Transgenic expression of VpreB-3 under the control of the immunoglobulin heavy chain enhancer and SV40 promoter

Recently we isolated a novel gene, VpreB-3 gene from the cDNA library of a pre-B cell clone. This gene is selectively expressed in pre-B and bone marrow-derived B cell lines. Its products are associated with the immunoglobulin heavy (IgH) chain in pre-B cell line. In the present study, to address the role of VpreB-3 on B cell development, transgenic mice carrying the VpreB-3 gene under the control of the IgH enhancer and SV40 promoter were produced. The transgenic mice expressed the VpreB-3 gene in bone marrow and spleen at a high level compared with control mice. In the thymus, the expression of the transgene was also detected, although its level was low. Flow cytometry analysis revealed that the frequency of CD45R+ and mu+ B cells were reduced in the bone marrow of 2 of the 11 transgenic mice and also reduced in the spleen of 1 of the transgenic mice. Furthermore, the results of a stromal cell-dependent B cell culture assay suggested that early B cell development, the differentiation from CD45R- B progenitor cells to CD45R+ pro-B cells, was delayed in the bone marrow cultures of 3 of the 5 transgenic mice compared with the control mice. These results suggested that VpreB-3 products may play some role in early B cell development at the stage of CD45R- B progenitor cells before the expression of surface mu chains.     

Mice with an inactivated joining chain locus have perturbed IgM secretion

A mouse with an inactivated joining chain locus was produced by gene targeting in embryonic stem cells by deleting the first exon. Heterozygote (J+/-) and homozygote (J-/-) offspring from these mice showed normal total serum immunoglobulin levels and a normal peripheral B cell compartment when compared to wild-type littermates. The distribution of serum immunoglobulin isotypes in serum was different; IgA levels were elevated while IgM levels were reduced in J-/- mice as compared to wild-type mice. High molecular weight serum IgM was reduced in J+/- and J-/- mice and instead found in oligomeric form of undefined structure. Furthermore, serum IgM from J+/- and J-/- mice showed a reduced ability to activate complement. The number of splenic and bone marrow IgM plaque-forming cells were reduced in unimmunized J+/- as well as in J-/- mice. Furthermore, the number of plaque-forming cells was reduced in B cells from both J+/- and J-/- mice after stimulation with lipopolysaccharide in vitro. The perturbation of IgM production in J-/- mice appears to affect a late stage of differentiation, because cells with intracellular IgM were readily detected both in vivo and in vitro. Finally, after immunization with T-dependent or T-independent antigens the IgM component of the immune response was reduced in J-/- mice while only a marginal reduction of the IgG response was detected.     

Enhanced virus clearance by early inducible lymphocytic choriomeningitis virus-neutralizing antibodies in immunoglobulin-transgenic mice

Following infection of mice with lymphocytic choriomeningitis virus (LCMV), virus-neutralizing antibodies appear late, after 30 to 60 days. Such neutralizing antibodies play an important role in protection against reinfection. To analyze whether a neutralizing antibody response which developed earlier could contribute to LCMV clearance during the acute phase of infection, we generated transgenic mice expressing LCMV-neutralizing antibodies. Transgenic mice expressing the immunoglobulin mu heavy chain of the LCMV-neutralizing monoclonal antibody KL25 (H25 transgenic mice) mounted LCMV-neutralizing immunoglobulin M (IgM) serum titers within 8 days after infection. This early inducible LCMV-neutralizing antibody response significantly improved the host's capacity to clear the infection and did not cause an enhancement of disease after intracerebral (i.c.) LCMV infection. In contrast, mice which had been passively administered LCMV-neutralizing antibodies and transgenic mice exhibiting spontaneous LCMV-neutralizing IgM serum titers (HL25 transgenic mice expressing the immunoglobulin mu heavy and the kappa light chain) showed an enhancement of disease after i.c. LCMV infection. Thus, early-inducible LCMV-neutralizing antibodies can contribute to viral clearance in the acute phase of the infection and do not cause antibody-dependent enhancement of disease.     

Requirement for increased IL-10 in the development of B-1 lymphoproliferative disease in a murine model of CLL

Malignant B-1 cells derived from NZB mice, a murine model of spontaneous autoimmunity and B cell lymphoproliferative disease, produce significantly higher levels of IL-10 mRNA than normal B-1 or B cells. IL-10 may act as an autocrine growth factor for the expansion of malignant B-1 cells. In order to determine if elevated endogenous production of IL-10 was a required element for the malignant transformation of B-1 cells in NZB mice, backcross animals were studied for the linkage between elevated IL-10 expression and the presence of lymphoid malignancy. The phenotypes of aged (NZB x DBA/2)F1 x NZB animals were determined and a strong correlation was found between the elevated levels of IL-10 mRNA and the development of B-1 malignant clones. In contrast, an increased level of IL-10 message was not associated with elevated serum IgM or the presence of anemia or reticulocytosis which is mainly seen in response to autoantibody production. These results indicate that, at least in NZB, the autoimmunity and lymphoproliferation phenotypes are not linked genetically. IL-10 may enhance proliferation and the development of B-1 cell malignancy rather than antibody production by the B-1 cell subpopulation. Thus, IL-10 plays an important role in B-1 malignancies, and downregulation of IL-10 could be a likely site for intervention in B cell malignancies.     

Enhanced B-1 cell development, but impaired IgG antibody responses in mice deficient in secreted IgM

The role of endogenous natural IgM in promoting the adaptive Ab response was investigated in newly constructed mutant mice in which B cells do not secrete IgM but still express surface IgM and IgD and undergo class switching to express other Ig isotypes. While the mutant mice had relatively normal numbers of conventional B (B-2) cells in all tissues examined, unexpectedly, B-1 cells in the peritoneum and spleen were approximately threefold more abundant. The elevated levels of B-1 cells were already detectable at 4 wk of age and were stably maintained throughout life. The levels of serum IgG2a, IgG3, and IgA were also elevated in the mutant mice at an early age. IgG2a response to a T cell-independent Ag was augmented, whereas IgG Ab responses to suboptimal doses of a T cell-dependent Ag were impaired. The latter defect was associated with fewer splenic germinal centers, impaired Ab affinity maturation, and less Ag trapping on follicular dendritic cells. Together, these findings demonstrate a physiologic role of natural IgM in the feedback regulation of B-1 cell development, the regulation of IgG2a production, and the promotion of efficient B-2 cell Ab responses.     

Overexpression of natural killer T cells protects Valpha14- Jalpha281 transgenic nonobese diabetic mice against diabetes

Progression to destructive insulitis in nonobese diabetic (NOD) mice is linked to the failure of regulatory cells, possibly involving T helper type 2 (Th2) cells. Natural killer (NK) T cells might be involved in diabetes, given their deficiency in NOD mice and the prevention of diabetes by adoptive transfer of alpha/beta double-negative thymocytes. Here, we evaluated the role of NK T cells in diabetes by using transgenic NOD mice expressing the T cell antigen receptor (TCR) alpha chain Valpha14-Jalpha281 characteristic of NK T cells. Precise identification of NK1.1(+) T cells was based on out-cross with congenic NK1.1 NOD mice. All six transgenic lines showed, to various degrees, elevated numbers of NK1.1(+) T cells, enhanced production of interleukin (IL)-4, and increased levels of serum immunoglobulin E. Only the transgenic lines with the largest numbers of NK T cells and the most vigorous burst of IL-4 production were protected from diabetes. Transfer and cotransfer experiments with transgenic splenocytes demonstrated that Valpha14-Jalpha281 transgenic NOD mice, although protected from overt diabetes, developed a diabetogenic T cell repertoire, and that NK T cells actively inhibited the pathogenic action of T cells. These results indicate that the number of NK T cells strongly influences the development of diabetes.     

Lateral cephalometric analysis of asymptomatic volunteers and symptomatic patients with and without bilateral temporomandibular joint disk displacement

Anecdotal evidence links silicone gel breast implants with the development of autoimmune connective tissue disease in women. To investigate whether silicone gel is capable of directly inducing and/or enhancing the development of autoimmune disease, female BALB/cAnPt (BALB/c) and New Zealand Black (NZB) mice were injected subcutaneously with silicone gel, pristane, a nonmetabolizable substance that can cause plasmacytomas in BALB/c and NZB mice, or saline and monitored for the development of glomerulonephritis and autoantibody production. NZB, but not BALB/c, mice spontaneously develop autoantibodies and an autoimmune hemolytic anemia by 12 months of age. Over a period of 10 months, biweekly screening for proteinuria revealed increases in urinary protein in NZB mice that received multiple injections of either silicone gel or pristane. In contrast, urinary protein was unaffected in identically treated BALB/c mice. Although, silicone gel had no effect on serum titers of antierythrocyte antibodies in NZB mice, the hematocrits were significantly decreased. Moreover, silicone gel both increased the concentration of IgM anti-type I collagen antibodies and skewed the immunofluorescent staining pattern of serum autoantibodies on HEp-2 cells. In contrast, silicone gel failed to induce the production of anti-erythrocyte or antinuclear antibodies in BALB/c mice and induced only slight increases in IgG anti-type I collagen antibodies. These results suggest that silicone gel can exacerbate the development of autoimmune disease in autoimmune NZB mice, but fails to induce disease in normal BALB/c mice. This is consistent with several epidemiological studies failing to demonstrate an increase in the incidence of autoimmune disease in women with breast implants. However, because silicone gel was able to exacerbate autoimmune disease in NZB mice, it may play a similar role in the development of autoimmune disease in a small percentage of women who are genetically susceptible to such diseases.     

Transgenic expression of Fas in T cells blocks lymphoproliferation but not autoimmune disease in MRL-lpr mice

Fas is a member of the TNF receptor family. Binding of Fas ligand to Fas induces apoptosis in Fas-bearing cells. Fas is expressed in various cells, including thymocytes, peripheral T cells, and activated B cells. The mouse lpr mutation is a loss of function mutation of Fas. MRL-lpr/lpr mice develop lymphadenopathy and splenomegaly, and produce multiple autoantibodies, which results in autoimmune disease. In this report, we describe the establishment of a line of Fas transgenic MRL-lpr mice in which mouse Fas cDNA was expressed using the T cell-specific murine lck promoter. The transgenic mice expressed functional Fas in thymocytes and peripheral T cells, but not in B cells. The transgenic mice did not accumulate abnormal T cells (Thy-1+ B220+), but still accumulated B cells (Thy-1- B220+); they produced a large quantity of Igs (IgG1 and IgG2a), including anti-DNA Abs, and developed glomerulonephritis. These results suggest that autoreactive or activated B cells must be killed through Fas expressed in the B cells by the Fas ligand expressed in activated T cells.     

B cells are anergic in transgenic mice that express IgM anti-DNA antibodies

B lymphocytes in individuals with systemic lupus erythematosus (SLE) secrete pathogenic autoantibodies to DNA which cause clinical nephritis. (NZB X NZW) F1 (BW) female mice also secrete pathogenic anti-DNA autoantibodies, and therefore are considered to be an animal model of SLE. The rearranged immunoglobulin (Ig) genes that encode an anti-DNA antibody from a diseased BW mouse have been cloned, and transgenic (Tg) mice have been created by microinjection of these constructs into fertilized eggs from normal mice. As we reported previously, when the construct contains the C gamma 2a heavy chain constant (CH) region, the mice spontaneously secrete anti-DNA IgG and they develop mild nephritis. This demonstrated that the Ig encoded by the transgene is pathogenic. In contrast, here we report that when the construct contains the same anti-DNA Ig variable (V) regions used previously, along with the C mu region, the autoreactive B cells are rendered tolerant. Most B cells in the Tg mice express the mu transgene product on their surface, and rearrangement of endogenous light chain genes is partially suppressed. Furthermore, most hybridomas made from Tg B cells secrete IgM anti-DNA. Despite this, the Tg mice have reduced levels of total serum Ig and they do not secrete anti-DNA IgM either spontaneously or following immunization with DNA. We conclude that most B cells in the Tg mice have been rendered anergic. Anergy is however reversible in vitro; lipopolysaccharide stimulation of Tg B cells leads to the production of a significant amount of IgM anti-DNA antibody. The studies demonstrate that in this line of Tg mice on a normal mouse genetic background potentially pathogenic B cells that express a high-affinity Ig specific for a natural autoantigen are subject to tolerance by induction of anergy.     

Local expression of transgene encoded TNF alpha in islets prevents autoimmune diabetes in nonobese diabetic (NOD) mice by preventing the development of auto-reactive islet-specific T cells

Lately, TNF alpha has been the focus of studies of autoimmunity; its role in the progression of autoimmune diabetes is, however, still unclear. To analyze the effects of TNF alpha in insulin-dependent diabetes mellitus (IDDM), we have generated nonobese diabetic (NOD) transgenic mice expressing TNF alpha under the control of the rat insulin II promoter (RIP). In transgenic mice, TNF alpha expression on the islets resulted in massive insulitis, composed of CD4+ T cells, CD8+ T cells, and B cells. Despite infiltration of considerable number of lymphoid cells in islets, expression of TNF alpha protected NOD mice from IDDM. To determine the mechanism of TNF alpha action, splenic cells from control NOD and RIP-TNF alpha mice were adoptively transferred to NOD-SCID recipients. In contrast to the induction of diabetes by splenic cells from control NOD mice, splenic cells from RIP-TNF alpha transgenic mice did not induce diabetes in NOD-SCID recipients. Diabetes was induced however, in the RIP-TNF alpha transgenic mice when CD8+ diabetogenic cloned T cells or splenic cells from diabetic NOD mice were adoptively transferred to these mice. Furthermore, expression of TNF alpha in islets also downregulated splenic cell responses to autoantigens. These data establish a mechanism of TNF alpha action and provide evidence that local expression of TNF alpha protects NOD mice from autoimmune diabetes by preventing the development of autoreactive islet-specific T cells.     

Expression level of a transgenic lambda2 chain results in isotype exclusion and commitment to B1 cells

Two new lambda2 chain-transgenic mouse lines were established, both of which showed stable transgene expression during aging of the mice. The line L23, which expressed the transgene at low levels, exhibited normal B cell development, antibody responses and serum Ig levels. Most of the B cells in this mouse line co-expressed the transgenic lambda2 chain together with an endogenous kappa chain, thus showing poor allelic exclusion of endogenous L chains. On the other hand, high expression of the transgenic lambda2 chain in the other mouse line, L2, resulted in nearly complete exclusion of endogenous L chain isotypes. In this line, the lambda2 transgene was already detectable in the cytoplasm of all preB-II cells and some pro/preB-I cells. Its expression during these early phases obviously inhibited development of conventional B2 cells, since the B cells in the periphery of these mice were almost exclusively of the B1 type. This finding was confirmed by adoptive transfer of transgenic bone marrow into lethally irradiated recipients. Very few B cells were present in the spleen of such recipients. The serum IgM levels of L2 mice were close to normal and the majority of these IgM were associated with the transgenic lambda2 chain. Antibody responses to thymus-dependent antigens in such mice were almost exclusively found to be of IgM class. Together, these findings indicate a developmental bias leading to a predominance of B1 cells in the L2 line.     

Recipient polyclonal B cell activation and immunoglobulin production induced by priming with a retroviral superantigen

The superantigen vSAG-7 (or MIs 1a) is a membrane glycoprotein encoded by the endogenous retrovirus mouse mammary tumor virus 7 (MMTV-7) and is highly stimulatory for V beta 6/CD4+ T cells. Priming of adult MMTV-7-negative mice with vSAG-7-expressing cells initially results in the activation of the peripheral V beta 6/CD4+ T cell compartment and is followed by T cell tolerance to the superantigen. During the course of tolerance induction the number of recipient B lymphocytes increases in the lymph nodes, but not the spleen, of vSAG-7-primed recipients. These B cells also express increased levels of class II MHC and present passively acquired superantigen. In this study we asked if these effects on the host B cell compartment are followed by the production of immunoglobulin. Priming of MMTV-7-negative BALB/c or CB.17 mice with vSAG-7-expressing cells from DBA/2 mice induced increases of both IgM and IgG2a in the serum. Use of Igh congenic CB.17 (IgMb) mice as recipients of the vSAG-7-presenting cells from DBA/2 (IgMa) donors indicated that the IgM and IgG produced were entirely of host origin. Priming with vSAG-7 also amplified (four- to fivefold) the antibody-producing cell response induced to a suboptimal dose of sheep RBC. Priming with purified B cells from vSAG-7 donors resulted in recipient V beta 6/CD4+ T cell activation and increased numbers of recipient B cells in the lymph nodes, but did not induce immunoglobulin production. In contrast, priming with purified CD8+ T cells resulted in increased quantities of serum IgM but not vSAG-7-reactive T cell activation or increased numbers of recipient B cells in the lymph nodes. These results indicate that the T cell activation and increased B cell number with upregulated class II MHC expression initially observed following vSAG-7 priming of adult MMTV-7-negative recipients are not linked to the induction of the recipient-derived immunoglobulin production.     

GD1alpha-replica peptides functionally mimic GD1alpha, an adhesion molecule of metastatic tumor cells, and suppress the tumor metastasis

Fluoxetine, a novel selective serotonin reuptake inhibitor utilized in the treatment of depression, is avidly bound to serum albumin and alpha-1-acid glycoprotein (AAG). AAG is an acute phase protein, and its serum levels are elevated in a variety of pathophysiological conditions including inflammation, depression, cancer, and acquired autoimmune deficiency syndrome. Further, the pharmacokinetic disposition and pharmacological activity of several highly bound drugs have been reported to be significantly altered as a result of elevated serum AAG. We investigated the effects of elevated serum AAG levels on the pharmacokinetic disposition, antidepressant activity, and steady state profile of fluoxetine and its demethylated metabolite, norfluoxetine. This was approached utilizing a novel strain of transgenic mice that expressed genetically elevated serum AAG levels severalfold over those of control mice. Serum and brain drug concentrations were determined by HPLC after fluoxetine administration. In transgenic mice, the volume of distribution and the terminal elimination half-life of fluoxetine were significantly reduced. Further, significant reductions in brain-to-serum fluoxetine concentration ratios and antidepressant activity were observed in transgenic mice, despite having higher serum drug levels than control mice. This trend in the serum continued at steady state, and brain fluoxetine levels were significantly lower in transgenic mice. The results of this study provide valuable insights regarding the consequences of elevated serum AAG levels, often seen in several disease states, on the pharmacokinetic disposition of fluoxetine.     

IL-12 is involved in the induction of experimental autoimmune myasthenia gravis, an antibody-mediated disease

IL-12 has been shown to be involved in the pathogenesis of Th1-mediated autoimmune diseases, but its role in antibody-mediated autoimmune pathologies is still unclear. We investigated the effects of exogenous and endogenous IL-12 in experimental autoimmune myasthenia gravis (EAMG). EAMG is an animal model for myasthenia gravis, a T cell-dependent, autoantibody-mediated disorder of neuromuscular transmission caused by antibodies to the muscle nicotinic acetylcholine receptor (AChR). Administration of IL-12 with Torpedo AChR (ToAChR) to C57BL/6 (B6) mice resulted in increased ToAChR-specific IFN-gamma production and increased anti-ToAChR IgG2a serum antibodies compared with B6 mice primed with ToAChR alone. These changes were associated with earlier and greater neurophysiological evidence of EAMG in the IL-12-treated mice, and reduced numbers of AChR. By contrast, when IL-12-deficient mice were immunized with ToAChR, ToAChR-specific Th1 cells and anti-ToAChR IgG2a serum antibodies were reduced compared to ToAChR-primed normal B6 mice, and the IL-12-deficient mice showed almost no neurophysiological evidence of EAMG and less reduction in AChR. These results indicate an important role of IL-12 in the induction of an antibody-mediated autoimmune disease, suggest that Th1-dependent complement-fixing IgG2a anti-AChR antibodies are involved in the pathogenesis of EAMG, and help to account for the lack of correlation between anti-AChR levels and clinical disease seen in many earlier studies.     

Aging-related changes in calcium oscillations in fertilized mouse oocytes

In vivo studies of lymphocyte biology have used intravenous (i.v.) injection as the primary mode of cell transfer, a protocol consistent with the anatomic distribution of most lymphocytes. However, for study of peritoneal cavity B cells, i.v. injection does not correlate with anatomical localization. This report describes the restoration of B-cell function in B lymphocyte-defective X-chromosome-linked immune-defective (XID) mice after intraperitoneal transfer of immunoglobulin heavy chain (Igh)-disparate peritoneal cavity (PerC) cells. In contrast to i.v. transfer, intraperitoneal (i.p.) transfer restored B-cell function in young, but not adult (> 8 weeks), XID mice. When host and donor Igh allotype matched, PerC B-cell engraftment was noted in older recipients; this reconstitution however, was also age-dependent. Migration from the peritoneum to systemic circulation was necessary for serum IgM production as shown by the presence of donor antibody-secreting cells in the host spleen. Host lymphocytes also influenced the success of i.p. transplantation as severe combined immune-deficient mice, regardless of age, exhibited donor serum IgM production. Recipient age, Igh allotype, and immune-deficiency were found to have an impact on the ability of i.p.-transferred PerC B cells to restore B-cell function in XID mice.     

A critical role of natural immunoglobulin M in immediate defense against systemic bacterial infection

To evaluate the role of natural immunoglobulin (Ig)M in the immediate response against microbial infection, we tested mutant mice that are deficient in secreted (s)IgM in an acute peritonitis model induced by cecal ligation and puncture (CLP). 20% of wild-type mice died within 32 h of CLP, whereas 70% of sIgM-deficient mice died within the same time period. The increased susceptibility was associated with a reduced level of tumor necrosis factor (TNF)-alpha, a decreased neutrophil recruitment and an increased bacterial load in the peritoneum, and elevated levels of endotoxin and proinflammatory cytokines in the circulation. Resistance to CLP by sIgM-deficient mice was restored by reconstitution with polyclonal IgM from normal mouse serum. Reconstitution with a monoclonal IgM specific to phosphatidylcholine, a conserved cell membrane component, has a modest effect but a monoclonal IgM specific to phosphocholine is not protective. These findings demonstrate a critical role of natural IgM in the immediate defense against severe bacterial infection.     

CD40 ligand gene defects responsible for X-linked hyper-IgM syndrome

The ligand for CD40 (CD40L) is a membrane glycoprotein on activated T cells that induces B cell proliferation and immunoglobulin secretion. Abnormalities in the CD40L gene were associated with an X-linked immunodeficiency in humans [hyper-IgM (immunoglobulin M) syndrome]. This disease is characterized by elevated concentrations of serum IgM and decreased amounts of all other isotypes. CD40L complementary DNAs from three of four patients with this syndrome contained distinct point mutations. Recombinant expression of two of the mutant CD40L complementary DNAs resulted in proteins incapable of binding to CD40 and unable to induce proliferation or IgE secretion from normal B cells. Activated T cells from the four affected patients failed to express wild-type CD40L, although their B cells responded normally to wild-type CD40L. Thus, these CD40L defects lead to a T cell abnormality that results in the failure of patient B cells to undergo immunoglobulin class switching.     

Resting B cells from autoimmune lupus-prone New Zealand Black and (New Zealand Black x New Zealand White)F1 mice are hyper-responsive to T cell-derived stimuli

To determine whether B cells from New Zealand Black (NZB) and (New Zealand Black x New Zealand White)F1 (NZB/W) mice possess intrinsic defects that lead to altered immune responsiveness, we purified resting B cells from these mice and compared their surface phenotype and function with those of resting B cells isolated from BALB/c and DBA/2 nonautoimmune mouse strains. Flow cytometric analysis of freshly isolated resting B cells revealed that NZB and NZB/W resting B cells are conventional B2-type cells similar to their nonautoimmune counterparts. Despite this, resting B cells from young NZB and NZB/W mice express lower levels of CD23 on their surface and aberrant levels of intracellular IgM. Upon stimulation, resting B cells from young NZB and NZB/W mice demonstrate increased proliferation, IgM secretion, or enhanced expression of costimulatory molecules in response to a variety of different T cell-derived stimuli, including cytokines and signals generated through CD40. Therefore, B cell hyper-responsiveness to T cell stimuli is immunodominant or codominant in NZB/W mice. Taken together, our results suggest that intrinsic B cell hyper-responsiveness may play a role in the pathogenesis of autoimmune disease in NZB and NZB/W mice. The increased clonal expansion of these B cells together with increased Ig production and enhanced costimulatory capacity serve to amplify the immune response. In the context of normal but incomplete T cell tolerance, B cell hyperresponsiveness to the limited signals provided by partially tolerant T cells may be sufficient to yield an autoantibody response.