Natural autoantibodies (NAAs) arise naturally without exogenous Ag stimulation. They constitute a substantial proportion of the normal Ab repertoire. They are mainly of the IgM class, unmutated, and typically polyreactive. It remains controversial whether NAA contribute to or protect from autoimmune diseases. Using site-directed transgenic mice expressing a prototypic NAA, named ppc1-5, we found that the expression of NAA in the autoimmune MRL-lpr mice protect from autoimmune manifestations. To demonstrate that the protection is, at least in part, via the secreted NAA per se, we administered purified ppc1-5 IgM to the wt MRL-lpr mice. We observed substantial reduction in proteinuria and kidney IC deposition, and prolonged survival in mice injected with ppc1-5 IgM as compared to those that received either PBS or control IgM. The ppc1-5 IgM injected mice had decreased levels of anti-Hep2 ANA and anti-dsDNA of the IgG3 subclass, although the total IgM and IgG levels did not alter significantly. We demonstrated that the ppc1-5 NAA, but not the control IgM, bound to apoptotic cells with a high capacity. Examination of cytokine production showed that the administration of ppc1-5 NAA significantly reduced the production of IFNgamma and IL-10 by CD4+ T cells. Taken together, our data demonstrate that the low-affinity, self-reactive NAAs can protect from lupus nephritis, and suggest that they may do so by promoting removal of apoptotic cells, and by regulating T-cell function.

Patients with gestational diabetes (GDM) develop polyhydramnios more frequently than normal. Although amniotic fluid (AF) volume complications have been associated with maternal hyperglycemia, the underlying AF transport mechanisms have not been explored. Five aquaporin (AQP) water channels have been implicated as mediators of AF transport across the amnion. We determined the relationships between AF volume and AQP1,3,8,9 and 11 expression in the amnion to test the hypothesis that AQP levels in the amnion are normal in GDM when AF volume is normal. Subjects at term with normal (n=20) pregnancies and GDM (n=9) were recruited. Placental and reflected amnion samples were collected at cesarean delivery. Amnion RNA was reverse transcribed and amplified by real time PCR for AQP1,3,8,9 and 11. AQP mRNA was referenced to18S and quantified using comparative CT method. AQP proteins were quantified by Western blotting and normalized to β-actin. Data were analyzed by ANOVA and regression. In normal subjects, placental and reflected amnion showed significant differences among AQP mRNA levels with AQP1,3 and 9 greater than AQP8 and 11. AQP1 and 3 were higher in placental than reflected amnion (P<0.001). Pattern and regional differences in AQP were similar in GDM patients. Although overall mean AQP mRNA levels in placental and reflected amnion were lower in GDM than normal, the differences were not significant. AQP1,3,9 and 11 proteins in placental and reflected amnion were not different between GDM and normal. AFI of GDM group was 15±2, not different from normal (12±1). There were no correlations between AFI and individual AQP levels in placental or reflected amnion of normal and GDM patients. The distinct expression pattern among the 5 AQPs in placental and reflected amnion suggests that individual AQPs may contribute differentially to amniotic water transport. The lack of difference in AQP levels between normal and GDM patients with normal AFI suggests that the AQP mediated AF transport may be normalized in GDM.

Natural autoantibodies (NAA) and their associated B cells constitute a substantial proportion of the normal Ab and B cell repertoire. They often have weak reactivity toward a variety of self-Ags such as DNA, nucleoproteins, and phospholipids. It remains controversial whether NAA contribute to or protect from autoimmune diseases. Using site-directed transgenic (sd-tg) mice expressing a prototypic NAA, we investigated the effect of NAA and NAA-producing B cells in disease development in the autoimmune-prone MRL/MpJ-Fas(lpr) (MRL-lpr) mice. We found that the expression of NAA in MRL-lpr mice prevented proteinuria and reduced kidney immune complex formation. The mice had significantly improved survival. Administration of the IgM NAA to MRL-lpr mice also delayed the onset of nephritis. The sd-tg MRL-lpr mice had decreased levels of anti-dsDNA Abs, anti-Hep2 nuclear Abs, and anti-Sm/ribonucleoprotein Abs. There is a shift in the IgG subclass profile from IgG2a and IgG3 to IgG1 in the sd-tg MRL-lpr mice. The CD4(+) T cells from the sd-tg MRL-lpr mice had increased expression of the negative costimulatory molecule CTLA-4 and increased production of IL-10 as compared with those from the wild-type mice. Furthermore, the NAA B cells produced large amounts of IL-10 upon TLR stimulation. These results indicate that NAA and NAA-producing B cells play an important role in protection from lupus nephritis and suggest that the NAA B cells may have an immune regulatory function via the provision of IL-10.

In healthy individuals, a substantial proportion of circulating Abs exhibit polyreactivity and self-reactivity. These Abs are referred to as natural autoantibodies (NAAs). As part of the innate immunity, NAAs play an important role in eliminating pathogens. However, inherent to their poly/autoreactivity is the potential for NAAs to differentiate to high-affinity autoantibodies during an immune response. We recently generated site-directed transgenic mice that express a prototypic NAA, ppc1-5, which binds a variety of self- and non-self-Ags including DNA and phosphocholine. We have shown previously that B cells expressing the ppc1-5 NAA are positively selected during their primary development. In this study, we demonstrate that following immunization with the T-dependent Ag, phosphocholine conjugated to keyhole limpet hemocyanin, ppc1-5 NAA B cells mounted a quick IgM Ab response and entered germinal centers, but they failed to differentiate to IgG-producing cells during late primary and memory responses. Hybridomas and cDNA clones derived from the immunized mice included many IgM NAA-producing cells, but IgG NAA clones were extremely rare. Instead, many of the IgG B cells replaced their IgH transgene with an endogenous V(H) gene and produced non-autoreactive Abs. These results indicate that although NAA B cells are positively selected in the preimmune repertoire and can participate in early IgM Ab response, they are subjected to regulatory mechanisms that prevent them from developing to high-affinity IgG autoantibody production. This would explain, at least in part, why NAAs do not cause autoimmunity in most individuals.

Despite stringent regulation of disease-associated autoantibodies, a substantial proportion of circulating Abs in sera of healthy individuals exhibit self-reactivity. These Abs are referred to as naturally occurring or natural autoantibodies (NAAs). To understand the origin and function of NAAs, we have generated a new site-directed transgenic mouse model in which a prerearranged VDJ gene coding for the H chain of a typical polyreactive NAA, ppc1-5, is inserted into the IgH locus. This H chain, when combined with its original L chain, the lambda 1 L chain, yields a NAA that characteristically binds a variety of self and non-self Ags including ssDNA, actin, ubiquitin, and nitrophenyl phosphocholine. Despite their autoreactivity, B cells expressing ppc1-5H/lambda 1 NAA are not negatively selected, but rather are overrepresented in the transgenic mice. The shift toward lambda 1 expression mainly occurs during the transition of immature to mature B cells in the spleen, suggesting a BCR selection process. The ppc1-5H/lambda 1 B cells exhibit a phenotype that is different from those of the known mature B cell populations, and they are located predominantly in the lymphoid follicles of the spleen and the lymph nodes. These B cells are functionally active, producing high levels of Abs in vivo and responding well to BCR stimulation in vitro. The findings indicate that the ppc1-5/lambda 1 natural autoantibodies originate from a distinct B cell subset that may be positively selected by virtue of its poly/autoreactivity.

Abs to DNA and nucleoproteins are expressed in systemic autoimmune diseases, whereas B cells producing such Abs are edited, deleted, or inactivated in healthy individuals. Why autoimmune individuals fail to regulate is not well understood. In this study, we investigate the sources of anti-dsDNA B cells in autoimmune transgenic MRL-lpr/lpr mice. These mice are particularly susceptible to lupus because they carry a site-directed transgene, H76R that codes for an anti-DNA H chain. Over 90% of the B cells are eliminated in the bone marrow of these mice, and the few surviving B cells are associated with one of two Vκ editors, Vκ38c and Vκ21D. Thus, it appears that negative selection by deletion and editing are intact in MRL-lpr/lpr mice. However, a population of splenic B cells in the H76R MRL-lpr/lpr mice produces IgG anti-nuclear Abs, and these mice have severe autoimmune organ damage. These IgG Abs are not associated with editors but instead use a unique Vκ gene, Vκ23. The H76R/Vκ23 combination has a relatively high affinity for dsDNA and an anti-nuclear Ab pattern characteristic of lupus. Therefore, this Vκ gene may confer a selective advantage to anti-DNA Abs in diseased mice.