were found to produce natural thymocytotoxic autoantibody in high prevalence and antibody titre. This autoantibody in NZB mice was detectable by the cytotoxicity test at both 4°C and 37°C; the prevalence and antibody titre were generally higher at 4°C. Mice of other strains also produced natural thymocytotoxic autoantibody although in lower prevalence and antibody titre and in some instances the activity was greater at 37°C than at 4°C. Natural thymocytotoxic autoantibody in NZB mice reacted equally with the thymocytes of virtually all strains of mice tested but to a lesser degree with the thymocytes of SJL/J mice. A serum pool obtained from old NZB mice had an extremely high titre of natural thymocytotoxic autoantibody (1:1024 at 4°C). Nevertheless, the cells in lymph nodes, spleen and blood leucocytes were only partially sensitive to this serum pool, and bone marrow cells were for the most part negative. By absorption, the antigen reacting with natural thymocytotoxic autoantibody was found in thymus, lymph node, spleen and brain of adult mice, thymus of newborn mice and some leukaemias. Natural thymocytotoxic autoantibody in NZB mice was an IgM-globulin as determined by sensitivity to 2-mercaptoethanol treatment and by Sephadex G-200 column chromatography in contrast to other natural antibodies (antinuclear, antierythrocyte and G antibodies) of IgG-globulin class. NZB mice also produced natural antibodies against thymocytes of the rat and the hamster; these antibodies were species-specific and did not react with the thymocytes of any but the homologous species.     

Abnormal thymic expression of epithelial cell adhesion molecule (EP-CAM) in New Zealand Black (NZB) mice

New Zealand Black (NZB) mice have been well documented to have a variety of thymic epithelial cell microenvironmental abnormalities, including disruption of corticoepithelial cell networks and medullary cell clusters. These abnormalities of the thymic stromal network are particularly important because similar observations have been noted in other models of murine lupus. Thymic epithelial cells, a key component of the microenvironment, play an important role in selection of the mature T cell receptor repertoire. Recently, a homotypic calcium-independent human and murine epithelial cell adhesion molecule, Ep-CAM, has been described which is located at the thymocyto-cortical cell junction. The function of Ep-CAM is still unclear but its unique location within the thymus suggests that it is critical in the process of providing maturation signals. Consequently, we examined the thymic expression of Ep-CAM in a series of autoimmune prone mice by thymic distribution of Ep-CAM in NZB, NZW, NZB/W, BXSB-Yaa, MRL- lpr/lpr, C3H- gld/gld and the control strains BALB/c, C57BL6, C3H and MRL(+/+), by immunohistology and flow cytometry. Interestingly, NZB mice are similar to control mice from day 4 to 2 weeks of age, having a very low expression of Ep-CAM at the thymocyto-cortical junction. In control strains, there is a marked increased in expression of Ep-CAM beginning at 5 weeks of age. In contrast, NZB mice fail to show significant expression of Ep-CAM even well into adulthood. This abnormality of NZB mice was also noted in NZB/W F1 and BXSB mice, but not MRL- lpr/lpr or C3H- gld/gld mice. Given the potential importance of Ep-CAM in thymic selection, this study provides important evidence that a defective stromal microenvironment is likely to be of etiological significance in the susceptibility of NZB to autoimmune disease.     

Studies on the NZB mouse thymus. I. Thymus weight relationships to age and body weight from birth to old age

The relationship of age and body weight to thymus weight in mature NZB mice is reported. Multivariate analysis of data obtained from nearly 1000 mice shows that the decrease in the weight of the thymus, which occurs during thymic involution, may be represented by a smooth curve which flattens out about the twentieth lunar month of age. NZB mouse thymus weight does not appear to be influenced by Coombs test positivity and involution patterns are similar to those of non-autoimmune mouse strains.     

The disease of the NZB mouse: examination of exchange ovum transplantation derived NZB and CFW mice

Exchange ovum transplantation has been employed between the autoimmune NZB and normal CFW mice. The features of the disease in these mice have been examined together with a small group of milk fostered animals.

Ovum transplanted NZB uterine nurtured to term from the CFW develop a positive Direct Coombs' Test (DCT) and, like normally derived NZB, this reaction, once positive, is usually maintained. Positive DCT reactions were also, on occasions, seen in the CFW uterine nurtured or milk fostered from the NZB, but these were always transient. Although anaemia and reticulocytosis characterize normal, ovum transplantation derived and milk fostered NZB, these changes could not be demonstrated in either the uterine nurtured or milk fostered CFW mice. Histological examination of the thymus, kidney and spleen of these CFW mice also failed to reveal any morphological feature present in either ovum transplantation or normally derived NZB.

The fact that the disease in the NZB was not altered in spite of being uterine nurtured from a normal strain, suggests that the stimulus to develop the disease must be present prior to the stage of implantation. The transient nature of the positive DCT reactions in the CFW, and anaemia and morphological changes not being demonstrated, suggests that stimulation to develop autoantibodies might be common in mice but this process is normally suppressed and it is the age related failure of this recognition and suppression process that accounts for the progressive disease of the aged NZB.

     

B Cells are Selectively Associated with Thymic Cortical but not Medullary Pathology in NZB Mice

Abnormal expansion of autoantibody-synthesizing B cells and self-reactive T cells, which most likely escape negative selection within the thymus, have both been characterized and reasoned to play a role in the pathogenesis of autoimmunity in NZB mice. Support for this thesis has been our observation that NZB mice have severe cortical and medullary thymic microarchitectural defects. As a means to dissect the roles of T and B cells in the induction of such abnormalities, B cell-deficient NZB mice were bred by backcrossing the Igh6(null)allele on to the NZB background (NZB-muMT mice). Such mice showed undetectable levels of autoantibodies. NZB-muMT mice, as compared to wild-type NZB mice, had lower absolute numbers of CD4(+)T cells. Furthermore, thymic abnormalities in NZB-muMT mice were restricted to the medulla. These data suggest that, while B cells may play a role in thymic cortical abnormalities, the medullary abnormalities are induced by other mechanisms.     

Comparison of T cell-mediated immune responsiveness of NZB, (NZB x &NZW)F1 hybrid and other murine strains.

The age-dependent capacity of NZB and (NZB x NZW)F1 hybrid, BALB/c, DBA/2, C57BL/6 and C3H mice to generate T cell-mediated immune responses was assessed qualitatively and quantitatively by measuring the following effector functions: (a) the time course of alloreactive cytotoxic T-cell activity triggered in vitro was comparable for NZ and other mouse strains; cell reactivity generated in vivo against EL4 tumour cells was low in young (NZB x NZW)F1 mice and in DBA/2 mice but was comparable for older (NZB x NZW)F1, NZB and other mouse strains; (b) the time-dependent, vaccinia virus-specific, cytotoxic T-cell activity after systemic infection was similar for all mouse strains; (c) the T cell-dependent primary footpad swelling after local injection with lymphocytic choriomeningitis virus was within the same range for all mouse strains tested with respect to size and kinetics of the reaction; (d) the cell-mediated immune protection against Listeria monocytogenes after systemic infection revealed that NZ mice are, independent of age, more susceptible than C3H or C57BL/6 mice and comparable to A strain mice. Therefore, these responses in young, or clinically relatively normal older, NZB or (NZB x NZW)F1 strains that are affected by a lupus-like autoimmune disease did not differ markedly from the range of responses of other mouse strains of 2-14 months of age, which are not known to be similarly diseased. Thus, overall cell-mediated immunity of NZ mice as assessed quantitatively and kinetically in these functional models is within normal ranges. Possible T-cell defects may therefore be selective and either do not occur or were not detected in these models.     

The genetic contribution of the NZB mouse to the renal disease of the NZB x NZW hybrid.

The occurrence of lupus nephritis in (NZB x NZW)F1 mice appears to depend on the action of at least two dominant or co-dominant genes (at least one gene from each parent) as neither of the inbred parental strains shows the disorder. Identifying affected animals by antemortem determinations of renal function, using improved methods of measuring proteinuria and renal clearance, we have studied the incidence of the renal disease in 230 (NZB x NZW)F1 x NZW backcross mice. The incidence was 49-6% which indicates that NZB strain contributes only one gene, or cluster of closely linked genes, to the renal disorder of the F1 hybrid. The gene(s) must be dominant or co-dominant, as it expresses its effect in the heterozygous state. Study of the H-2 status of the backcross mice showed a loose linkage of the NZB renal disease gene(s) to the D end of the H-2 complex, the crossover frequency being 32-6+/-3-1%.     

Lymphocyte subpopulations in the thymus of NZB x NZW mice: phenotypic characterization by flow cytofluorometry analysis.

The composition of the thymocyte population was investigated as a function of age in the autoimmunity-prone NZB x NZW F1 (NZB x W) female mice and in control BALB/c female mice. Single- and two-colour flow cytofluorometry analyses were used to quantitate the cell surface binding of fluorochrome-conjugated antibodies directed against various lymphocyte markers and of fluoresceinated peanut agglutinin (PNA). In both mouse strains, two major phenotypically distinct thymocyte subpopulations were thus identified. The predominant subpopulation was characterized as bright Thy-1+, Lyt-1 + 2+ and bright PNA+, and the other one as dull Thy-1+, Lyt-1 + 2- and dull PNA+. The relative frequencies of these two subpopulations were similar in NZB x W and BALB/c mice at 3 months of age. However, from 6 months onwards, slight but significant differences became detectable between the two strains. Thus, in BALB/c mice, both thymocyte subpopulations regressed at approximately the same rate during ageing so that their relative proportions remained constant. In contrast, in NZB x W mice, while the number of bright Thy-1+ cells diminished as in BALB/c mice, the number of dull Thy-1+ cells barely varied from 3 to 12 months of age, which resulted in a proportional increase of this latter subpopulation. Moreover, elevated frequencies of surface immunoglobulin-bearing cells were recorded in the thymus of 8-12 month old NZB x W mice but not in BALB/c mice. Therefore, the development of autoimmunity in NZB x W mice appears associated with an abnormal age-dependent evolution of the intrathymic lymphocyte population.     

Age related changes in auto-erythrocyte rosettes in the C57BL/6J and NZB/BINJ mice.

The changes in the auto-erythrocyte rosetting thymic and splenic lymphocytes and the induction of autoimmunity was followed with age in C57BL/6J and NZB/BINJ mice. The auto-erythrocyte rosetting cells (auto-RFC) showed shifts in their pattern in both thymus and spleen in C57BL/6J and NZB/BINJ mice. Both strains had approximately the same percentage (approximately 3%) of thymic auto-RFC at 1 month of age. In C57BL/6J mice the rosette population increased to 6.6% by 2 months, declined after 3 months and subsequently increased gradually with age. In contrast, the NZB/BINJ thymic auto-rosettes peaked at 4 months and gradually declined thereafter. Both the NZB/BINJ and C57BL/6J strains were tested for the presence of anti-erythrocyte antibodies by the direct Coombs' agglutination test. The results showed that at 6 and 10 months 50% and 90% of the NZB/BINJ mice were positive for antibodies, respectively, and the thymic and splenic auto-RFC dramatically decreased in numbers. In the C57BL/6J mice during this same period, very low incidence of auto-antibodies was detected by the Coombs' test and auto-RFC increased in numbers.     

A study of mast cells in autoimmune NZB/W F1 mice: possible relationship between mast cells and increased vascular permeability in the thymus of NZB/W F1 mice.

We examined the possible relationship between thymic mast cells and increased vascular permeability in the thymus of autoimmune NZB/W F1 mice. Light-microscopic observation of tissue sections showed that non-autoimmune BDF1 mast cells increased with age. In contrast, autoimmune NZB/W F1 mast cells did not increase in the thymic parenchyma at the age of 9 weeks. However, NZB/W F1 mast cells resumed the age-associated increase from the age of 12 weeks and exceeded the number of BDF1 mast cells at the age of 30 weeks. Blood histamine levels of 9-week-old NZB/W F1 mice were higher than those of BDF1 mice of comparable age. Furthermore, peritoneal mast cells of NZB/W F1 mice were more sensitive to compound 48/80 than those of BDF1 mice. Increased blood histamine levels of NZB/W F1 mice seem to be due to the enhanced histamine release from mast cells. These results suggest a possible correlation between the high histamine levels by degranulation of mast cells and increased vascular permeability in the thymus of NZB/W F1 mice.     

Thymic function in NZB mice. I. Duration of thymic function in New Zealand Black (NZB) mice

Ageing 6–9-month-old NZB mice have been grafted with a syngeneic new-born thymus. Rapid normalization of the initially low level of circulating thymic factor (TF) and of responsiveness of spleen cells to phytohaemagglutinin (PHA) and concanavalin A (con A) was observed. However, this restoration was transient, both serum TF and mitogen responsiveness returning to the initially low levels within a month. These data support the idea that NZB mice present an early depression of thymus endocrine function, independent of anti-thymus antibodies and that recovery of mitogen responsiveness may be transient, directly dependent on serum TF level.     

Immunologic abnormality in NZB/W F1 mice. Thymus-independent expansion of B cells responding to interleukin-6.

We have previously reported that B cell abnormality in NZB/W F1 mice developed independently of thymus. Here we examined further whether B cells from NZB/W F1 mice respond to interleukin-6 (IL-6), a factor for terminal differentiation of B cells. When freshly isolated splenic B cells were incubated for 5 days in the presence of human IL-6, an increased production of both IgM and IgG, including anti-DNA antibody, was evident in NZB/W F1 mice; there was no increase in BALB/c mice. A magnitude of augmentation in IgG but not IgM production by IL-6 became more apparent in older NZB/W F1 mice. The increased immunoglobulin production seen with IL-6 was neutralized by treatment with rabbit anti-recombinant human IL-6 antibody. As B cells from athymic NZB/W F1 nude mice also responded to IL-6, it was suggested that B cells in NZB/W F1 mice differentiated into the IL-6-responding state in a thymus-independent manner. This B cell abnormality may be associated with the pathogenesis of autoimmune disease in NZB/W F1 mice.     

Lashley maze learning deficits in NZB mice.

In a prior study we found excellent Lashley III maze learning in BXSB mice and poor learning in NZB mice, despite the fact that both strains are autoimmune and develop cortical ectopias. This prompted us to examine NZB Lashley maze performance in detail, including comparisons to other strains and attempts to improve performance by giving additional trials with or without additional intramaze visual cues. In conventional Lashley testing (10 trials), RF mice (non-autoimmune and nonectopic) and BXSBs performed well in the Lashley maze. They had high learning indices and few errors. NZB mice performed poorly, with low learning indices and many errors. Even with additional trials or additional trials plus intramaze cues, NZB performance remained poor. The number of backward and forward errors stayed high; learning indices were low. Since both BXSB and NZB mice develop autoimmune disorders and cortical ectopias, it is unlikely that differential Lashley performance is the result of the presence of these phenomena. NZB mice are known to have alterations in their hippocampal morphology, and this is a possible mediator of the Lashley deficit.     

Requirement of H-2 heterozygosity for autoimmunity in (NZB X NZW)F1 hybrid mice

In the F1 hybrid of autoimmune New Zealand Black (NZB) and phenotypically normal New Zealand White (NZW) mice, there occurs a severe systemic lupus erythematosus (SLE)-like autoimmune disease more fulminant than that found in the parental NZB mice. To determine the role of the H-2 complex in the pathogenesis of autoimmune disease of the (NZB X NZW)F1 hybrid, we developed H-2-congenic NZB (NZB.H-2z) and NZW (NZW.H-2d) strains, and compared the degree of autoimmune features between congenic H-2d/H-2d and H-2z/H-2z homozygous F1 hybrids and the original H-2d/H-2z heterozygous (NZB X NZW)F1 hybrid. We found that autoimmune features such as productions of IgG class anti-DNA antibodies and retroviral gp70 immune complexes and the development of renal disease were to a great extent reduced in both H-2 homozygous F1 hybrids, as compared with the H-2 heterozygous (NZB X NZW)F1 hybrid. It would thus appear that the heterozygosity of H-2d haplotype derived from NZB and H-2z from NZW is essential for the autoimmune disease characteristic of the (NZB X NZW)F1 hybrid.     

Spontaneous antiidiotypic antibodies to the NZB Coombs autoantibody.

We have further investigated the phenomenon of spontaneous anti-(Coombs) antiidiotypic antibodies in the F1 hybrids of New Zealand black (NZB) and CBA mice. These mice show an age-related increase in incidence of such antiidiotype during the first year of life. There is no difference between males and females in the occurrence of antiidiotype. Reciprocal hybrids are both affected, so that maternal influence from the NZB strain is not critical. The antiidiotype also occurs in spite of the xid gene. We have so far detected such spontaneous antiidiotype only in (CBA X NZB)F1 hybrids and not in hybrids of NZB with two other strains or in a variety of recombinant inbred strains between NZB and C58. Our results to data suggest extensive shared idiotypy among NZB mice and a limited number of total idiotypes.     

Evidence for elevated prothymocyte activity in the bone marrow of New Zealand Black (NZB) mice. Elevated prothymocyte activity in NZB mice.

New Zealand Black (NZB) mice spontaneously develop an autoimmune syndrome similar to that of systemic lupus erythematosus. Numerous abnormalities in T lymphocyte development have been reported in NZB mice, and the autoimmune syndrome can be adoptively transferred to naive recipients using bone marrow cells. In the present study, we have used an adoptive transfer system to study quantitatively the relative prothymocyte activity of marrow in either young (4-6 weeks of age) or older (8-10 months of age) NZB mice. Our results demonstrate that NZB marrow has approximately 7-fold more prothymocyte activity than that of marrow in SEA mice, a histocompatible, non-autoimmune prone normal strain of mice. This was ascertained by a competitive repopulation assay, in which mixtures of NZB and SEA prothymocytes were compared directly for their ability to repopulate the thymus of adoptive recipients. This increase in prothymocyte activity in the primary recipient of NZB marrow was associated with an increased competitive advantage of NZB marrow prothymocytes over that of SEA marrow prothymocytes in repopulating the hemopoietic (bone marrow) compartment of the primary host. These findings suggest that elevated prothymocyte activity in NZB mice, along with our previously presented evidence for abnormalities of thymocytopoiesis in NZB mice, may be important in their predisposition for autoimmunity.     

Accelerated autoimmune disease and lymphoreticular neoplasms in F1 hybrid PN/NZB and NZB/PN mice

This report describes the first studies of inheritance of autoimmunity in inbred Palmerston North (PN) mice, a model of systemic lupus erythematosus (SLE). Mating of PN mice with the nonautoimmune DBA/2 strain produced evidence that PN disease had a recessive mode of inheritance. When PN mice were crossed with autoimmune NZB mice, female offspring from both crosses developed anti-DNA antibodies and died prematurely with vasculitis, renal disease, and lymphomas. In contrast, reciprocal hybrid males had different patterns of mortality; PN/NZB males from the PN female X NZB male mating had moderately prolonged life spans, whereas NZB/PN males from the opposite cross (NZB female X PN male) had prolonged survival to the mean age of 104 weeks. To determine if testicular hormones were solely responsible for increased longevity in hybrid males, PN/NZB and NZB/PN mice were castrated at 2 weeks of age and compared to sham-operated littermate controls. Prepubertal castration did not influence longevity in PN/NZB males, but loss of gonadal hormones significantly reduced life spans in reciprocal NZB/PN males. Female hybrids were not affected by oophorectomy. Because castration changed disease expression only in male hybrids from the NZB female X PN male cross, it was concluded that parentage influenced sensitivity to the protective effects of male hormones. Although surgical sterilization had disparate effects on males, castrated PN/NZB and NZB/PN males consistently outlived oophorectomized females. The lack of clear-cut reversal of disease in males subjected to early castration suggested that nonhormonal, possibly genetic, factors contributed to longevity in both groups of male hybrids.     

Strain differences in the immune response of mice: III. A raised tolerance threshold in NZB thymus cells

Irradiated (NZB×BALB/c)F1 mice were injected with syngeneic bone marrow cells, syngeneic or parental thymus cells, and sheep red cells. The antibody plaque-forming cell response depended on the number of sheep cells and the age and strain of the thymus cells. Young or adult BALB/c thymus, and young hybrid thymus, responded best to low numbers of sheep cells; with higher numbers they became tolerant. Adult hybrid thymus, and young or adult NZB thymus, responded better to high numbers of sheep cells. Hybrid mice irradiated and restored with BALB/c bone marrow developed thymus cells with the reactivity of BALB/c thymus. It is argued that NZB mice, and older hybrids, may develop autoimmunity because of an abnormality of tolerance induction manifested in their thymus cells, but of bone marrow origin.     

Increased vascular permeability of Brucella abortus bacilli in the thymus of NZB/W F1 mice.

Various amounts of the bacterium, Brucella abortus (BA) were injected intravenously into autoimmune NZB/W F1 mice and non-autoimmune BDF1 mice and then the localization of BA in the thymus was traced using an immunohistochemical method at 30 min and 3 h after injection. The results showed that a greater amount of BA became consistently localized in the thymic parenchyma in a free form or in a phagocytized form in NZB/W F1 mice in comparison with BDF1 mice, indicating a marked increase of vascular permeability in the thymus of NZB/W F1 mice. The extravascular leakage of BA was clearly dose-dependent. The significance of invasion of bacterial antigens from the general circulation into the thymic parenchyma is discussed in relation to autoimmune states.