Reprints Available Directly from the Publisher Photocopying Permitted by License Only Antigenic Stimuli Do Not Influence Thymic B Lymphocytes: a Morphological and Functional Study in Germ-free and Conventionally Reared Piglets

We have recently reported that thymic B lymphocytes (TBL) are the first B-cell subpopulation undergoing isotype switching to IgG and IgA during embryonic life. The aim of this study is to analyze the influence of antigenic stimulation on TBL location and activity using a germ-free (GF) newborn pig model, in which maternal antibodies and antigens do not affect B-cell development. Immunohistological analysis showed that TBL were disseminated mainly in the thymic medulla. There were no differences in the distribution of TBL, both in GF newborn piglets before and after colonization with Escherichia coli and in older conventionally reared (CONV) piglets. The number of immunoglobulin (Ig)-secreting cells measured by the ELISPOT method was not influenced by microflora and food antigens. IgM-positive cells secreting IgM and CD45RC-positive cells spontaneously producing IgM, IgG, and IgA were detected in newborn thymus. Our findings suggest that TBL differentiation and Ig switching to IgG and IgA-secreting cells is not influenced by external antigens and that the thymic microenviroment plays an important role in this process.


.The importan
e of direct T-cell interaction and cytokines in their matura- tion has been recently confirmed by functional analyses of murine and human TBL (Inaba et al.,  1990, 1995; Punnonen and de Vries, 1993).

To investigate the characteristics of TBL, we used a unique pig model.In contrast to mice and humans, in which B-cell development is influenced by mater- nal antibodies and antigens (Kearney and Vakil,  1986), pigs have a special six-layered placenta preventing the transfer of maternal regulatory factors to the embryo.Transfer of maternal Igs occurs after suckling maternal colostrum (,terzl et al., 1965).For this reason, pig fetuses and colostrum-deprived piglets reared in germ-free (GF) conditions are very useful experimental models that allow to differentiate between innate factors and factors arising under the influence of passively acquired antibodies and exter- nal antigenic stimuli (Tlaskalov-Hogenov et al., 1983).We have observed, using the ELISPOT method, that the fetal pig thymus contains Igsecreting cells very early in embryonic life (Prokeov et al., 1981; Cukrowska et al., 1996a).In contrast to fetal splenic B cells, which have been found to produce exclusively IgM, fetal thymic B cells of the same age undergo spontaneous and after in vitro polyclonal activation isotype switching into IgG and IgA (Cukrowska et al., 1995, 1996a).

In the present study, we analyze the influence of extern l antigenic stimuli on spontaneous Ig produc- tion by TBL and on the location of B cells in the pig thymus.


RESULTS

Location of Thymic B Cells in GF Newborns, GF

Piglets Colonized with Escherichia coli and Conventionally Reared (CONV) Piglets

The newborn pig thymus displayed sparsely scattered lymphocytes expressing membrane-bound Igs (Fig. 1, left).Most cells were seen in the medulla, without an apparent accumulation or follicular structures.There was no agglomeration around the Hassall's bodies.Single contoured cells were noted in the cortex in lower numbers than in the medulla (by 1 to 2 orders of magnitude).The reaction with the pig anti-rabbit antibody was negative.The deep cortex and medulla harbored larger cells with coarse cytoplasmic DABreactive granules (macrophages).This staining of macrophages was unchanged after omission of the antibodies (Fig. 1, right).

The adult pig thymus exhibited a similar distribu- tion of Ig-positive lymphocytes as in GF newborn piglets; macrophages were concentrated in the corti- comedullary junction (data not shown).The stimula- tion of GF piglets by Escherichia coli 086 elicited an increase in endogenous peroxi ase-reactive macro- phages, whereas Ig-positive lymphocytes remained scarce and no follicular agglomeration was found (Fig. 2, left).In contrast to the scattered d

of TBL, splenic Ig-positive cells were assembled to follicles (Fig. 2, fight).


Spontaneo
s Ig Production Measured by the ELISP T Assay

As shown by immunohistological studies, the location and organization of TBL was not altered by antigenic stimuli; we also tried to analyze the functional state of these cells by measuring spontaneous Ig production.

Conventional microflora and food ant gens influenced neither the number of spot-forming cells nor the isotype pattern of Ig production by TBL.In contrast to splenic B cells of CONV piglets that showed a significant increase in Ig-secreting cells of all iso- topes, the thymus of CONV piglets and GF newborns contained comparable numbers of IgM-, IgG-, and IgA-secreting cells (Figure 3).The experimental /IgM [--] IgA 7-/J IgG FIGURE 3 Spontaneous Ig production in (A) thymus and (B) spleen of GF newborns, GF piglets colonized by E. coli 086 and CONV piglets.Ig production in thymic-and splenic-cell suspensions was measured by the ELISPOT method.The results are determined as the number of Ig-secreting cells (Ig-SC) per 106 nucleated cells; arithmetical means _+ SD from four animals are shown.GF, GF newborns; EC, GF piglets colonized by E. coli; CONV, conventionally reared piglets.

role in immune response against microflora, the problem of their bi

ogical functions is still open.The role of TBL as antige
-presenting cells in shaping of T-cell repertoire has been proposed (Inaba et al.,  1991, Mazda et al., 1991).Mouse thymic B cells, but not splenic cells were found to induce tolerance by a deletion mechanism.However in the light of recent findings on the importance of natural autoantibodies in selection processes (Holmberg and Kearney 1994), it cannot be excluded that Igs spontaneously secreted by TBL locally influence T-cell development.Analy- ses of the antibody specificity produced by TBL during ontogeny is still lacking.Interestingly, we have recently reported a pronounced reactivity of "preimmune" Igs, that is, isolated from fetal and GF newborn sera with pig thymocytes (Tlaskalov-Hogenov et al., 1994; Cukrowska et al., 1996b).


MATERIALS AND METHODS


Animals

Colostrum-deprived Miniature Minnesota pig new- borns day old were used.Some fetuses were kept in isolators after a gnotobiotic hysterectomy and were given a sterilized milktype diet (Travniek et al.,  1966).The germ-free state was controlled twice a week by an aerobic and anaerobic cultivation of a 1 000 800 600 400 FIGURE 4 Spontaneous Ig production by thymic IgM-positive and CD45RC-positive cells.Cells were separated from GF newborn thymocytes on miniMACS column

pig CD45
C mAb.Ig production was measured by the ELISPOT method.The results are determined as the number of Ig-secreting cells (Ig-SC) per 5 l05 nucleated cells; arithmetical means _+SD from five animals are shown.TS, thymic-cell suspension.rectal swab.GF piglets received 5 108 non- pathogenic E. coli 086 in the milk from the fifth day after the birth.On day 7 after colonization, animals were exsanguinated under anesthesia and lymphoid organs were taken for cell isolation.CONV piglets, 2 to 3 months old were also used.
200 TS IgM + CD45RC + IgM [--] IgA F IgG

Isolation of Cells

The lymphoid organs were gently minced in RPMI-1640 medium, filtrated trough a nylon mesh, washed three times in the medium, and than resuspended in the complete RPMI-1640 medium supplemented by 10% fetal calf serum and gentamycin.lmmunohistochemistry Pig Ig-positive cells were detected on acetone- extracted cryostat sections (6 m) of unfixed frozen pig thymus by one-step direct immunoenzyme test using the HRP-labele

rabbit anti-swine a
tibody (RASW-HRP) diluted 1:40 (Sevac, Prague, Czech Republic).The incubation was hr at room temperature and the bound enzyme was developed with DAB-H202 reagent.The control test involved incubation with an antibody of different specificity (HRPcoupled swine anti-rabbit) or incubation with DAB-H202 alone.


Detection of Spontaneously Ig-Secreting Cells

The number of spontaneously Ig-secreting cells of all isotypes was determined by the ELISPOT method using anti-pig isotype-specific mAb as previously described (Cukrowska et al., 1996a).The following murine anti-pig mAb were used: anti-IgM (LIG-4) (Dvorak et al. 1986), anti-IgG (23.3 a), anti-IgA (27.9.1), anti-light chain (27.7.1 and 27.2.1) (van Zaane and Holst, 1987) (the kind gift from A

.J. Bianchi, Lelystad, the Netherlands).In ord
r to confirm that spot formation was constituted as a result of Ig synthesis during the in vitro incubation, an inhibition of protein synthesis by cycloheximide (Sigma, St.Louis) was conducted (Cukrowska et al.,  1996a).This treatment abrogated the spot formation of all isotypes, thus demonstrating that this assay detects cells synthesizing and actively secreting Igs.

Enrichment of B Lymphocytes from Thymus A positive separation procedure by the magnetic miniMACS column (Miltenyi Biotech, Bergish-Gladbach, Germany) was performed using anti-pig IgM and anti-pig CD45RC mAb (Saalmtiller 1996) to enrich B lymphocytes from newborn thymus accord- ing to manufactural instructions.Separated lymphocytes were spun down, resuspended in 10% RPMI-1640 medium, and used for detection of Ig secretion by the ELISPOT method.The purity of separated cells was >90% as assessed by flow cytometry.Magnetic microbeads and/or mAb had no effect on spontaneous Ig secretion (data not shown); thus, we could rule out the possible activation of cells by mAb and magnetic micr