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.     

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.     

Recirculating and sessile B cell populations in normal and CBA/N mice

Chromosomally distinguishable syngeneic mice were parabiosed and the resultant chimerism was followed for 6 weeks in the lymphoid organs, by culturing their cells with polyclonal mitogens, lipopolysaccharide (LPS) for B cells and phytohemagglutinin (PHA) for T cells. As expected of a recirculating population, the T cells equilibrated completely. The B cells in lymph nodes (LN) and Peyer's patches (PP) also equilibrated completely, suggesting that they too are recirculating. B cells in the spleen and blood, however, did not equilibrate over this period. After separation of parabiosed mice, the percentage of partner cells in both the recirculating T and B lymphocyte populations declined steadily, but it continued to rise in the LPS-responsive populations in spleen and peripheral blood suggesting that they were derived from precursor populations which were themselves chimeric. Injection of lymphocytes into CBA/Ca or CBA/N mice showed that LPS-responsive populations in LN and spleen localized differently. These results have been interpreted as demonstrating two major populations of LPS-responsive B lymphocytes in the mouse, one recirculating and the other sessile. The recirculating population appears to be the only LPS-responsive population in LN and PP. In the spleen, however, the recirculating cells constitute about a quarter of the LPS-responsive cells, while the rest are sessile cells. The relationship between these two populations has yet to be clarified. CBA/N mice are deficient in both populations but the sessile one appears to be more severely depleted.     

Evidence for a loss of recirculation capacity of T-cells after antigenic stimulation

The distribution of i.v. injected ⁵¹Cr-labelled normal or antigen-activated thymic or thymus-derived cells (T-cells) was studied in isologous recipients.

Activated lymphocytes were obtained from the spleens and lymph nodes of lethally X-irradiated allogeneic mice infused 5 days earlier with lymphoid cells. Practically all of the cells of these organs were found to be of donor type origin and they exhibited an increased mean cell volume. Both activated and non-activated T-cells exhibited a high accumulation to spleen and liver within 40 hr after injection into isologous recipients. The non-activated cells also exhibited a high tendency to seek to lymph nodes whereas this capacity was largely absent in the activated cell populations. Since the lymph node seeking capacity of lymphocytes is considered to be a specific trait for the recirculating pool of lymphocytes, it is possible that T-cells may temporarily lose their recirculating capacity when activated by an antigen.

     

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.     

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.     

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.     

STUDY OF CONGENITALLY IMMUNOLOGIC MUTANT NEW ZEALAND MICE: V. B CELL FUNCTION OF NZB-Xld MICE

NZB mice bearing the CBA/N X chromosome linked defect were generated by repetitive backcrossing and selection of the X^ld gene. The male offspring resulting from the cross of NZB with CBA/N were selected as being X^ldY on the basis of sera IgM and IgG₃ levels and responsiveness to DNP-Lys-Ficoll. Following this inbreeding protocol, 6th generation backcross NZB X^ldY mice were compared to littermate controls with respect to B cell function. Sera immunoglobulin levels of IgG₁, IgG_2a, IgG_2b and IgA were similar in X^ldY and XY mice. In contrast, levels of IgM and IgG₃, from X^ldY mice were approximately 15% and 50%, respectively, of values found in littermates. Furthermore, X^ldY mice failed to respond to DNP-Lys-Ficoll and had less than 3% splenic Lyb 5.1-bearing cells. Splenic immunoglobulin cell surface profiles, obtained by the fluorescent activated cell sorter, indicated a significant reduction in the frequency of Ig bearing cells in X^ld animals. Such profiles were similar to those obtained for spleen cells from reference control CBA/N mice. Finally, an elevated number of splenic, lymph node and bone marrow background and lipopolysaccharide-induced B cell clones in semi-solid phase agar was found in NZB but not C57BL/6, C3H, BALB/c and DBA/2 controls. In contrast, NZB X^ldY mice had virtually no detectable B cell colonies. This data, obtained on significantly inbred X^ldY NZB mice, suggests that the X^id gene is dominant over several aspects of polyclonal B cell activation in NZB mice and indicates that serial observation of these mice will be valuable in understanding the interactions of genetic immunologic mutations and cellular function in autoimmunity.     

Cytotoxic potential of different lymphoid cell populations against chromium-51 labelled tumour cells

Release of chromium-51 (⁵¹Cr) from pre-labelled target cells was used as an assay of cytotoxicity. Different immune and non-immune lymphoid cell populations were tested for in vitro cytotoxicity against pre-labelled tissue culture target cells (Ha 3) which originated from a tumour induced by murine sarcoma virus (MSV) in a CBA mouse. Anti-Ha 3 peritoneal exudate and spleen cells showed significant specific cytotoxicity against these target cells. This was true for cells taken from animals immunized against either strong (H-2 plus non-H-2) or weak (non-H-2) antigenic determinants on the Ha 3 cells. Exogenous complement was not required for this cytotoxic activity. Tissue culture cells derived from a methylcholanthrene-induced tumour (MBE) of CBA origin were not damaged by peritoneal exudate cells from mice immunized against non-H-2 antigens on the Ha 3 cells. Anti-Ha 3 lymph node cells were relatively ineffective in these experiments although some cytotoxic activity was detected with lymph node cells sensitized against strong antigens. Direct morphological observation confirmed the cytotoxicity of anti-Ha 3 peritoneal exudate cells measured by ⁵¹Cr release.     

Lymphocyte migration patterns in autoimmune MRL-lpr/lpr mice: relationship to age, disease manifestations and lymphocyte homing receptor expression

We have previously reported that lymphoid cells from systemic lupus erythematosus (SLE) mice with established disease migrate aberrantly. This study evaluates the abnormal lymphocyte migration patterns found in MRL-lpr/lpr (MRL/l) mice in relation to age, disease manifestations and the expression of lymphocyte homing receptors. 51chromium-labelled lymph node cells from MRL/l and from normal histocompatible CBA mice of different ages were injected i.v. into age and sex-matched CBA recipients. Diminished lymph node and increased hepatic uptake of MRL/l compared to CBA cells was evident as early as 6 weeks of age. Abnormalities in lymphocyte migration antedated the appearance of elevated antihistone antibody (AHA) levels but not the development of lymphadenopathy. Using the monoclonal antibody MEL-14, no differences in the expression of lymphocyte homing receptors between MRL/l and CBA lymph node cells were found at any age. Thus abnormalities in lymphocyte migration in MRL/l mice appear as early as six weeks and are not related to changes in homing receptor expression.     

Permanent hapten-specific tolerance in B lymphocytes.

Tolerance to the hapten TNP was induced in mice congenic with CBA but bearing the Ig-b allotype (Ig-b mice). To induce a high degree of tolerance it was necessary to give five injections of TNP-sulphonate followed by an immunogenic challenge (alum precipitate of TNP-BSA with pertussis adjuvant). Lymph node or spleen cells from these mice were transferred, with or without an equal number of non-tolerant CBA spleen cells, to irradiated CBA recipients and these were challenged with a different TNP-protein conjugate. Anti-TNP antibody bearing the Ig-b allotype was then assayed separately from total anti-TNP, as a measure of the contributions made by tolerant and non-tolerant B-cell populations respectively. Tolerant lymph node cell did not depress the response of normal cells, nor did the normal cells 'break' the tolerance of the Ig-b population even when the latter had been treated with anti-T-cell serum and complement. No response was obtained from tolerant lymph node cells when the recipients were challenged at different time up to 12 weeks after transfer. By this time the control non-tolerant lymph node cells had also lost the capacity to respond. It is concluded that: (1) effectively permanent tolerance, which is not maintained by afferent mechanisms, can be induced in lymph node B cells; (2) B-cell tolerance can be greatly enhanced by immunogenic challenge; (3) spleen may contain a distinct population of B cells which is less susceptible to tolerance; and (4) the life-span of virgin lymph node B cells is probably less than 12 weeks.     

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.

     

Reduced Capacity to Produce Specific 'Effector' Cells after Injection of CBA Mice with C3H Cells

Lymphocytes from mice of strain CBA are strongly MLC-responsive to lymphocytes from the H-2 compatible- but M-antigen-incompatible strain C3H. This strong reactivity disappears after infusion of CBA mice with C3H lymphocytes. This study shows that the host-versus-graft reactivity (swelling of local lymph node after antigen injection) is specifically reduced after injection of CBA mice with C3H × CBA spleen cells However, lymphocytes from such mice showed a specifically increased GVH reactivity (inhibition of erythroid cell growth) compared with lymphocytes from unimmunized mice. Lymphocytes from normal CBA mice showed a high proliferative rate in the spleens of irradiated C3H × CBA mice. Such 'educated' cells showed strongly increased specific GVH reactivity. Lymphocytes from CBA mice previously injected with C3H×CBA cells showed reduced capacity to proliferate when injected into irradiated C3H × CBA hybrids and a poor capacity to develop new 'effector' cells reactive against C3H × CBA bone marrow target cells. The results indicate that the presence of specifically 'MLC responsive' lymphocytes in a lymphoid cell population is a prerequisite of its production of 'effector' cells able to respond in this GVH assay     

Modulation of B-cell abnormalities in lupus-prone (NZB x NZW)F1 mice by normal bone marrow-derived B-lineage cells.

(NZB x NZW)F1(NZB/WF1) mice spontaneously develop an autoimmune disease characterized by abnormality of haemopoietic stem cells. The present study examined a possible regulatory cell interaction between NZB/WF1 and normal bone marrow cells using radiation-induced chimeras. We demonstrated that the ability of NZB/WF1 bone marrow cells to transfer the typical disease with hypergammaglobulinemia including autoantibodies into lethally irradiated normal recipients was prevented by cotransfer of bone marrow from normal CBA/J mice but not from xid CBA/N mice carrying a selective defect in B-cell function. Flow cytometric analysis revealed that the generation of NZB/WF1 cells was reduced in the mixed chimeras given CBA/J but not CBA/N bone marrow cells. Interestingly, radiation chimeras reconstituted with a mixture of NZB/WF1 bone marrow and CBA/J splenic B cells did not show elevation of serum immunoglobulin levels, although most of the spleen cells were dominated by NZB/WF1 cells. On the other hand, NZB/WF1 B cells maturated in vivo in the presence of CBA/J bone marrow or splenic B cells lost the hyper-responsiveness to lipopolysaccharide (LPS) in the autoantibody production in vitro. These results suggest that radiosensitive normal B-lineage cells have the regulatory activity to ameliorate the hypergammaglobulinemia of NZB/WF1 mice by reducing the generation of NZB/WF1 B cells and/or by correcting their hyper-responsiveness, and that NZB/WF1 mice may have a defect(s) in the regulatory cell function. In addition, CBA/J splenic B cells were shown to modulate the B-cell abnormality even when injected into non-irradiated NZB/WF1 mice manifesting autoimmunity.     

Selective migration of lymphocytes within the mouse small intestine

The factors which determine the migration of lymphoid cells to lamina propria or Peyer's patches of mouse small intestine have been investigated by autoradiographic tracing of intravenously injected spleen, thymus and lymph node cells. The numbers of labelled cells found in antigen-free grafts of foetal small intestine were compared with the numbers in normally sited gut. Thymus, normal spleen and B spleen lymphocytes, labelled with [³H]adenosine or [5-³H]uridine, were confined to Peyer's patches in normal and grafted gut. [³H]Thymidine-labelled lymphoblasts from the mesenteric nodes of young (19–22 days) mice and mice infected with Nippostrongylus brasiliensis were found in the lamina propria of both graft and normal small intestine, but [³H]thymidine-labelled lymphoblasts from oxazolone-primed lymph nodes did not migrate to the villi. The possible roles of intraluminal antigens, source of cells and changes in cell surface receptors during differentiation, in determining the selective migration of cells to the lamina propria and Peyer's patches, are discussed.     

Enumeration of polyclonal mitogen-responsive cells in different lymphoid tissues of the mouse.

The relative number of cells capable of responding to Con A, PHA and LPS in the spleen, blood, lymph node and Peyer's patches of CBA mice has been quantified by means of a cytological analysis technique. No difference has been found between Con A- and PHA-responsive cells in spleen and lymph node. The lymphoid tissues of T cell-deprived mice have a reduced content of PHA responsive cells, but LPS responsiveness is within normal limits. Pretreatment of peripheral lymphocyte populations with high concentrations of anti-O antiserum and complement abolishes the response of the treated cells to PHA, but not to LPS, whereas similar treatment with a cytotoxic anti-immunoglobulin serum, which has no effect on PHA-responsive cells, only partially reduces the response to LPS. The results for mitogen responsiveness are discussed with reference to other methods of quantifying T and B cells using cell-surface markers.     

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.     

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 palestaining 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.     

Quantitative analysis of the chimeric state in mice: III. Comparison of the colonizing capacities of syngeneic cell suspensions from different sources

Cytological analysis of the lymphoid organs of mice neonatally injected with syngeneic cell suspensions derived from different sources showed that the proportions of donor cells in the host spleen, thymus and bone marrow were low, irrespective of the type of suspension administered at birth. In contrast, the proportions of donor cells found in the host lymph nodes were for all inocula demonstrably higher than those found in other lymphoid organs. Various types of inocula, however, displayed different lymph node colonizing capacities depending upon the tissue from which the inoculum was prepared. The order of the lymph node colonizing properties showed a fair correlation with the known order of tolerance-conferring capacities of these inocula. Both phenomena are in accord with the hypothesis that the higher the content in recirculating small lymphocytes in a given tissue, the higher the capacity of the inoculum prepared from that tissue to colonize the host lymph node system and to induce tolerance.     

An impairment of antibody production in adoptively restored, lethally irradiated thymectomized mice

Lethally irradiated, bone marrow protected, normal and thymectomized adult mice were given 140–250 × 10⁶ syngeneic spleen and lymph node cells from normal or sensitized donors. These mice were tested at intervals following irradiation for their ability to reject allogeneic and xenogeneic skin grafts and to produce haemagglutinins. The results show that adoptively restored, thymectomized mice are capable of responding normally to skin homografts of varying antigenic disparity and of producing haemagglutinins to xenogeneic antigens. However, the majority of the mice which received lymphoid cells from normal donors were unable to produce haemagglutinating antibody in response to allogeneic antigens. Both 19S and 7S antibody were produced by the thymectomized mice.