Demonstration of active suppressor cells in spleens of young NZB mice

NZB mice, a strain prone to the development of autoimmune disease, have during the first 2 weeks of life suppressor cells in their spleens which can in coculture with adult spleen cells suppress the antibody response to sheep red blood cells (SRBC) generated in culture by the adult cells. The suppressive activity of spleen cells from NZB mice in the first week after birth is similar to that of spleen cells from 4-day-old C57BL/6 mice, a strain which does not spontaneously develop autoimmune disease. As in “normal” strains of mice, suppressor cell activity in NZB mice is diminished at 2 weeks and undetectable at 3 weeks of age. The data indicate that there is no defect inherent in the suppressor cells detected in the spleens of newborn and young NZB mice and suggest that the development of autoimmune responses does not result from a lack of suppressor cells in the young animals.     

Developmental abnormalities of terminal deoxynucleotidyl transferase positive bone marrow cells and thymocytes in New Zealand mice: effects of prostaglandin E1

The enzyme TdT was used as a marker with which to study the ontogeny of primitive lymphopoietic cells in NZ strain mice. A marked accumulation of abnormally large, rapidly proliferating TdT+ cells was seen in the subcapsular region of the thymus cortex in the NZB and NZB/W mice. This abnormal accumulation of TdT+ thymocytes was most pronounced in the NZB/W hybrid and persisted for at least the first 16 wk of life. In addition, significantly elevated percentages of TdT+ bone marrow cells (presumptive prothymocytes) were present in NZB, NZW, and NZB/W mice between 1 and 4 wk of age, with the highest mean peak levels occurring in the NZB strain. Treatment of both normal and adrenalectomized BALB/c and NZB/W mice with pharmacologic doses (7 to 10 mg/kg) of PGE1 caused a marked, dose-dependent decrease in thymus weight and thymus cell number within 12 to 18 hr. Histologic and cell separation studies showed that this was due to the selective depletion of PNA+ TdT+ cortical thymocytes. Similarly, PGE1 caused a reversible, dose-dependent decrease in the percentage of TdT+ bone marrow cells. In contrast, PGF2 alpha, which is not therapeutically active against autoimmunity in NZB/W mice, had no detectable effect on TdT+ bone marrow cells or thymocytes in BALB/c or NZB/W mice. These results directly document the existence of abnormalities in the development of lymphopoietic precursor cells in the bone marrow and thymus cortex of NZ strain mice prior to the onset of autoimmune phenomena. The results also raise the possibility that the therapeutic efficacy of exogenous PGE1 in autoimmune NZ strain mice may be related, at least in part, to its ability to rectify the abnormal development of these early lymphoid cells.     

Characteristics of a spontaneous monoclonal thymocytotoxic antibody from New Zealand Black mice: recognition of a specific NTA determinant

Naturally occurring thymocytotoxic autoantibodies (NTA) have been described both in humans and in mice with SLE, and have been reported to be preferentially reactive with T suppressor as compared to T helper cells. However, although NTA has been shown by some groups of investigators to induce autoantibodies in normal strains of mice, other researchers have suggested that NTA has only a minor, if any, role in murine lupus. We have been studying the characteristics of a monoclonal antibody (TC-17) derived from the fusion of 4-mo-old NZB spleen cells with P3-X63-AG8.653 plasmacytoma cells. This monoclonal IgM reagent is cytotoxic for approximately 40% of total thymocytes, 50% of cortical thymocytes, less than 1% of cortisol-resistant thymocytes, 10% of splenocytes and lymph node cells, and less than 3% of bone marrow and fetal liver cells. The thymocytotoxicity can be absorbed by thymocytes but not by brain cells. Although NZB, NZW, NFS, and BALB/c thymocytes all manifest reactivity with TC-17, there was considerable difference between strains with respect to antigen density; NZB thymocytes have the highest density. By FACS analysis, TC-17 occurs independently of Lyt-1, Lyt-2, and T helper cell-specific antigens, and is more prevalent on larger proliferating thymocytes. TC-17 augments the response to SRBC but does not influence responses to TI-1 (TNP-BA) or TI-2 (DNP-Ficoll) antigen and production of LPS-induced B cell colonies. We believe that TC-17 recognizes a new T cell antigen, probably one involved in T cell differentiation. Because this monoclonal NTA reacts with only 40% of thymocytes, and is not absorbed with brain, it would not have been detected in mouse sera by using previously published methods. NTA are a heterogeneous group of autoantibodies; some specificities such as TC-17 went unrecognized in the past, and may be important either for disease pathogenesis or for secondary immunologic abnormalities.     

Characteristics of in vitro production of antibodies to DNA in normal and autoimmune mice.

The in vitro production of antibodies to dsDNA was studied with spleen cells from normal and autoimmune mice. After culture for 4 days, the binding of dsDNA in the culture supernatant was measured by a radioimmunoprecipitation assay. The production of antibodies to dsDNA by spleen cells appeared at 15 hr after culture and reached a plateau at 24 hr. No antibodies were produced by thymus cells or splenic T cells. The specificity for dsDNA was shown by competitive inhibition with nonradioactive nucleic acids. Autoimmune strains of mice (NZB/NZW, BXSB, MRL/1) produced more antibodies to dsDNA than did several control strains. Young B/W mice and control strain mice produced mainly IgM antibodies, whereas older B/W mice produced predominantly IgG antibodies to dsDNA. The in vitro production of antibodies to dsDNA by aged B/W spleen cells was macrophage and T cell dependent.     

Failure of NZB spleen to respond to prethymic bone marrow suppressor cells.

Mouse bone marrow contains theta-negative lymphocytes that can suppress an in vitro plaque response by spleen cells primed in vivo with burro red blood cells (BRBC). These bone marrow cells are radiosensitive and can be induced with thymosin fraction 5 or alpha 1 thymic peptides to express the theta antigen. Enrichment for these suppressor pre-T lymphocytes can be achieved by a one-step density centrifugation, macrophage depletion, or a combination of both procedures. NZB mice, which spontaneously develop an autoimmune disorder, have a suppressor abnormality revealed by this assay system. Upon analysis, they have normal BM pre-T suppressor cells but their spleen cells are refractory to the BM suppressor signal. NZB BM suppressor cells inhibit the response by DBA/2 spleen cells, but DBA/2 BM suppressor cells do not inhibit NZB spleen. This resistance to suppression is a property of the B cell fraction recovered from NZB spleen.     

Role of the chemokine stromal cell-derived factor 1 in autoantibody production and nephritis in murine lupus

In normal mice, stromal cell-derived factor 1 (SDF-1/CXCL12) promotes the migration, proliferation, and survival of peritoneal B1a (PerB1a) lymphocytes. Because these cells express a self-reactive repertoire and are expanded in New Zealand Black/New Zealand White (NZB/W) mice, we tested their response to SDF-1 in such mice. PerB1a lymphocytes from NZB/W mice were exceedingly sensitive to SDF-1. This greater sensitivity was due to the NZB genetic background, it was not observed for other B lymphocyte subpopulations, and it was modulated by IL-10. SDF-1 was produced constitutively in the peritoneal cavity and in the spleen. It was also produced by podocytes in the glomeruli of NZB/W mice with nephritis. The administration of antagonists of either SDF-1 or IL-10 early in life prevented the development of autoantibodies, nephritis, and death in NZB/W mice. Initiation of anti-SDF-1 mAb treatment later in life, in mice with established nephritis, inhibited autoantibody production, abolished proteinuria and Ig deposition, and reversed morphological changes in the kidneys. This treatment also counteracted B1a lymphocyte expansion and T lymphocyte activation. Therefore, PerB1a lymphocytes are abnormally sensitive to the combined action of SDF-1 and IL-10 in NZB/W mice, and SDF-1 is key in the development of autoimmunity in this murine model of lupus.     

Induction of autoimmune phenomena in normal mice treated with natural thymocytotoxic autoantibody

Natural thymocytotoxic autoantibody (NTA) developed spontaneously in New Zealand Black (NZB) mice consists of two autoantibodies in terms of target cell specificity. One of the autoantibodies, NTA-2, is strongly cytotoxic only against desialized lymphocytes, whereas the other one, NTA-1, is cytotoxic against both intact thymocytes and asialolymphocytes. To study the pathogenic role of NTA in murine autoimmunity, DBA/2 mice were injected every other day with affinity-purified NTA (NTA-1, NTA-2). Control mice received normal mice sera (NMS) or saline. After 20 days of treatment, spleen cells from DBA/2 mice treated with NTA-1 or NTA-2 showed a significant increase in the number of anti-ssDNA plaque-forming cells and IgM-producing cells. Sera from NTA-treated mice showed greater DNA binding than sera from control mice did. The levels of proteinuria were moderately increased in NTA-2-treated mice. Con A responsiveness of thymocytes was markedly reduced in NTA-2-treated mice. On the other hand, Con A-activated spleen cells from both control and NTA-treated mice equally suppressed anti-SRBC antibody production in vitro, suggesting that NTA treatment didn't affect the direct precursors of suppressor T cells. Finally, prior absorption of NTA-1 by thymocytes prevented its ability to induce anti-DNA antibodies; however, prior absorption of NTA-2 by thymocytes didn't affect its activity.     

Responses of B cells from autoimmune mice to IL-5

Three strains of mice (NZB/W F1 X NZW (NZB/W), BXSB, and MRL/Mp-lpr/lpr (MRL/lpr] develop an autoimmune disease that is clinically and immunologically similar to human SLE. A characteristic of these mice is polyclonal B cell hyperactivity. To explore whether this may be related to hyper-responsiveness to B cell stimulatory factors, we investigated the proliferative and secretory responses of B cells from these mice to semi-purified natural and rIL-5, a major regulator of B cell development in the mouse. As this lymphokine stimulates growth and differentiation of activated B cells, attention was focused on in vivo-activated B cell populations, obtained from the interface of 50/65% Percoll density gradients, from normal or autoimmune mice. This cell population from NZB/W mice secreted IgM and incorporated [3H]TdR at significantly higher levels in response to IL-5, and was more sensitive to IL-5, than a comparable population from several normal murine strains. NZB/W female and male mice displayed heightened responses to IL-5, indicating that this is characteristic of the strain in general and is not associated with the accelerated severe disease of the females. Small resting B cells from NZB/W and normal mice were insensitive to IL-5 stimulation. In contrast to NZB/W mice, no difference was observed in the magnitude of either proliferative or Ig secretory responses between in vivo-activated B cell populations from autoimmune BXSB and MRL/lpr or normal mice. Thus, B cell hyper-responsiveness to IL-5 is a characteristic of NZB/W mice but not of two other lupus-prone murine strains. As one unique feature of NZB/W mouse B cells compared to normal and other autoimmune B cells is an elevated proportion of Ly-1+ B cells, the possibility of IL-5 hyper-responsiveness being associated with this B cell subpopulation was investigated. Fluorescence-activated cell sorter sorted Ly-1+ and Ly-1- B cells both responded to IL-5, however Ly-1+ B cells consistently showed a higher stimulation index in both proliferative and Ig secretory responses to this lymphokine.     

Effects of short-term administration of sex hormones on normal and autoimmune mice

The effects of short-term administration (2 to 4 wk) of sex hormones on the immune system of normal (C57BL/6) and autoimmune (C57BL/6-lpr, C3H/lpr, B/W) strains of mice were investigated. Both estrogen (E2) and testosterone (Te) had significant effects on the numbers of T and B cells as well as on the density of cell surface antigens as demonstrated by flow cytometry. For example, Te depleted Thy-1.2+ thymocytes in normal mice and brought about a shift to lower density cells. Lyt-2+ cells appeared to be the main target cells of hormonal modulation in normal and autoimmune mice. Both sex hormones significantly depleted these cells in the thymus but had differential effects in the peripheral lymphoid organs, particularly in the spleen. In general, E2 depleted Lyt-2+ cells, whereas Te increased or maintained this subpopulation of cells in spleen and lymph nodes. Similarly, the suppressor cell activity and IL 2 production on a per cell basis in E2-treated animals was diminished, whereas Te-treated animals had normal or enhanced activity. The relevance of these findings to differential sex susceptibility in autoimmune diseases is discussed.     

Properties of fractionated spleen cells from NZB/W mice.

The unit gravity sedimentation technique was used to separate spleen cells from sevveral strains of mice. Settling patterns (plot of cell number against settling rate) were similar for BALB/c, DBA/2, C3H/He, and NZB/W mice of different ages. In particular, no subpopulation was found by this technique to be missing from the spleens of old NZB/W mice.A number of functional studies performed with the separated cells proved more informative than the settling patterns themselves. Fractions of cells which sedimented at a rate of between about 6 mm/hr and 10 mm/hr were enriched in responsiveness to PHA, Con A, and allogeneic cells. These fractions obtained from old NZB/W mice lacked such activities. However, the active fractions from young NZB/W spleens, which were enriched in θ-bearing cells, could restore the responsiveness of old NZB/W mice to primary immunization with sheep erythrocytes. These studies indicate that functional separation of spleen cells from NZB/W mice is possible and that activities lacking in whole spleens from old NZB/W mice are also lacking in the separate fractions. The ability to restore helper T cell function in old NZB/W mice with active fractions from young NZB/W mice has implications for further study and treatment of their autoimmune disease.     

Regulation of primary cytotoxic T lymphocyte responses generated during mixed leukocyte culture with H-2d identical Qa-1-disparate cells

Cytotoxic lymphocyte (CTL) responses are not usually generated during primary mixed leukocyte culture (MLC) with H-2 identical cells. Thus NZB mice are unusual in that their spleen cells do mount CTL responses during primary MLC with H-2d identical stimulator cells; the predominant target antigen for these NZB responses is Qa-1b. Considering the numerous immunoregulatory defects in NZB mice, we postulated that these NZB anti-Qa-1 primary CTL responses were due to an abnormality in T suppressor cell activity. Cellular interactions capable of suppressing NZB anti-Qa-1 primary CTL responses were investigated by using one-way and two-way MLC with spleen cells from NZB mice and other H-2d strains. Although H-2d identical one-way MLC with the use of NZB responders resulted in substantial CTL responses, only minimal CTL responses were detected from two-way MLC with the use of NZB spleen cells plus nonirradiated spleen cells from other H-2d mice. Thus the presence of non-NZB spleen cells in the two-way H-2d identical MLC prevented the generation of NZB CTL. Noncytotoxic mechanisms were implicated in the suppression of the NZB CTL responses during two-way MLC, because only minimal CTL activity was generated when NZB spleen cells were cultured with semiallogeneic, H-2d identical (e.g., NZB X BALB) F1 spleen cells. The observed suppression could be abrogated with as little as 100 rad gamma-irradiation to the non-NZB spleen cells. The phenotype of these highly radiosensitive spleen cells was Thy-1+, Lyt-1+, Lyt-2-, L3T4+. The functional presence of these cells in the spleens of semiallogeneic, H-2d identical F1 mice indicated that their deficiency in NZB mice was a recessive trait. These data suggest that NZB mice lack an L3T4+ cell present in the spleens of normal mice that is capable of suppressing primary anti-Qa-1 CTL responses. This model system should facilitate additional investigations of the cellular interactions and immunoregulatory mechanisms responsible for controlling primary CTL responses against non-H-2K/D class I alloantigens. The model may also provide insight into the immunoregulatory defects of autoimmune NZB mice.     

In vitro immune response of cells of various lymphoid tissues in (NZB X NZW)F1 mice: evidence for abnormality of the mesenteric lymph node cells

Autoimmune-prone (NZB X NZW)F1 (B/W) mice have been shown to have a variety of immunologic perturbations. However, most studies have been performed with spleen cells. By using the Mishell-Dutton culture system, we examined the in vitro immune response of the various lymphoid tissue to determine whether an imbalance at a selective lymphoid site may exist in B/W mice. It was shown that the ability of mesenteric lymph node (MLN) cells of B/W mice to generate plaque-forming cells (PFC) in response to sheep red blood cells was consistently less than that of the spleen cells. This relationship held true in the aged mice. In contrast, the ability of the MLN cells of other strains not prone to develop autoimmunity to generate PFC was higher than that of the spleen cells. No significant difference in the mitogenic response of the lymphoid cells from various lymphoid tissue in the young B/W mice was seen, as compared with normal lymphoid cells from control mice. However, it was demonstrated that a relative decrease of B cells and immunoregulatory Lyt-123+ cells in the MLN in the B/W mice occurred early in life, and it was concluded that this abnormality may account for the low PFC response observed.     

Synthesis of prostaglandin E by peritoneal macrophages from NZB/W mice

Peritoneal macrophages from NZB/W (murine lupus) mice spontaneously produce less prostaglandin E (PGE) than peritoneal macrophages from immunologically normal mice. Reduced PGE synthesis is seen as early as 2 months of age and becomes more profound as disease progresses. It is suggested that impaired production of PGE by peritoneal macrophages from NZB/W mice may account in part for abnormal macrophage function observed in these animals.     

Studies of reticuloendothelial function in the mouse with model immune complexes. II. Serum clearance, tissue uptake, and reticuloendothelial saturation in NZB/W mice.

We have extended the findings in the accompanying paper by characterizing the serum clearance and tissue uptake of model soluble immune complexes and the saturation of the reticuloendothelial system (RES) by these complexes in normal mice and in mice with murine lupus (NZB/W F1 females). Adult NZB/W or young C3H mice were injected with radiolabeled stable site-specifically cross-linked mouse anti-DNP oligomers as model immune complexes to probe RES function. Blood clearance and uptake by liver, spleen, and kidney were unimpaired in NZB/W mice. To determine if the RES exhibits partial saturation in the NZB/W mice, we deliberately induced a state of RES blockade with heat-aggregated human gamma-glubulin (HAG). With increasing doses of HAG (1 to 4 mg/20 g body weight) both strains similarly showed a progressive increase in RES saturation as measured by reduced liver uptake of model oligomers. Recovery from saturation was complete by 120 min in both strains. At maximal liver saturation there was only a small increase in oligomer uptake by kidney or spleen, the majority of complexes remaining within the circulation. Thus, RES capacity for handling soluble model immune complexes appears unimpaired in NZB/W mice.     

Abnormal polyclonal B cell activation in NZB/NZW F1 mice

Spleen cells from autoimmune (10-mont-old) NZB/NZW (B/W) mice failed to generate appreciable numbers of antibody-forming cells (AFC) in vitro to TNP-substituted sheep erythrocytes in response to the polyclonal B cell activators (PBA), LPS and PPD, despite normal DNA synthetic responses to these agents and normal AFC responses to TNP-Ficoll. The failure to respond to PBA in old B/W mice was not due to suppressor T cells since anti-brain-associated-theta-treated spleen cells still failed to generate AFC in response to PBA. The defect was age-related since cells from young B/W mice generated vigorous AFC responses to PBA. It is suggested that the failure of the spleen cells of old B/W mice to generate AFC is a result of in vitro polyclonal B cell activation in the course of autoantibody formation.     

Natural thymocytolytic autoantibodies in NZB and other strains of mice

Spontaneously occurring autoantibodies to thymus cells were detected in NZB and other strains of mice by means of cytolysis in agar gel. Serum antibodies were detected by a spot test in which circular zones of lysed thymus cells were observed after the diffusion of serum and complement. In addition, cells forming antithymocyte antibodies were detected in the spleens of tested animals by means of a plaque assay in which antibody-forming cells could be enumerated as plaque-forming cells. The thymocytolytic antibodies were of the IgM class, they resisted heating at 56 °C for 30 min and showed optimal binding activity at 4 °C, even though they were active in temperatures up to 37 °C. They could be detected in a small proportion of mice 1–2 months old, but they had a higher incidence in mice older than 8 months.Studies performed with thymocytes originating from various murine strains indicated that the antibodies under study combined with an antigen which was present on thymocytes of all murine strains tested, including syngeneic and autologous thymocytes. Absorptions demonstrated the presence of the antigen on murine thymus, spleen, and brain cells. Absorptions with L5178Y lymphoma cells suggested that there might be two different antigens involved in the reactions with these thymocytolytic antibodies. Properties of the thymocytolytic antibodies suggested that they are identical with natural thymocytotoxic autoantibodies described by Shirai et al.The possible pathogenic role of these antibodies in autoimmune disorders of NZB mice, particularly in the Coombs'-positive hemolytic anemia, was investigated and discussed.     

Physical separation of "suppressor" from "helper" thymocytes.

Thymocytes from 1-month-old NZB/W mice were separated at unit gravity and then studied for helper or suppressor effects in a GVH assay. Thymocytes with a settling rate of 4 to 6 mm/hr suppressed but did not help. In contrast, help but no suppression characterized thymocytes settling at 6 to 10 mm/hr. Thus helper and suppressor thymocytes in this GVH assay were physically separated.     

Regulation of an in vitro anti-DNA antibody response by thymocytes and T cells from NZB/W and normal mice

The spontaneous in vitro anti-DNA antibody response generated by preautoimmune and many normal mouse spleen cells was suppressed by the addition of syngeneic thymocytes or splenic T cells. Suppressive activity was found in normal mice (DBA/2J) and to an equivalent degree in the autoimmune (New Zealand Black X New Zealand White)F1 (B/W) strain. The suppressor cells were cortisone-resistant, radiosensitive and carried Lyt 1 and Lyt 2 markers. Nonspecific suppression was not involved since the primary and primed in vitro anti-sheep erythrocyte (anti-SRBC) responses were unaffected. Both spontaneous and lipopolysaccharide-stimulated anti-DNA antibody responses could be suppressed. There was no difference in the suppressive activity of cells from young or old, normal or autoimmune mice. These T cells may therefore play a role in preventing the anti-DNA antibody response in normal and young B/W mice, but evidently fail to influence the development of in vivo anti-DNA autoimmune responses in the old B/W mice.     

Treatment of NZB/NZW mice with total lymphoid irradiation: long-lasting suppression of disease without generalized immune suppression

We used total lymphoid irradiation (TLI; total dose = 3400 rad) to treat the lupus-like renal disease of 6-mo-old female NZB/NZW mice. Similar to our past studies, this treatment resulted in a marked prolongation of survival, decrease in proteinuria, and decrease in serum anti-DNA antibodies compared with untreated littermate controls. Although there was no evidence of disease recurrence in TLI-treated mice until after 12 mo of age, the in vitro proliferative response to phytohemagglutinin by NZB/NZW spleen cells recovered within 6 wk such that responses were greater than control NZB/NZW animals. A similar recovery and overshoot after TLI were evident in the primary antibody response to the T cell-dependent antigen sheep red blood cells (SRBC). Both the total and IgG anti-SRBC antibody responses after TLI were greater than those of untreated NZB/NZW controls, and were comparable with those of untreated non-autoimmune mice. Despite this increased response to mitogens and antigens after TLI, we noted a decrease in spontaneous splenic IgG-secreting cells and a decrease in IgG but not IgM antinuclear antibody production. Nonspecific suppressor cells of the mixed leukocyte response were detectable in the spleens of NZB/NZW mice early after TLI. However, the disappearance of suppressor cells was not associated with recrudescence of disease activity. Furthermore, transfer of large numbers of spleen cells from TLI-treated NZB/NZW mice did not result in disease suppression in untreated age-matched recipients. In summary, treatment of NZB/NZW mice with TLI results in a prolonged remission in autoimmune disease, which is achieved in the absence of generalized immunosuppression.     

Impairment of primary in vitro response of New Zealand mouse spleen cells to a thymic-dependent antigen.

New Zealand Black (NZB) and NZB by New Zealand White (NZW) F₁ hybrid ($\text{B}\text{W}$) mice develop clinical signs of autoimmune disease between 6 and 10 months of age but spleen cells from these strains have a greatly reduced in vitro response to sheep erythrocytes (SRBC) as early as 5–6 weeks of age. This hyporesponsiveness can be only partially restored with 2-mercaptoethanol, allogeneic macrophages or spleen cells, or allogeneic factor. The response of NZB and $\text{B}\text{W}$ spleen cells to the thymic independent antigen DNP-Ficoll is nearly normal. The reduced in vitro SRBC response was found to be attributable to splenic T and B cells rather than macrophages. Macrophages from NZB mice were found to function normally. The in vitro behavior of NZB lymphocytes is very similar to non-autoimmune mice infected with common murine viral pathogens. NZB and $\text{B}\text{W}$ mice may be making an active immune response as early as 5 weeks of age.