Reprints Available Directly from the Publisher Photocopying Permitted by License Only Presence of Germline and Full-length Iga Rna Transcripts among Peritoneal B-1 Cells

Next to conventional B cells (or B-2 cells), peritoneal B-1 cells have been shown to contribute significantly to the production of IgA-secreting plasma cells in the gut. Evidence for this was mainly based on studies comprising manipulated animals, including lethally X-irradiated and transgenic mice. To examine the ability of peritoneal B-1 cells from untreated mice to switch actively to IgA in vivo, we performed RT-PCR analysis on FACS-sorted peritoneal B-cell subsets from untreated BALB/c mice in order to examine the presence of germline Ca mRNA and mature Ca mRNA transcripts. Germline Ca and mature Ca transcripts were readily detectable in peritoneal B-1 cells (defined as IgMbright/IgDOU), but not, or very little, in peritoneal B-2 cells (defined as IgMaul/IgDbrigrt). Moreover, by subdividing the B-l-cell population in CD5 B-la cells and CD5-B-lb cells, it was shown that in vivo expression of germline Ca and mature Ca transcripts was largely restricted to the B-lb-cell lineage. These results indicate that peritoneal B-1 cells indeed are capable to switch to IgA under normal physiological conditions and hereby further support the view that B-1 cells contribute significantly to the mucosal IgA response, albeit this function appears to be restricted to the B-lb-cell subset.


ventional B-c
ll populations with respect to cell-surface-marker expression, localization, development, and antibody repertoire (Stall et al., 1996).Conventional B cells may undergo somatic hypermutation in the germinal centers and are responsible for high-affinity antibody responses to various antigens, whereas B-1 cells, which show a very low frequency of hypermutation of their Vh genes (Kantor, 1996), primarily produce low-affinity IgM immunoglobulins, most of which are cross-reactive with bacteria-related and self-antigens (reviewed in Kroese et al., 1996).

Evidence that B-1 cells are involved in lgA production was demonstrated in different chimeric mouse models and B6-Sp6 /x,t transgenic mice (reviewed in Kroese et al., 1995).Additionally, it was   shown that several B-l-cell lines showed a high frequency of switching to IgA (Whitmore et al.,  1991).However, all these studies comprised manipu- lated in vivo mouse models or in vitro experiments that left many questions regarding the B-l-cell lineage in normal untreated mice unanswered.lgA class switching of IgM B lymphocytes is preceded by the synthesis of germline Ca mRNA transcripts (Lebman et al., 1990).Germline IgA transcription starts 5' of an Ice exon, proceeds through the switch region, and terminates downstream of the Cce exons.This leads to splicing of the Ice exon to the Cce exon, which forms the germline IgA transcript (Lebman et al., 1990).Downregulation of germline IgA transcripts has been shown to inhibit IgA isotype switching that implies a role for IgA germline transcripts prior to the expression of full-length IgA transcripts (Lin and Stavnezer, 1992).

To study the ability of peritoneal B-1 cells (both Bl a and B-lb) to switch to IgA under normal physiological conditions, we sorted peritoneal B-cell subpopulations from untreated BALB/c mice and examined those populations for the presence of germline IgA transcripts and full-length IgA mRNA transcripts.


RESULTS AND DISCUSSION

Peritoneal Washout Cells Express Germline Cce Transcripts The presence of germline Cce mRNA transcripts and mature IgA heavy-chain mRNA transcripts was determined by RT-PCR in unsorted peritoneal cells from 3-month-old untreated BALB/c mice.cDNA was synthesized from mRNA extractions of perito- neal washout cells as well from unsorted Peyer's patch cells, which already have been shown to express Cce and mature IgA transcripts (Weinstein et al.,   1991).Sorted splenic T cells served as negative controls.Additionally, all cell suspensions were tested for mature C/z transcripts.The Cce germline transcripts were revealed by the use of the Ice-leader and Ccel-Cce2 primers, whereas full-length lgA and IgM transcripts were identified by usage of an universal Vh primer in combination with Cce or C/z primers, respectively.In each set of experiments, cDNAs of various different cell suspensions were normalized by means of serial dilutions with fi-actin mRNA (650 bp).The specificity of the primers was tested on RNA derived from mouse IgA and IgM secreting hybridoma cell lines 2F7 (Bos et al., 996)  and NEO4211 (Bos and Meeuwsen, 1989), respec- tively.The germline primer did not result in a PCR product in either cell lines, whereas the universal Vh primer in combination with the Cce primer and primer did result in the correct PCR product for the corresponding hybridoma (Table I).Both unsorted p

itoneal cells and Peyer
s patches cells express Note: + denotes mRNA l

els detectab
e after 40 cycles of RT-PCR using cDNA from the different sorted and unsorted lymphocyte populations.

germline Ca RNA (445 bp) and mature Cc transcripts (490 bp) (Table I).


Germline IgA Transcript Expression Is

Restricted To Peritoneal B-1 Cells

To specify the phenotype of peritoneal B cells that switch to IgA, peritoneal B-cell subpopulations were sorted based on differences in IgM and IgD expression.The IgM bright and IgD au" populations are formed by the B-1 cells, whereas the B-2 cells are found within the IgM au" and IgD bright populations.

Figure 1 shows a typical example of the staining used and the sorting gates set.The purity of the two sorted peritoneal B-cell subsets was approximately 95%.

RNA was extracted and cDNA was synthesized from the sorted B-cell fractions and analyzed for mature IgM, mature IgA, and germline Ca transcripts.

Ca germline transcript and mature IgA expression occurred mainly in the peritoneal B-1-cell subpopulation (Figure 2 and Table I).In some B-2-cell sorts, a 20-50-fold lower expression of Ca germline tran- script and/or mature IgA transcripts compared to the B-1-cell sorts could be detected.This could be due to contaminating B-1 cells, since in these sorts higher percentages of B-1-cell contamination was observed.

Our parameter for determining switching to IgA is the expression of germline Cc and full-length IgA mRNA transcripts.Germline transcripts are translated preceding the actual isotype switching.A strong correlation between germline transcript expression and isotype switching has been shown (Lebman et al.,  1990).However, the function of germline transcripts is less clear, since mice lacking the c exon are still able to switch to IgA (Harriman et al., 1996).

Our data do not provide information on the proportion of B cells expressing germline or mature IgA transcripts.Previously, we have found by FACS analysis only a very low surface IgA expression on peritoneal B-1 cells (<1% IgA B-cells) (Kroese et   al., 1989).The mouse peritoneal cavity might thus provide the required microenvironment for B-1 cells to initiate switching to IgA in vivo, but most l kely PerC IgD CD5 -


IgM


FIGURE

Identification and sorting of peritoneal B-cell sub- populations by flow cytometry.Peritoneal washout cells of 3-month-old untreated BALB/c mice were stained with anti-IgM (FL) [331], anti-IgD (PE) [11-26], and anti-CD5 (APC) [53-78].Shown gates for sorting were set on the basis of IgM, IgD, and CD5 expression.

-actin
C -GLT B2 B1 Blb Bla 650 bp 460 bp FIGURE 2
Germline Cc transcript expression in sorted peritoneal B-cell subpopulations.Expression of germline Ca transcripts was determined by RT-PCR analysis of RNA from 2 105 sorted B cells.The amount of cDNA of the different B-cell populations used in the PCR reactions were equalized by comparing with/3-actin PCR products derived from serial diluted cDNAs.The size of the expected PCR products for/3-actin and germline Ca transcripts RNA are 650 and 445 bp, respectively.they differentiate and mature to IgA plasma cells after migration out of the peritoneal cavity.For Peyer's patch B cells, it is known that they leave those sites after initiation of isotype switching and migrate through the circulation toward the lamina propria while they further differentiate to IgA plasma cells (Tseng, 1984).Where and how B-1 cells migrate from the periton al cavity and differentiate to IgA plasma cells in the lamina propria remain to be established.Also the factors that are involved in the initiation of isotype switching of B-1 cells in the peritoneal cavity are still unknown.Different interleukins, such as IL-4, IL-5, and TGF-/3, already have been shown to play a role in the regulation of IgA switching and secretion (Harriman et al., 1988; Lebman and Coffman, 1988).Additionally, CD40 ligation by direct B-cell-T-cell interaction seems to be important for isotype switch- ing (Jumper et al., 199

.Both peritoneal T cells as well as other cell
ypes, such as mesothelial cells, might produce the correct cytokines and provide the stimuli for B-1 cells to start isotype switching to IgA.

Whether peritoneal B-1 cells also can switch to other isotypes such as IgG and IgE remains also to be established.


IgA Expression Within the Peritoneal Cavity Is

Confined to the B-lb-Cell Population The B-l-cell population consists both of CD5 B-la cells and CD5-B-lb cells.To examine the ability of both peritoneal B-1 a-and B-lb-cell subpopulations to switch to IgA, the subsets were sorted (Figure 1), RNA was isolated and cDNA was synthesized.Both B-a and B-lb cells express mature IgM transcripts, however, the expression of germline Cc and mature IgA transcripts appeared to be largel confined to the peritoneal B-1 b cells (Table I and Figure 2).The PCR products of the B-lb-cell population has also been cloned and sequenced, which confirmed the identity of the product as germline Cc transcripts compared to the EMBL sequence databank (data not shown).In one out of four experiments also, a very low expression of germline Cc transcripts was found in the B-la subset, which was most probably due to contaminating B-lb cells in

Two alte
native explanations can explain this preferential expression of IgA transcripts among B-lb cells.First, it might be argued that B-la cells dow

egulate CD5 expre
sion after switching to IgA.This might explain reconstitution experiments with purified B-1 a cells, which resulted in IgA plasma cells in the lamina propria (Beagley et al., 1995) and our own preliminary experiments with sorted peritoneal B 1 cells showing that both B-la and B-lb cells may contribute to the pool of intestinal IgA plasma cells (Kroese et al., unpublished observations).Alterna- tively, B-la and B-lb cells might belong to closely related, but separate B cell linea

s, whereby the switching t
IgA is mainly restricted to the B-lb-cell lineage.

In conclusion, our experiments show that peritoneal B cells actively switch toward IgA in vivo, thereby confirming our previous data in manipulated animals that B-1 cells contribute to the IgA production in the mouse.The function of B-1-cell-derived IgA might be important in the establishment and maintenance of the normal gut flora, since we have shown that B-l-cell- derived monoclonal IgA antibodies primarily react with normal gut bacteria (Bos et al., 1996).


MATERIAL AND METHODS


Animals

Bo

male and f
male BALB/c mice at 3 to 4 months were studied.Mice were bred in the animal facility at the Stanford Department of Genetics.


Cell Preparation

Peritoneal washout cells were obtained from the mouse peritoneal cavity by injection of chilled deficient RMPI 1640 medium (Irvine Scientific, Santa Ana, CA) supplemented with 10 mM Hepes, 3% newborn calf serum and 0.1% NAN3.Single-cell suspensions from spleen and Peyer's patches w

e prepa
ed by mincing tissue fragments in the same medium between the frosted ends of micoscope slides.All cell suspensions were treated with red- blood-cell lysing buffer to eliminate erythrocytes.


Staining and Cell Sorting

The B-cell subpopulations were sorted on an exten- sively modified fluoresence-activated cell sorter (FACS II; Becton-Dickinson, Mountain View, CA), as described (Hardy et al., 1984).For sorting subpopulations, single-cell suspensions were stained in tubes on ice with optimal concentrations of conjugated antibodies.Biotinylated antibodies were detected with avidin-Texas Red.Dead cells were stained with propidium iodide and were excluded from sorting.After sorting, 30,000 viable sorted cells were reanalyzed to test the purity of the sorted cells.


Antibodies

Rat and mouse monoclonal antibodies used in this study were as follows: Rat anti-mouse IgD (11-26), rat anti-mouse IgM (331), rat anti-mouse Ly- (CD5; 53-78), mouse anti-mouse Igh-6a (IgM of "a" allo- type, DS-1), and mouse anti-mouse Igh-5a (IgD of "a" allotype, AMS 9.1).Purification and conjugation of antibodies to biotin, fluorescein, phycoerythrin, and allophycocyanin (APC) are described elsewhere (Hardy et al., 1984).


RT-PCR

Total RNA, isolated from different cell suspensions by use of TRIzol (Life Technologies) according to the manufacturer' instructions, was used as a template for cDNA synthesis in a 30-/zl reaction mix containing 1.6 /zg oligo-dT 12-18 (Pharmacia), 10 mM dNTP mix (Pharmacia), MilliQ-DEPC, First Strand Buffer (Life Technologies), 0.1 mM DTT (Life Technolo- gies), 30 U RNA quard (Pharmacia), and 200 U Superscript TM II (Life Technologies).The reaction proceeded at 45C for 30 min, followed by heating to 94C for 5 min.For the RT-PCR, an aliquot of cDNA w