[Frontiers in Bioscience 3, a58-65, November 1, 1998]

	[Frontiers in Bioscience 3, a58-65, November 1, 1998] Reprints PubMed CAVEAT LECTOR
	EARLY INDUCTION AND AUGMENTATION OF PARASITIC ANTIGEN-SPECIFIC ANTIBODY-PRODUCING B LYMPHOCYTES IN THE NON-PEYER’S PATCH REGION OF THE SMALL INTESTINE Ching-Hua Wang¹, Elizabeth M. Richards¹, Robert D. Block², Enio M. Lezcano¹ and Ricardo Gutierrez¹ 1 Biology Department, California State university, 5500 University parkway, San Bernardino, CA 92407, USA, ² Department of Neurology and Neural Science, School of Medicine, University of Southern California, Los Angeles, CA 90033, USA Received 10/12/98 Accepted 10/20/98 5. DISCUSSION The experiments reported here quantitatively examined the non-Peyer patch tissues of the small intestine, Peyer’s patch, mesenteric lymph node, and spleen of rats for the presence of B lymphocytes, expressing IgM, IgA, IgE, IgG1, IgG2a, IgG2b, and IgG2c antibody isotypes that were specific for monoclonal antibody affinity purified T. spiralis 9D4 antigen using a double labeling immunofluorescence assay. The results showed that merely 1 day after infection, 9D4 antigen-specific IgG1-PC first appeared in significant numbers in the lamina propria of the non-Peyer’s patch tissue of the small intestine (figure 4). This was followed by drastic increases of antigen-specific IgM- (figure 1), IgE- (figure 3), and the rest of the subtypes of IgG-PC (figures 5-7) 24 hr later. This is the first piece of evidence demonstrating such an almost immediate B cell response after T. spiralis infection that is directly taking place right in the tissues of the small intestine that are not associated with the Peyer’s patches. It has been demonstrated that it takes some 12 hr before the first T cells to become activated in these tissues (25), and the 9D4 antigen is a glycosylated protein preparation (28, 29). Therefore, the B cells examined here most likely need the stimulation from the T cells due to the antigen being T cell-dependent. The fact that the 9D4-specific IgG1-PC increased 16 times (figure 4) over the control within 12 hr after T cells became activated, strongly demonstrates the immediacy of the interaction between the T and B cell populations in the tissues and the efficiency of this immune response. The proliferative response by IgE-PC increased 18 times (figure 3), and that of the IgM-, IgG2a-, IgG2b- and IgG2c-PC climbed up 7, 6, 10, and 13 times, respectively (figures 1, 5-7), all by 24 hr, over the controls. This further reveals the rapidity of the immune response in a much broader scope. When the kinetics are scrutinized in more detail, one can see that IgG2b-PC reached its peak only 2 days after infection, which was 10 times of the control. This was followed by a decline on day 5 and then a rebound by day 7 (figure 6). Whether this suggests a short half-life or a dissemination or migration of these cells needs to be further investigated. IgM- and IgG1-PC reached their peak 24 hr after IgG2b-PC, producing a 10x and 44 x augmentation, respectively (figures 1, 4)). IgE-PC climbed to the peak on day 5, which was 45 times over the control (figure 3), while IgG2c-PC produced the highest proliferation on day 7 (19 times of the control), the last day examined (figure 7). Although these kinetic curves do not necessarily correlate with the quantities of antibodies ultimately secreted by these B cells, the magnitude of enhancement in the numbers of these Ab-PC only days after infection once again demonstrates the immunological competency of the non-Peyer's patch tissues of the small intestine. Our results correspond with previous evidence of high levels of serum IgG and IgE antibodies (9-11, 14, 18, 19) and evidence of rapid increase of IgE in intestinal fluids (15, 16) after T. spiralis infection. Therefore, it is more than likely that there is such correlation between the numbers of Ab-PC and the quantities of antibodies produced in vivo. In fact, when examining these double labeled cells as shown in figures 8a and 8b, one cannot help but noticing that aside from the labeled cells, there are quite a few small aggregated clumps within the lamina propria showing both the green and red color. Most likely, these are immune complexes formed between the secreted IgE antibodies and the 9D4 antigens, indicating that these IgE molecules are indeed produced by the IgE-bearing B cells in response to the parasitic challenge in the small intestine. No proliferation of 9D4-specific IgA-PC was observed in the intestine until 3 to 4 days later as compared to the other isotypes of Ab-PC (figure 2). This shows that at least in this immune response, e. g. a multiple isotypic B cell response, IgA is not as important as IgG- and IgE-PC. This result is contrary to many of previous studies, in which IgA has always been found to be a vital immunological factor in the mucosal immune response against various pathogens and stimuli (1-6). Since IgA-PC has been associated with the function of the Peyer's patches, and Peyer's patches are not the tissue sites taken by T. spiralis as its niche, it is reasonable to believe that when Peyer's patches are not stimulated directly by a pathogen, IgA-PC are not generated in abundance. In this study, the numbers of Ab-PC were quantified in two separate regions in the Peyer's patches, the germinal centers and the non-germinal tissue. It has been revealed that the latter region of the Peyer's patches played no role in all responses by B cells during the entire period studied (figures 1-7). However, in the germinal centers, a totally different kinetics appeared. Barely one day after infection, 9D4-specific IgM- and IgG2a-PC increased significantly over the controls by 17 and 7 times, respectively (figures1, 5). IgE-PC increased 29 times by day 2 (figure 3), which tapered off during the rest of the study period. A comparison of the patterns of kinetics of IgE-PC found in the intestine and in the PP-GC reveals a seemingly opposite trends, in which the former tissue generated a significant ascending curve, whereas the latter produced a gradual descending curve with days post infection (figure 3). First, this suggests that the T. spiralis antigen can quickly diffuse into the germinal centers of the Peyer's patches even though they are not the preferred sites for the parasitic invasion. Once the antigenic stimulation occurs, activated by T cells, Ab-PC like IgM-, IgG2a- and IgE-PC begin proliferating. Second, no statistical difference was found when comparing the numbers of IgE-PC in these two tissues on days 2 and 3. However, on days 5 and 7, intestinal tissue showed significantly higher numbers of IgE-PC than PP-GC. On day 5, the number found in the intestine per VCU was nearly 4x that of the PP-GC per field of view, and on day 7, it was nearly 2x that of the PP-GC (figure 3). The PP contains far higher density of lymphocytes than the non-Peyer's patch tissues of the small intestinal tissues. The data collected reflect numbers of Ab-PC per VCU in the intestine and that of Ab-PC per field of vision in the PP. As tissues, the former contains loosely associated and various types of cells, including lymphocytes, and the latter harbors densely packed lymphocytes. This shows that in this particular immune response, the intestine plays a leading role in generating Ab-PC against the invading parasite, not the Peyer's patches. The responses elicited by the other Ab-PC in the PP-GC were also weaker and significantly delayed as compared to that of the intestine, e.g., IgG2b- and IgG1-PC occurred 3 and 4 days later than those in the intestine, respectively (figures 4, 6). Once again, no IgA-PC response was observed in the PP-GC throughout the period studied (figure 2). Substantiating the point that IgA is not important in this immune response, these results also indicate that whatever IgA-PC produced in the intestine, they were produced by the intestinal tissues, not first by the PP, then migrated intestinally into the lamina propria. They also suggest that the parasitic antigen used is not an IgA inducing antigen. In the MLN, no IgA- and IgG2a-PC were elicited (figures 2, 5). A significant response, yet 3x as weak as that of the intestine, was generated by IgM-PC in the MLN on day 3 only (figure 1). All the other Ab-PC produced significant yet weaker responses against the parasitic antigen, too (figures 3, 4, 6, 7). One interesting bi-peak phenomenon was observed in the response curves of IgE-, IgG2c-, IgG1, and IgG2b-PC. The first two Ab-PC showed a peak on day 2 and a peak on day 7, separated by a dip, and the last two Ab-PC had their first peak on day 3 and second peak on day 7, intervened with a similar decline (figures 3, 4, 6, 7). Similar pattern of response was also demonstrated in the IgM-PC in the intestine (figure 1) and the IgG2a-PC response in the spleen (figure 5). The difference between the peak and the valley is statistically significant. One reasonable explanation for this phenomenon is that these B cells, at least a proportion of them, undergoes migration from the intestine to the general circulation via the lymphatics and blood. Similar pattern of migration has been noticed among intestinally activated T cells using the same experimental system (25), and it is not surprising that these B cells may also hold the same pattern. When the responses by Ab-PC were examined in the spleen, it is demonstrated that although all isotypes of Ab-PC augmented their responses, they were weaker and delayed than the intestine (figures 1-7). Since the spleen is anatomically away from the antigenic stimulation site, these results are understandable. Comparing the kinetics of appearance of Ab-PC in all tissues examined, it appears that nearly all of the different isotypic B lymphocytes become activated in non-Peyer’s patch regions of the intestine either at the same time, or significantly earlier than in the Peyer’s patch regions, except IgM-, and IgG2a-PC. These results strongly support the idea that most of the T. spiralis antigen-specific B lymphocytes are not derived from the Peyer’s patches. In stead, they are originated, stimulated, and activated within the lamina propria of the intestine. The significance of this study is that this is the first time intestinal B lymphocytes specific for a parasitic antigen have been visualized and quantified at such an early stage of a primary infection. The antigen-specific IgE-PC first appeared as early as 48 hr, and IgG1-PC just 1 day after infection, both occurred not only sooner than the appearance of IgA-PC, but also in greater overall numbers than IgA-PC in the Peyer’s patch, mesenteric lymph node and the spleen. The study, therefore, demonstrates that the small intestine, when challenged, is capable of exerting an immediate and yet highly efficient immune response encompassing multiple cellular populations to protect its host.

[Frontiers in Bioscience 3, a58-65, November 1, 1998]
Reprints
PubMed
CAVEAT LECTOR

EARLY INDUCTION AND AUGMENTATION OF PARASITIC ANTIGEN-SPECIFIC ANTIBODY-PRODUCING B LYMPHOCYTES IN THE NON-PEYER’S PATCH REGION OF THE SMALL INTESTINE

Ching-Hua Wang¹, Elizabeth M. Richards¹, Robert D. Block², Enio M. Lezcano¹ and Ricardo Gutierrez¹

1 Biology Department, California State university, 5500 University parkway, San Bernardino, CA 92407, USA, ² Department of Neurology and Neural Science, School of Medicine, University of Southern California, Los Angeles, CA 90033, USA

Received 10/12/98 Accepted 10/20/98

5. DISCUSSION

The experiments reported here quantitatively examined the non-Peyer patch tissues of the small intestine, Peyer’s patch, mesenteric lymph node, and spleen of rats for the presence of B lymphocytes, expressing IgM, IgA, IgE, IgG1, IgG2a, IgG2b, and IgG2c antibody isotypes that were specific for monoclonal antibody affinity purified T. spiralis 9D4 antigen using a double labeling immunofluorescence assay. The results showed that merely 1 day after infection, 9D4 antigen-specific IgG1-PC first appeared in significant numbers in the lamina propria of the non-Peyer’s patch tissue of the small intestine (figure 4). This was followed by drastic increases of antigen-specific IgM- (figure 1), IgE- (figure 3), and the rest of the subtypes of IgG-PC (figures 5-7) 24 hr later. This is the first piece of evidence demonstrating such an almost immediate B cell response after T. spiralis infection that is directly taking place right in the tissues of the small intestine that are not associated with the Peyer’s patches.

It has been demonstrated that it takes some 12 hr before the first T cells to become activated in these tissues (25), and the 9D4 antigen is a glycosylated protein preparation (28, 29). Therefore, the B cells examined here most likely need the stimulation from the T cells due to the antigen being T cell-dependent. The fact that the 9D4-specific IgG1-PC increased 16 times (figure 4) over the control within 12 hr after T cells became activated, strongly demonstrates the immediacy of the interaction between the T and B cell populations in the tissues and the efficiency of this immune response. The proliferative response by IgE-PC increased 18 times (figure 3), and that of the IgM-, IgG2a-, IgG2b- and IgG2c-PC climbed up 7, 6, 10, and 13 times, respectively (figures 1, 5-7), all by 24 hr, over the controls. This further reveals the rapidity of the immune response in a much broader scope. When the kinetics are scrutinized in more detail, one can see that IgG2b-PC reached its peak only 2 days after infection, which was 10 times of the control. This was followed by a decline on day 5 and then a rebound by day 7 (figure 6). Whether this suggests a short half-life or a dissemination or migration of these cells needs to be further investigated. IgM- and IgG1-PC reached their peak 24 hr after IgG2b-PC, producing a 10x and 44 x augmentation, respectively (figures 1, 4)). IgE-PC climbed to the peak on day 5, which was 45 times over the control (figure 3), while IgG2c-PC produced the highest proliferation on day 7 (19 times of the control), the last day examined (figure 7). Although these kinetic curves do not necessarily correlate with the quantities of antibodies ultimately secreted by these B cells, the magnitude of enhancement in the numbers of these Ab-PC only days after infection once again demonstrates the immunological competency of the non-Peyer's patch tissues of the small intestine. Our results correspond with previous evidence of high levels of serum IgG and IgE antibodies (9-11, 14, 18, 19) and evidence of rapid increase of IgE in intestinal fluids (15, 16) after T. spiralis infection. Therefore, it is more than likely that there is such correlation between the numbers of Ab-PC and the quantities of antibodies produced in vivo. In fact, when examining these double labeled cells as shown in figures 8a and 8b, one cannot help but noticing that aside from the labeled cells, there are quite a few small aggregated clumps within the lamina propria showing both the green and red color. Most likely, these are immune complexes formed between the secreted IgE antibodies and the 9D4 antigens, indicating that these IgE molecules are indeed produced by the IgE-bearing B cells in response to the parasitic challenge in the small intestine.

No proliferation of 9D4-specific IgA-PC was observed in the intestine until 3 to 4 days later as compared to the other isotypes of Ab-PC (figure 2). This shows that at least in this immune response, e. g. a multiple isotypic B cell response, IgA is not as important as IgG- and IgE-PC. This result is contrary to many of previous studies, in which IgA has always been found to be a vital immunological factor in the mucosal immune response against various pathogens and stimuli (1-6). Since IgA-PC has been associated with the function of the Peyer's patches, and Peyer's patches are not the tissue sites taken by T. spiralis as its niche, it is reasonable to believe that when Peyer's patches are not stimulated directly by a pathogen, IgA-PC are not generated in abundance.

In this study, the numbers of Ab-PC were quantified in two separate regions in the Peyer's patches, the germinal centers and the non-germinal tissue. It has been revealed that the latter region of the Peyer's patches played no role in all responses by B cells during the entire period studied (figures 1-7). However, in the germinal centers, a totally different kinetics appeared. Barely one day after infection, 9D4-specific IgM- and IgG2a-PC increased significantly over the controls by 17 and 7 times, respectively (figures1, 5). IgE-PC increased 29 times by day 2 (figure 3), which tapered off during the rest of the study period. A comparison of the patterns of kinetics of IgE-PC found in the intestine and in the PP-GC reveals a seemingly opposite trends, in which the former tissue generated a significant ascending curve, whereas the latter produced a gradual descending curve with days post infection (figure 3). First, this suggests that the T. spiralis antigen can quickly diffuse into the germinal centers of the Peyer's patches even though they are not the preferred sites for the parasitic invasion. Once the antigenic stimulation occurs, activated by T cells, Ab-PC like IgM-, IgG2a- and IgE-PC begin proliferating. Second, no statistical difference was found when comparing the numbers of IgE-PC in these two tissues on days 2 and 3. However, on days 5 and 7, intestinal tissue showed significantly higher numbers of IgE-PC than PP-GC. On day 5, the number found in the intestine per VCU was nearly 4x that of the PP-GC per field of view, and on day 7, it was nearly 2x that of the PP-GC (figure 3). The PP contains far higher density of lymphocytes than the non-Peyer's patch tissues of the small intestinal tissues. The data collected reflect numbers of Ab-PC per VCU in the intestine and that of Ab-PC per field of vision in the PP. As tissues, the former contains loosely associated and various types of cells, including lymphocytes, and the latter harbors densely packed lymphocytes. This shows that in this particular immune response, the intestine plays a leading role in generating Ab-PC against the invading parasite, not the Peyer's patches. The responses elicited by the other Ab-PC in the PP-GC were also weaker and significantly delayed as compared to that of the intestine, e.g., IgG2b- and IgG1-PC occurred 3 and 4 days later than those in the intestine, respectively (figures 4, 6). Once again, no IgA-PC response was observed in the PP-GC throughout the period studied (figure 2). Substantiating the point that IgA is not important in this immune response, these results also indicate that whatever IgA-PC produced in the intestine, they were produced by the intestinal tissues, not first by the PP, then migrated intestinally into the lamina propria. They also suggest that the parasitic antigen used is not an IgA inducing antigen.

In the MLN, no IgA- and IgG2a-PC were elicited (figures 2, 5). A significant response, yet 3x as weak as that of the intestine, was generated by IgM-PC in the MLN on day 3 only (figure 1). All the other Ab-PC produced significant yet weaker responses against the parasitic antigen, too (figures 3, 4, 6, 7). One interesting bi-peak phenomenon was observed in the response curves of IgE-, IgG2c-, IgG1, and IgG2b-PC. The first two Ab-PC showed a peak on day 2 and a peak on day 7, separated by a dip, and the last two Ab-PC had their first peak on day 3 and second peak on day 7, intervened with a similar decline (figures 3, 4, 6, 7). Similar pattern of response was also demonstrated in the IgM-PC in the intestine (figure 1) and the IgG2a-PC response in the spleen (figure 5). The difference between the peak and the valley is statistically significant. One reasonable explanation for this phenomenon is that these B cells, at least a proportion of them, undergoes migration from the intestine to the general circulation via the lymphatics and blood. Similar pattern of migration has been noticed among intestinally activated T cells using the same experimental system (25), and it is not surprising that these B cells may also hold the same pattern. When the responses by Ab-PC were examined in the spleen, it is demonstrated that although all isotypes of Ab-PC augmented their responses, they were weaker and delayed than the intestine (figures 1-7). Since the spleen is anatomically away from the antigenic stimulation site, these results are understandable.

Comparing the kinetics of appearance of Ab-PC in all tissues examined, it appears that nearly all of the different isotypic B lymphocytes become activated in non-Peyer’s patch regions of the intestine either at the same time, or significantly earlier than in the Peyer’s patch regions, except IgM-, and IgG2a-PC. These results strongly support the idea that most of the T. spiralis antigen-specific B lymphocytes are not derived from the Peyer’s patches. In stead, they are originated, stimulated, and activated within the lamina propria of the intestine.

The significance of this study is that this is the first time intestinal B lymphocytes specific for a parasitic antigen have been visualized and quantified at such an early stage of a primary infection. The antigen-specific IgE-PC first appeared as early as 48 hr, and IgG1-PC just 1 day after infection, both occurred not only sooner than the appearance of IgA-PC, but also in greater overall numbers than IgA-PC in the Peyer’s patch, mesenteric lymph node and the spleen. The study, therefore, demonstrates that the small intestine, when challenged, is capable of exerting an immediate and yet highly efficient immune response encompassing multiple cellular populations to protect its host.