Characteristics of histiocytic lesions in the reticuloendothelial system of NZB mice

The so-called Potter's lesion, previously described as preneoplastic in the lymph nodes of C58 mice, develops frequently in autoimmune NZB mice. These lesions were characterized in the present study by bands or sheets of pale-staining histiocytic cells in the cortex and medulla of the lymph node, and multiple small nodules of the same cells were found in the red pulp of the spleen and the liver. Electron microscopically, the cells had pleomorphic cytoplasm with long processes, electron-dense bodies, abundant mitochondria, and a characteristic labyrinth structure with many C-type viruses. Mac-1 antigen, IgG-Fc receptor, ferritin, and ACPase activity were identified on these cells. Intraperitoneally-injected iron colloids were found in the lesions of the spleen and liver but not in those of the lymph nodes. The lymph node lesions appeared when the mice were about 3 months of age and enlarged until the mice were around 10 months old, after which they gradually receded and were replaced by small vessels and fibroblastic cells. These data indicate that the lesions represent reactive hyperplasia of the macrophage system and may have no direct association with the development of malignant lymphoma in NZB mice.     

Studies on Lymphocyte Homing in Autoimmune Prone NZB Mice

Lymphocyte homing patterns in young (3-5 months old) and old (10-12 months old) autoimmune prone NZB mice were investigated by transferring ⁵¹Cr labelled lymphoid cells into syngeneic and H-2 compatible allogeneic recipients. We confirmed that non H-2 alloantigens as well as H-2 alloantigens can be important determinants of apparent abnormalities of cellular distribution with the techniques employed. No gross abnormalities of lymphocyte traffic were present in the young NZB mice as compared to the autoimmune resistant strains of mice when syngeneic cells are used. Spleen of older NZB mice appeared to be less attractive to lymph node cells than was the spleen from young NZB mice. Splenocytes of older NZB mice localized significantly more in the liver and less in the lymph nodes as compared with splenocytes from young NZB mice. The mechanisms underlying abnormalities of lymphoid cell distribution which feature the autoimmune-prone NZB mice are not yet clear and further studies will be necessary before they can be characterized definitively. Our findings, using syngeneic cells, are in disagreement with those of Zatz and Lance since evidence of abnormal distribution of lymphocytes in young NZB mice were not seen when syngeneic cells were employed.     

Age-decrease of cells sensitive to an autoantibody-specific for thymocytes and thymus-dependent lymphocytes in NZB mice

NZB mice naturally produce an autoantibody which in the presence of complement is specifically cytotoxic for thymocytes and thymus-dependent lymphocytes (T-cells) in the peripheral lymphoid tissues (lymph nodes and spleen) and the circulation of mice. Using a direct cytotoxicity test with a NZB mouse serum pool which contained the high titred autoantibody, a progressive decrease was observed with age in the proportion of the autoantibody-sensitive cells in mesenteric lymph node, spleen, and blood of NZB mice in comparison with mice of other strains (C57BL/6J and NZW). The numerical decrease in the population of autoantibody-sensitive cells was evident at younger age and greater degree in the peripheral blood than in the lymph node and spleen. The age-decrease in the number of autoantibody-sensitive cells in lymph node and spleen contrasted with the numerical increase in nucleated cells in these organs. The age-decrease in the proportion and number of the autoantibody-sensitive cells in the blood exceeded the decrease in the blood lymphocyte count. This finding indicated that T-cells in the blood are selectively depleted with the ageing of NZB mice. A similar observation was made on the blood lymphocytes of (NZB × NZW)F₁ hybrid mice. The depletion of T-cells in the blood in association with the production of natural thymocytotoxic autoantibody is termed autoimmune thymus-dependent lymphocytopenia.     

Changes in lymphoid populations of ageing CBA and NZB mice

Changes in subpopulations of lymphoid cells of normal (CBA) and autoimmune (NZB) mice were studied as a function of age, by observing migration patterns of ⁵¹Cr labelled lymph node, spleen and thymus cells from donors aged 8 days to 12 months. The method permits analysis of the proportions and numbers of recirculating and non-recirculating lymphocytes in lymphoid compartments. Changes in the lymphoid populations of CBA mice were found, which could be attributed to the normal processes of maturation and senescence. In NZB mice relative and absolute decreases in the recirculating cell content of lymph node and spleen were observed which coincided with the time of development of autoimmunity. The significance of these results, in relation to altered immunocompetence with age, is discussed.     

The appearance of immunological competence at an early age in New Zealand black mice

Adult and 5-day-old New Zealand Black (NZB) and Ju control strain mice were injected with sheep red blood cells. At various times after injection they were killed and the spleens, lymph nodes and thymus tested for haemolysin-producing cells. When the response was expressed as plaque-forming cells (PFC) per million viable cells, the response curve of the spleens of baby NZB mice was very similar to the response in the spleens of the adults, and the response in the lymph nodes of the babies was as high and more sustained than that of the adults. In Ju control strain baby mice of this age, the response in the spleen and the lymph nodes was both reduced and delayed compared with the adults. Neither strain gave a significant response in the thymus. The spleen and lymph nodes of adult NZB mice showed a response which was delayed but not reduced as compared with the adults of the Ju control strain, whereas in baby NZB mice the spleen and lymph nodes showed a response which was advanced and increased (particularly in the lymph nodes) compared with control strain babies (Ju, Swiss, C57B1, CBA). The NZB mice did not reach this level of responsiveness until they were 4–5 days old.     

Studies on thymus products. IV. Absence of serum `thymic activity'' in adult NZB and (NZB × NZW) F1 mice

New Zealand Black (NZB) mice and (B/W) F1 hybrids have a normal level of serum `thymic activity' (TA) at birth but this level decreases prematurely between the third and sixth weeks of life. At 2 months, NZB and NZ (B/W) F1 mice have no significant TA, whereas TA is still at birth's level in six control mouse strains and remains at this level until the fourth to the sixth month. Six weeks after the decline of serum TA, NZB mice show disappearance of theta-positive lymph node rosette-forming cells (RFC) and 2 weeks later, progressive decrease in spleen RFC sensitivity to anti-theta serum (AΘS) and azathioprine (AZ) as in neonatally thymectomized CBA mice. Normal AZ and AΘS sensitivity of spleen and lymph node RFC is reconstituted by in vitro or in vivo treatment by thymic extracts. The early thymic abnormalities found in NZB mice are in keeping with the thymic medullar epithelium atrophy reported in newborn NZB mice.     

Autoimmune reactions and malignant changes in New Zealand black mice

Although NZB mice were bred and maintained in a germ-free environment their spleens enlarged and showed a sequence of histological events concomitant with the advent of positive antiglobulin (Coombs) reactions at 8–10 months which were similar to, but less intense than, those of their conventional NZB counterparts. The numerous large follicles with prominent germinal centres which developed in the white pulp and the proliferations of large pyroninophilic cells in the red pulp thus represented a humoral autoimmune reaction uncomplicated by external microbial antigenic stimuli. This burst of immunological activity in the spleen was followed by a reticulum cell neoplasia (apparently originating within the follicles and from the perifollicular mantles) which was transferable by intraperitoneal injection of spleen cell suspensions to syngeneic and allogeneic (BALB/c) recipients. By comparison, the inguinal lymph nodes of these same germ-free NZB mice were both immunologically inactive and exempt from the malignant process. Lesions in the thymus, and kidney lesions resembling human membranous glomerulonephritis or lupus nephritis, were found in both germ-free and conventional mice of this strain. Possible relationships between the autoimmunity, malignancy and the virus-like particles known to be present in germ-free NZB mice are discussed.     

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.     

T and B lymphocytes in New Zealand Black mice: an analysis of the theta, TL and MBLA markers

The lymphocytes of thymus, blood, spleen, lymph nodes and bone marrow were studied in NZB mice between the ages of 1 and 14 months, and compared with lymphocytes of A and CBA strain mice of the same age. By standard cytotoxicity techniques, the proportion of cells possessing the θ, TL and MBLA markers was found to be similar in NZB in mice and controls. In 14-month old NZBs, whose spleen was largely replaced by reticulum cell sarcoma, and in younger recipients of the passaged tumour, there was a reduction in the percentage of θ and MBLA-positive cells in the spleen. In a few mice at 4 and 9 months, small numbers of MBLA-positive cells were present in the thymus and there was a corresponding decrease in θ-positive cells. TL-positive cells were not present outside the thymus, and θ-positive cells were not present in the bone marrow in unusual numbers. NZB peripheral lymphocytes appeared to have the same surface concentration of θ as those of A or CBA mice, as judged by anti-θ titration curves. The reticulum cell sarcoma was shown to be θ-negative and MBLA-negative, while an NZB thymoma was θ-positive, TL-positive and MBLA-negative. It was concluded that the peripheral lymphoid organs contain a large population of T lymphocytes of abnormal character.     

Particles resembling murine leukaemia virus in New Zealand Black mice

Particles which morphologically resembled murine leukaemia virus were detected by electron microscopy in the tissues (spleen, thymus, inguinal lymph nodes, bone marrow or pancreas) but not in serum or plasma pellets of untreated conventional New Zealand Black (NZB) mice aged 1–82 weeks. They were also found in the corresponding tissues of NZB mice which had been thymectomized shortly after birth. The presence of similar particles in the spleen, thymus or pancreas of conventional NZB embryos and, additionally, in the lymph nodes of NZB mice which had originally been introduced into a germ-free environment by Caesarian section and fostering on germ-free mice of another strain, suggests that the virus is transmitted `vertically' through the germ cells or placenta. Preliminary investigations showed similar particles in the organs of conventional F₁ (NZB × NZW) hybrid and New Zealand White (NZW) mice.

Large numbers of particles also resembling murine leukaemia virus were found in the spleen and in plasma or serum pellets of young conventional NZB mice which had developed reticulum cell neoplasia following serial passage of lymphoid cell suspensions from ageing conventional NZB donors.

The possible relationship of these particles to the autoimmune reactions and malignant changes which occur spontaneously in conventional NZB mice is discussed.

     

Hyperactive T-cell function in young NZB mice; increased proliferative responses to allogenic cells.

The one-way mixed lymphocyte reaction was employed to study proliferative responses to antigens by mature, immunocompetent T cells from NZB mice 3 weeks to 4 months old. Compared to cells from control mice of the same H-2 type, thymus, spleen and lymph node cells from NZB mice were hyperactive in this response. The results are discussed in relation to possible effects of chronic stimulation by endogenous type C leukaemia virus upon differentiation of functional T cells or upon regulation by T cells of other T-cell functions, including augmentation of antibody responses.     

Small lymphocytes in peripheral lymphoid tissues of nude mice. Life-span and distribution.

The distribution of small lymphocytes according to life-span in the peripheral lymphoid tissues of the mouse mutant "nude" has been studied by means of auto-radiography and scintillation counting to evaluate the localization of B lymphocytes with varying life-span. The vast majority of the lymphocytes in this congenitally athymic mouse are relatively long-lived, although few cells live for 6 weeks or more. Differences in labelling percentages of blood, spleen and lymph node lymphocytes indicated a production of lymphocytes with a short residence time in the spleen. A similar production was not seen in the lymph nodes. While the lymphocytes in the spleen were evenly distributed according to life-span, the paracortical lymphocytes in lymph nodes were found to have a generally shorter life-span than those of the cortex, in opposition to findings in normal mice. The cortical cells which were by far the most numerous in the lymph nodes seemed to be more sessile than para-cortical lymphocytes. The life-span of these latter cells are comparable to those of thoracic duct lymphocytes, and the scarcity of cells in the paracortex reflects the small number of recirculating lymphocytes in nude mice.     

Malignant changes in New Zealand black mice

Ageing, Coombs positive, NZB mice, may spontaneously develop a neoplasia of the reticulum cell type which can be transferred by serial passage of their lymphoid tissues in young intact or neonatally thymectomized syngeneic recipients. The recipients (103/117) of cell suspensions prepared from the enlarged spleens and/or lymph nodes of four such donors developed an extensive and lethal reticulum cell neoplasia affecting the spleen, lymph nodes, lungs and liver but the bone marrow, thymus and kidneys were seldom involved. The recipients (16/17) of spleen cells from a fifth donor showed massive proliferations of eosinophils in all the organs examined.

Prematurely positive antiglobulin (Coombs) reactions were detected in only two recipients. Although there was an indication that the IgM content of the sera decreased as one of the passages progressed, the levels of IgG and IgA were not seriously distorted.

Particles resembling murine leukaemia virus were identified by electron microscopy in the spleen and in plasma or serum pellets of passage recipients. However, similar particles were also seen in the thymus and/or spleen or bone marrow of untreated NZB mice including an 18-day embryo and animals aged 1–56 weeks, although no particles were found in plasma and serum pellets of mice aged 6–70 weeks.

The theory that autoimmunity, malignancy and virus infection are directly related is discussed.

     

HBV转基因小鼠肝脏NKT细胞功能与PD1、CD28表达分析

目的研究HBV转基因小鼠肝脏中NKT细胞的功能与表面PD1、CD28表达的关系。方法分离小鼠肝脏、脾脏、胸腺和腹膜淋巴结单个核细胞,利用流式细胞检测技术,分别检测其淋巴细胞中NKT细胞的频率,同时检测肝脏NKT细胞PD1、CD28的表达及IFN-γ、IL-4的分泌功能,比较肝脏、脾脏、胸腺和腹膜淋巴结这几个主要免疫组织淋巴细胞中NKT细胞所占的比例,并分析肝脏NKT细胞PD1、CD28的表达与细胞功能的关系。结果与正常同品系小鼠比较,HBV转基因小鼠肝脏、脾脏、胸腺和腹膜淋巴结NKT细胞数量明显减少(P<0.05),与脾脏、胸腺和腹膜淋巴结相比,肝脏淋巴细胞中含有大量的NKT细胞;与正常同品系小鼠比较,HBV转基因小鼠肝脏NKT细胞PD1的表达明显增多(P<0.05),CD28的表达明显减少(P<0.05),肝脏NKT细胞IFN-γ、IL-4的分泌功能明显降低(P<0.05)。结论肝脏中含有大量的NKT细胞,HBV转基因小鼠肝脏NKT细胞的功能存在明显的缺陷,并提示PD1的增加和CD28的降低可能与NKT细胞功能的下调密切相关。     

Features of renal vasculitis in autoimmune MRL lpr/lpr mice: phenotypes and functional properties of infiltrating cells.

MRL lpr/lpr (MRL/1) mice spontaneously develop a widespread renal vasculitis. The majority of the cells in vasculitic lesions are bright Ly-1, L3T4 and la-positive in contrast to the cells found in lymph nodes and spleens of the old MRL/1 mice. However, despite differences in phenotypical patterns, B and T cells from arteritic lesions do not differ from mononuclear cells (MNC) eluted from MRL/1 lymph nodes with regard to the frequency of IgG secreting cells and the proliferative responses to Concanavalin A (Con A). Co-culture experiments with congeneic MRL+/+ (MRL/n) spleen cells indicate that the poor response to Con A of the MNC eluted from vasculitic lesions is, unlike the case of lymph node MNC, due to suppressive action of vasculitic cells on the indicator cell population. Further support for the activation status of infiltrating MHC in kidney vasculitic lesions, expressed by high in vivo uptake of 3H-thymidine, was obtained by autoradiography performed on frozen sections. The observed differences in phenotypic patterns and functional features between lymph node MNC and infiltrating vasculitic MNC indicate that different immune mechanisms may be responsible for the development of lymphadenopathy and vasculopathy, respectively in MRL/1 mouse.     

Spontaneous increase of plasma-like cells with high GANP expression in the extrafollicular region of lymphoid organs of autoimmune-prone mice

Autoimmune-prone mice bear a hyper-active B cell population generated spontaneously in peripheral lymphoid organs. Expression of beta RNA-primase GANP was shown to be an activation marker in lymphoid follicle germinal center (GC) B cells after immunization with T cell-dependent antigen (TD-Ag) in normal mice. In this study, we examined the expression of GANP in lymphoid tissues of autoimmune-prone mice. GANP expression was up-regulated in GC-B cells after stimulation with TD-Ags; however, highly GANP-positive (GANP(hi)) cells were also observed in lymph nodes of non-immunized MRL/lpr mice. GANP(hi)cells in lymph nodes as well as in spleens of the different autoimmune-prone strains, MRL/lpr, NZB, (NZBxNZW)F1 and BXSB, gradually increased with age. This population was detected only in small numbers in the red pulp region of the spleen after immunization with TD-Ag in normal C57BL/6 and BALB/c mice. GANP(hi)cells had a B220(-)IgM(+)Syndecan-1(+)phenotype, but were negative for PAS-staining and bromo-deoxyuridine incorporation. These results demonstrate that GANP(hi)plasma-like cells appear in lymph nodes of autoimmune mice during aging, suggesting that the new plasma cell population might be generated after hyper-activation of B cells during the course of autoimmune disease.     

Perforin mRNA expression in the inflamed tissues of NZB/W F1 lupus mice decreases with methylprednisolone treatment.

Perforin is one of the important cytolytic factors in cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. In this study, the authors examined perforin mRNA levels in the kidney, spleen, liver, lung, heart, and brain of NZB/W F1 lupus mice and NZW mice. Perforin mRNA levels in the kidney, spleen, liver, and lung of NZB/W F1 mice increased significantly with age, whereas those in the heart and brain of NZB/W F1 mice showed little change between 2 and 10 months of age. In all tissues examined in NZW, control mice perforin mRNA levels showed little change during the experimental period. In addition, the authors examined the effect of methylprednisolone (MPSL) on perforin gene expression in the tissues of NZB/W F1 mice. MPSL ameliorated the increase in perforin mRNA levels in the kidney, spleen, liver, and lung of NZB/W F1 mice. These findings suggest that perforin may contribute to tissue injuries in autoimmune lupus mice and that MPSL may be effective in lupus partly by decreasing perforin expression.     

The effect of cyclophosphamide and irradiation on cells which suppress contact sensitivity in the mouse.

Contact sensitivity was produced in mice by painting the skin with picryl chloride and was assessed by the increase in ear thickness following local challenge. Contact sensitivity was passively transferred by immune lymph node and spleen cells taken at 4 days. The mice were then challenged immediately and the reactions read at 24 and 48 hr. Immune lymph node and spleen cells taken at day 8 virtually fail to transfer. Experiment showed that they contain cells which suppress passive transfer. These are demonstrated by mixing approximately equal numbers of 4-day cells, which transfer contact sensitivity, and cells taken at later times and injecting them intravenously into recipients. These 'suppressor cells' can be demonstrated by day 6 and are still present at day 11 after immunization. The precursors of the suppressor cells are sensitive to cyclophosphamide. Irradiation of immune mice 2 days before taking cells also selectively inactivates the suppressor cells. When mice are pretreated with cyclophosphamide before immunization or irradiated 2 days before transfer, the lymph node and spleen cells taken on day 9 after immunization transfer contact sensitivity. In contrast the same number of cells from untreated mice were inactive. This suggests that the cells which mediate passive transfer or their precursors may occur in an inhibited form in lymph nodes and spleen at later times after immunization. These suppressor cells in immune mice differ from the T suppressor cells produced by the injection of picryl sulphonic acid--an agent which causes unresponsiveness: (1) the precursors of the T suppressor cells resist cyclophosphamide; (2) the T suppressor cells are found in mice treated so as to produce unresponsiveness while the other type of suppressor cells occurs in mice immunized for contact sensitivity. However, both types of suppressor cells are selectively inactivated by irradiation as compared with the cells which mediate contact sensitivity and both are able to act on the effector stage of contact sensitivity.     

Selective labeling of mesenteric lymph nodes: cell production and emigration of newly formed lymphocytes to other organs

Lymphocyte production by mesenteric lymph nodes of normal young pigs was studied by intranodal injections of either tritiated thymidine or tritiated deoxycytidine as DNA precursors. One or two days after selective labeling of the mesenteric lymph nodes the relative and absolute number of lymphocytes derived from mesenteric lymph nodes were determined autoradiographically in the following organs: mesenteric, cervical and inguinal lymph nodes, spleen, thymus, bone marrow, Peyer's patches, tonsil, different regions of the gut, lung and liver. The overall cell production of mesenteric lymph nodes, as derived from the sum of all labeled cells one day after labeling, was estimated to be about 7 X 10(9) lymphocytes. Up to 40% of all newly formed lymphocytes had already left the lymph nodes within one day and were found in all organs studied. There was a preferential homing to the mucosa of the small intestine, but a considerable number migrated to the spleen and even to the thymus and bone marrow. In lymphoid organs all labeled cells were small and medium-sized lymphocytes one and two days after labeling. In cervical lymph nodes, spleen, tonsil and Peyer's patches the relative distribution to T and B cell areas was determined. There was an obvious preference of newly formed lymph node cells to home to T cell areas. The differences of labeling between thymidine or deoxycytidine were surprisingly low.     

Selective labeling of mesenteric lymph nodes: Cell production and emigration ofnewly formed lymphocytes to other organs

Lymphocyte production by mesenteric lymph nodes of normal young pigs was studied by intranodal injections of either tritiated thymidine or tritiated deoxycytidine as DNA precursors. One or two days after selective labeling of the mesenteric lymph nodes the relative and absolute number of lymphocytes derived from mesenteric lymph nodes were determined autoradiographically in the following organs: mesenteric, cervical and inguinal lymph nodes, spleen, thymus, bone marrow, peyer'spatches, tonsil, different regions of the gut, lung and liver. The overall cell production of mesenteric lymph nodes, as derived from the sum of all labeled cells one day after labeling, was estimated to be about 7 × 10⁹ lymphocytes. Up to 40% of all newly formed lymphocytes had already left the lymph nodes within one day and were found in all organs studied. There was a preferential homing to the mucosa of the small intestine, but a considerable number migratedto the spleen and even to the thymus and bone marrow. In lymphoid organs all labeled cells were small and medium-sized lymphocytes one and two days after labeling. In cervical lymph nodes, spleen, tonsil and peyer's patches the relative distribution to T and B cell areas was determined. There was an obvious preference of newly formed lymph node cells to home to T cell areas. The differences of labeling between thymidine or deoxycytidine were surprisingly low.