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.

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-Faslpr (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.

The region of the amnion overlying the placenta plays an active role in fluid exchange between amniotic fluid and fetal blood perfusing the surface of the placenta, whereas little transfer occurs across the reflected amnion that contacts the membranous chorion. Because aquaporins (AQPs) facilitate rapid movement of water across cells, we hypothesized that AQP gene expression in placental amnion is higher than in reflected amnion. Furthermore, because gestational diabetes mellitus (GDM) is often associated with polyhydramnios, we hypothesized that amnion AQP gene expression is reduced when amniotic fluid volume is elevated. Human placental and reflected amnion were obtained at cesarean delivery and subjected to relative quantitation of AQP mRNA by real-time RT-qPCR and proteins by western immunoblot. Amnion mRNA levels of five AQPs differed by up to 400-fold (P < 0.001), with AQP1 and AQP3 most abundant, AQP8 least and AQP9 and AQP11 intermediately expressed. Aquaporin proteins showed a similar profile. Aquaporin mRNA abundance was higher (P < 0.001) in placental than reflected amnion, whereas protein levels were lower (P < 0.01). In GDM pregnancies, neither AQP mRNA nor protein levels were different from normal. There was no correlation between AQP mRNA or protein levels with the amniotic fluid index in normal or GDM subjects. We conclude that there is a strong differential expression profile among individual AQPs and between regions of the amnion. These findings suggest differences in contribution of individual AQPs to water transport in the two regions of the amnion. Furthermore, AQP expression in the amnion is not altered in patients with GDM.

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 λ1 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/λ1 NAA are not negatively selected, but rather are overrepresented in the transgenic mice. The shift toward λ1 expression mainly occurs during the transition of immature to mature B cells in the spleen, suggesting a BCR selection process. The ppc1-5H/λ1 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/λ1 natural autoantibodies originate from a distinct B cell subset that may be positively selected by virtue of its poly/autoreactivity.

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 VH 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.