藤橋 浩太郎

We have directly compared enzyme-linked immunoassays (ELISAs) with bioluminescent immunoassays employing derivatives of the bioluminescent molecule aequorin, and have shown that detection of mucosal and serum antibodies is considerably more sensitive when detected by luminometry. Luminometry is based upon counting photons of light via phototubes and is generally similar to scintillation spectrometry. Current commercial luminometric technology employs a phototube which is most efficient for light emission in the 400-420 nm wavelength range. For this reason, we have chosen the bioluminescent molecule, aequorin, which upon the addition of Ca2+ undergoes a conformational change resulting in the emission of blue light at 469 nm, The high quantum yield is reflected by the fact that addition of Ca2+ to 1 ng of recombinant streptaequorin, a covalent conjugate of streptavidin and aequorin, resulted in the production of 7 x 10(8) relative light units, In this study, we show the superior sensitivity of biotin-streptaequorin when directly compared with biotin-streptavidin linked horseradish peroxidase commonly used for ELISA. For example, mice orally immunized once with cholera toxin (CT) did not exhibit detectable fecal IgA antibodies as determined by ELISA, whereas use of streptaequorin and the bioluminescent immunoassay revealed fecal IgA anti-CT-B subunit antibody titers of 1:24 500. In addition, no detectable anti-CT-B antibodies were noted in saliva samples by ELISA 7 days following oral immunization with CT, while IgA endpoint titers could be extrapolated to 1:393 000. The 21 day fecal IgA anti-CT-B titers were 1:512 by ELISA, whereas titers determined by luminometry reached 1:10(7) when Neutralite avidin and biotinylated aequorin were employed. In general, the bioluminescent immunoassay was > 10(4)-fold more sensitive when compared with ELISA for detection of mucosal and serum antigen- and isotype-specific antibody responses, Thus, the bioluminescent immunoassay is a more sensitive assay for detection of antibodies in dilute external secretions.

Elevated numbers of plasma cells are associated with localized and chronically inflamed gingiva of patients with adult periodontitis. However, only limited information is currently available as to how cytokines produced by CD4(+) T cells are involved in these increased B cell responses in affected gingival tissues. When gingival mononuclear cells (GMC) were isolated from inflamed tissues and examined by flow cytometry, similar to 20-30% of lymphocytes were CD4(+) T cells. For the analysis of Th1 and Th2 cytokine expression by these CD4(+) T cells, RNA was extracted and reverse transcriptase-polymerase chain reaction (RT-PCR) was performed by using specific 5' and 3' primers for interferon-gamma (IFN-gamma) and IL-2 (Th1), IL-4, IL-5, IL-6, IL-IO and IL-13 (Th2) and beta-actin (internal control). Two distinct cytokine profiles were noted based on the expression of selected Th1 and Th2 cytokines, where one pattern was represented by expression of mRNA for IFN-gamma, IL-6, IL-10 and IL-13, while the second consisted of mRNA for IFN-gamma, IL-6 and IL-13. In most samples, mRNA for IL-2, IL-4 and IL-5 were not detected by cytokine-specific RT-PCR. When RNA was isolated from CD4(+) T cells of concanavalin A-stimulated peripheral blood mononuclear cells (PBMC) of the same patients and examined by RT-PCR, mRNA for all Th1 and Th2 cytokines were detected. These findings suggest that although human CD4(+) T cells are capable of producing an array of Th1- and Th2-type cytokines, the CD4(+) T cells associated with periodontitis are limited to production of IFN-gamma, IL-6, IL-13 and in some instances IL-10. CD4(+) T cells from diseased periodontal tissues are divisible into two groups based upon whether or not IL-10 is produced, together with IFN-gamma, IL-6 and IL-13.

Escherichia coli labile toxin (LT) was assessed as mucosal immunogen and as adjuvant for tetanus toroid (TT) in mice, After oral administration of LT, C57BL/6 (H-2(b)) and BALB/c (H-2(d)) mice were high mucosal and serum antibody responders, while C3H/HeN (H-2(k)) mice were low responders. High responders exhibited mainly serum Ige (including IgG1, IgG2a, and IgG2b), as well as IgM and IgA, while mucosal responses were IgA. Analysis of LT-B-specific CD4(+) T helper (Th) cells from Peyer's patches (PP) or from spleen revealed a mixed Th1 (interferon-gamma) and Th2 (interleukin-4 and -5) cell pattern, Oral LT given with TT induced TT-specific response patterns identical to LT-B, Analysis of mRNA from TT-specific PP CD4(+) Th cells also revealed a mixed Th1- and Th2-type response. Thus, antibody response profiles induced by LT are regulated by both CD4(+) Th1 and Th2 cell types.

Inflamed gingival tissues are enriched in macrophages (MOs) and CD4-positive T cells; however, T helper-type cytokines such as interleukin (IL)-2 and IL-4 are absent Therefore, we investigated whether a relationship exists between IL-4 receptor (IL-4R) expression and MO persistence in the absence of exogenous IL-4, Gingival MOs, when compared with monocyte(MN)/MOs from peripheral blood mononuclear cells, expressed high levels of IL-4R mRNA. Furthermore, in vitro cultures of gingival MOs remained viable whereas identically treated peripheral blood MN/MOs rapidly lost viability, However, when gingival MOs were incubated with recombinant IL-4 (rIL-4), the cell viability was dramatically reduced, When the frequency of apoptotic cells was assessed in rIL-4-treated gingival MO cultures, higher numbers of apoptotic cells were noted in rIL-4-treated versus control cultures. Furthermore, rIL-4-treated MOs from inflamed gingiva showed DNA fragmentation as assessed by electrophoresis, These findings clearly, show that addition of exogenous rlL-4 to gingival MO cultures leads to cell death by apoptosis. This finding would suggest that topical application of rIL-4 may inhibit the persistence of MOs in adult periodontitis, which could then creased inflammation.

The mechanisms which regulate mucosal IgA responses to orally administered protein vaccines are not yet fully elucidated. We have used two delivery systems, soluble tetanus toroid (TT) with the mucosal adjuvant cholera toxin (CT) and recombinant Salmonella expressing Tox C, a fragment of TT, to assess the nature of CD4(+) T helper (Th) cells and derived cytokines which support mucosal IgA responses in both normal and cytokine knockout (interferon gamma knockout; IFN-gamma(-/-) and IL-4(-/-)) mice. Our results provide important new information regarding Th cell and cytokine regulation of mucosal IgA responses. Whereas TT coadministered with CT induces predominant TT-specific Th2-type responses, rSalmonella delivery of Tox C induced dominant Th1-type responses along with synthesis of the Th2-cytokine IL-10. Both vaccine regimen elicited high levels of mucosal S-IgA and IL-6 production by macrophages. Further, oral immunization of IFN-gamma(-/-) and IL-4(-/-) mice with rSalmonella Tox C also induced macrophage-derived IL-6 and Th2-derived IL-10 as well as S-IgA responses, suggesting that IFN-gamma from Th1-type cells as well as traditional Th2 cells producing IL-4 and IL-5 are not essential for mucosal IgA responses. Rather, induction of second level Th2 cells producing IL-10 together with high levels of IL-6 from other cell sources may be sufficient for mucosal IgA responses in the absence of traditional Th2 cells. These studies were facilitated by the development of a sensitive new luminometry assay which allowed detection of cytokines and cell surface molecules which are below the levels of detection by current solid phase assays.

Despite pathophysiologic effects including diarrhea, cholera toxin (CT) is a potent mucosal immunogen and adjuvant. We investigated the influence of CT on T helper (Th)-type 1 (Th1) and Th2 cell-regulated Ag-specific B cell isotype and IgG subclass Ab responses elicited when the toxin was co-administered orally with different protein Ags. When mice were orally immunized with tetanus toroid (TT) and CT as adjuvant, this regimen induced TT-specific secretory IgA responses in the gastrointestinal tract as well as serum IgG, including IgG1 and IgG2b subclasses, and IgA responses. This oral regimen also induced TT- and CT-B-specific IgE responses. In addition, CT also elicited adjuvant effects for Ag-specific IgG1, IgE, and IgA responses when two other protein Ags, OVA and hen egg white lysozyme, were given by the oral route. Quantitative reverse transcriptase-PCR was performed to assess levels of mRNA for Th1 (IFN-gamma) and Th2 (IL-4) cytokine expression in TT-stimulated CD4(+) T cell cultures. Both Peyer's patches and splenic CD4(+) T cells expressed markedly increased levels of IL-4-specific message, but did not result in changes in IFN-gamma mRNA expression. To determine whether the route of immunization influenced IgE responses, mice were immunized s.c. with TT and CT as adjuvant. Significant increases in total and TT-specific IgE Abs were Induced when CT was co-administered. Taken together, these results show that CT acts as a mucosal adjuvant to enhance Th2-type responses and in particular, the IL-4 produced results in a characteristic Ab isotype pattern associated with this cytokine.

Currently only limited information is available as to why dominant IgA isotype responses are supported by mucosal T cells in effector tissues. To address this issue directly, gamma delta and alpha beta T cells were isolated from the submandibular gland (SMG) of mice as an example of mucosal effector tissues. Freshly isolated CD3(+) T cells from this tissue contained relatively high numbers of activated cells [approximately 10% interleukin-2 receptor (IL-2R)(+) cells and 15 % of cells in cycle stages S and G(2) + M], of which 25 % and 75 % were gamma delta and alpha beta T cells, respectively. The cytokine-specific quantitative reverse transcriptase-polymerase chain reaction and enzyme-linked immunospot analyses revealed that, although both gamma delta and alpha beta T cells were capable of producing an array of Th1 or Th2 cytokines following stimulation via the T cell receptor-CD3 complex, these mucosal T cells were mainly committed to IL-5 and IL-6 expression in vivo (Th2 type). Both freshly isolated gamma delta and alpha beta T cells expressed mRNA and contained IL-5 and IL-6 spot-forming cells (SFC); however, only the latter exhibited high mRNA levels and SFC for a Th1 cytokine (interferon-gamma). Taken together, the results show that freshly isolated CD3(+) T cells from SMG contain activated gamma delta and alpha beta T cells which are programmed to produce IL-5 and IL-6. Thus, SMG, an example of an IgA effector tissue, can be characterized as a Th2-dominant site. However, although both gamma delta and alpha beta T cells express cytokine profiles consistent with a Th2 phenotype, only the latter subset with a CD4(+) CD8(-) phenotype provided effective help for mucosal B cell responses in vitro.

Intraepithelial lymphocytes (IELs) have been extensively studied in the murine small intestine. However, to date no studies have assessed IEL in the large intestine, despite the marked differences in function and lumenal environment. In the present study, we isolated IEL from both small and large intestine of three mouse strains (BALB/c, C3H/HeN, C57BL/6) and determined the frequency of CD2, CD4, and CD8 expression on CD3(+) IEL, as well as the frequency of alpha beta and gamma delta TCR usage and V beta distribution. Higher numbers of IEL/unit length were always isolated from the small intestine (20-30 x 10(6)/5 mice) compared with large intestine (1.1-2.5 x 10(6)/5 mice). Interestingly, IEL from the large intestine of all strains were predominantly alpha beta TCR(+) whereas gamma delta TCR(+) IELs predominated in small intestine. Large intestinal IELs were mainly CD4(+), in both BALB/c and C3H/HeN mouse strains. IELs from large intestine of C57BL/6 mice were mainly CD8(+); however, the CD4(+) subset was fourfold higher when compared with small intestine IEL. Potential functional differences between IEL subsets was assessed by determining the relative levels of mRNA for IL-1, 2, 4, 5, 10, IFN-gamma, TGF-beta, and TNF-gamma. Similar patterns for IL-1, IFN-gamma and TNF-alpha were seen while more IL-2, IL-4, IL-5, and IL-10 mRNA was noted in large intestinal IEL. Stimulation of C3H/HeJ IEL with anti-CD3 also resulted in higher levels of IL-3/GM-CSF, IL-4, and IL-6 by IEL from large intestine. These results show that marked differences occur among the T cell subsets present in IELs from mouse small and large intestine.

The major purpose of this study was to elucidate Th1 [interferon (IFN)-gamma and interleukin (IL)-2] and Th2 (IL-4, IL-5 and IL-6) cytokine-producing CD3+ T cells in salivary glands,which are the major mucosal effector tissues in the oral region. Thus, CD3+ T cells were isolated from salivary gland-associated tissues (SGAT) which consist of the submandibular gland (SMG: similar to 46%), the periglandular lymph node (PGLN: similar to 72 %), and the cervical lymph node (CLN: similar to 90 %). When SMG CD3(+) T cells were examined by Th1 and Th2 cytokine-specific ELISPOT and reverse transcriptase-polymerase chain reaction assay, high levels of both cytokine-specific spot-forming cells (SFC) and mRNA for IFN-gamma, and for IL-5 and IL-6 were noted as representative Th1 or Th2 cytokines, respectively. Following stimulation with concanavalin A (Con A), SMG CD3(+) T cells expressed mRNA and produced lymphokines for an array of Th1 (IFN-gamma and IL-2) and Th2 (IL-4, IL-5 and IL-6) cytokines. In comparison to the SMG CD3(+) T cells, PGLN and CLN contain lower numbers of IFN-gamma-, IL-5- and IL-6-producing T cells. When these two tissues were compared, PGLN CD3(+) T cells contained higher numbers of cytokine-secreting cells than CLN. Further, IL-2 and IL-4 SFC and mRNA were also noted in addition to IFN-gamma, IL-5 and IL-6 after Con A activation. These findings showed that CD3(+) T cells in SGAT, especially the SMG, are programmed to produce IFN-gamma, and IL-5 and IL-6 as Th1 and Th2 cytokines, respectively in vivo, although these cells are capable of producing other Th1 and Th2 cytokines after receiving appropriate T cell activation signals.

We have used the potent mucosal immunogen cholera toxin (CT) to assess antigen-specific CD4+ T-cell responses, including Th1- and Th2-type cells in mucosa-associated tissues, e.g. Peyer's patches (PP), and systemic tissue, e.g. spleen (SP), for their regulatory role in the induction of CT-specific B-cell antibody responses in the gastrointestinal (GI) tract as well as in systemic sites. The CT was given by either oral or intravenous (i.v.) routes and the mice orally immunized M,with CT exhibited brisk IgA anti-CT antibody responses in faecal extracts and elevated IgG anti-CT antibody responses in serum. Further, significant IgA anti-CT spot-forming cells (SFCs) were seen in lamina propria lymphocytes (LPLs) from mice orally immunized with CT. In contrast, i.v. immunization with CT induced IgM and IgG anti-CT SFC responses in SP, and serum anti-CT antibodies of these two isotypes, no anti-CT responses were induced in the GI tract after immunization by this route. The CD4+ T cells isolatedfrorn PP and SP of mice orally immunized with CT were stimulated in vitro with CT-B-coated latex microspheres for 1-6 days, and the induction of IL-2 and interferon gamma (IFN-gamma) (Th1-type) or IL-4 and IL-5 (Th2-type) producing SFCs were analysed by a cytokine-specific ELISPOT and cytokine-specific mRNA was detected by reverse transcriptase (RT)-PCR assays. Cultures of PP and SP CD4+ cells from orally immunized mice contained elevated numbers of IL-4- and IL-5-SFCs, significant levels of IL-4 and IL-5 mRNA, only small numbers of FN-gamma- and IL-2-SFCs and low to undetectable levels of IFN-gamma and IL-2 mRNA. On the other hand, SP CD4+ Th-cell cultures from mice immunized i.v. with CT included both IL-4- and IL-5- as well as IFN-gamma- and IL-2-SFCs; no CT-specific Th cells were detectable in PP. These results show that oral immunization with CT preferentially induces CD4+ T cells which produce IL-4 and IL-5 in both PP and SP. Furthermore, the presence of elevated numbersof these Th2-type cells directly correlated with significant IgA anti-CT-B antibody responses in LPLs isolated from the GI tract.

To study the biological consequences of cytokine production and apoptosis by intraepithelial lymphocytes (IEL), we have studied these characteristics in both the high and low density CD3(+) IEL populations. Stimulation of low- or high-density CD3(+) IEL via the T cell receptor (TCR)-CD3 complex using monoclonal anti-CD3, anti-alpha beta TCR or anti-gamma delta TCR antibodies resulted in opposing effects. In one case, a significant number of the high-density CD3(+) T cells entered cell cycle from the resting stage (DNA replication was observed) and anti-TCR-CD3 treatment enhanced the numbers of interferon-gamma and interleukin-5 spot-forming cells in this cell fraction. In contrast, when the low-density alpha beta TCR(+) or gamma delta TCR(+) T cells were activated via the TCR-CD3 complex, DNA fragmentation was observed. These results demonstrated that the activation signals transduced via the TCR-CD3 complex resulted in their entry into the cell cycle and subsequent interferon-gamma and interleukin-5 production in the high-density IEL T cell subset. However, identical signals induced apoptosis in the majority of the low-density fraction of CD3(+) IEL.

The realization that induction of immune responses at mucosal surfaces may prevent colonization, invasion or dissemination of pathogenic microorganisms has spurred intensive efforts to develop vaccines which elicit effective mucosal immunity. In this paper, recent results are discussed for mice given cholera toxin as both an immunogen and as an adjuvant for inducing both humoral and gastrointestinal mucosal immune responses. Oral administration of cholera toxin alone or with a co-administered protein vaccine tetanus toroid induces a strong T helper type 2 (TH2) cell response in both Peyer's patches and spleen. Both serum IgG and secretory IgA antibodies specific for cholera toxin or for the co-administered protein tetanus toroid were induced. When administered parentally, however, no mucosal antibody responses were evident and a mixed TH1- and TH2-type CD4(+) T cell response was noted in the spleen. Various vectors are being employed in an effort not only to induce mucosal immune responses but also to direct the response to a TH1-type response, thought to promote strong cell-mediated immune responses, or to a TH2-type response for maximum B cell antibody responses. The ability to manipulate the TH cell responses may provide a more rational approach for the design of vaccines. Although lymphoid tissues of the female reproductive tract differ from that of the gut, many of the strategies and evolving principles may be directly applicable to the development of vaccines designed to prevent sexually transmitted diseases.

To study virus-specific cytotoxic T lymphocyte (CTL) activity at the single cell level, an IFN-gamma specific ELISPOT assay was adapted to elucidate the frequency of influenza-specific CTLs together with a standard cytotoxic Cr-51-release assay. Peripheral blood mononuclear cells (PBMCs) from human volunteers were cultured with influenza virus-infected autologous cells; following 3 or 7 days of culture, T cell subsets were assessed for IFN-gamma production by IFN-gamma-specific ELISPOT and ELISA, while IFN-gamma mRNA expression was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). Influenza virus-specific CTL activity was measured in a 4 h Cr-51-release assay. Culture of PBMC with autologous A/Taiwan influenza (H1N1)-infected stimulator cells resulted in IFN-gamma spot forming cells (SFCs) at 3 days that increased after 7 days of incubation. Numbers of IFN-gamma SFCs directly correlated with levels of secreted IFN-gamma and higher levels were seen in supernatants from 7 day cultures. RT-PCR analysis (35 cycles of amplification) showed greater IFN-gamma mRNA in T cells isolated from 7 day cultures. Separate aliquots of T cells from these cultures were also assessed for virus-specific cytotoxicity and T cells from 7 day (but not from 3 day) cultures induced high Cr-51 release. Analysis indicated a significant direct correlation between level of cytotoxicity, number of IFN-gamma SFCs, and amount of IFN-gamma in culture supernatants. Studies with purified T cell subsets showed that elevated IFN-gamma SFCs, IFN-gamma synthesis, and cytotoxic activity were associated with CD4-CD8(+) T cells but not with the CD4(+)CD8(-) T cell subset. These results show that increased IFN-gamma production, including increased IFN-gamma mRNA and IFN-gamma SFCs directly correlate with increased antigen-specific, CD8(+) T cell mediated cytotoxicity. Thus, assessment of IFN-gamma SFCs may provide an alternative and quantitative means for assessment of influenza virus-specific CTL in humans.

Our previous studies have shown that murine alphabeta TCR+ intraepithelial lymphocytes (IEL) contained T cells that can provide B cell help. In this study, we have examined the three subsets of alphabeta TCR+ IEL for Ag-specific helper function and cytokine production because alphabeta TCR+ IEL are divisible into three subsets including CD4+, CD8- T cells, CD4+, CD8+ double positive (DP) T cells, and CD4-, CD8+ T cells. When these three subsets of alphabeta TCR+ IEL from C3H/HeN mice (H-2k) orally immunized with SRBC were cultured with splenic B cells, adherent cells, and SRBC, both CD4+, CD8- and DP T cell fractions supported IgM, IgG1, and IgA anti-SRBC responses, whereas the CD4-, CD8+ T cell subset did not exhibit helper function. Addition of anti-I-A(k) mAb resulted in the reduction of SRBC-specific PFC responses, whereas anti-H-2K(k) mAb did not affect the CD4+, CD8- and DP T cell-supported B cell responses. Furthermore, when these CD4-bearing T cells from mice orally immunized with SRBC were co-cultured with B cells and adherent cells in the presence of unrelated Ag (e.g., horse RBC), SRBC-specific B cell responses were not induced. When type 1 and type 2 Th cell cytokine production was examined by IFN-gamma-, IL-2-, IL-4-, and IL-5-specific enzyme-linked immunospot assays, increased numbers of IL-4- and IL-5-secreting type 2 Th cells, whereas lower numbers of IFN-gamma and IL-2-producing type 1 Th cells were seen in CD4+, CD8- and DP T cell fractions on in vitro stimulation with SRBC. In the case of CD4-, CD8+ T cells, approximately equal and low numbers of type 2 cytokine-producing cells were noted in both SRBC-stimulated and -unstimulated cultures. Furthermore, when different fractions of IEL T cells from Ag-stimulated and -unstimulated cultures were characterized for cytokine-specific mRNA by reverse transcription polymerase chain reaction, stronger bands for IL-4 and IL-5 were detected in both CD4+, CD8- and DP IEL subsets when compared with the unstimulated cells. in contrast, the change in intensity of the band of polymerase chain reaction product for IFN-gamma was slight or unchanged in Ag-stimulated CD4-bearing IEL when compared with unstimulated cells. These results suggest that subsets of alphabeta TCR+ IEL, and especially the CD4-bearing T cells, are capable of providing helper function for Ag-specific B cell responses. Of significant interest was the finding that DPT cells isolated from the epithelium of the gastrointestinal tract exhibited cytokine synthesis and helper function in addition to CD4+, CD8- IEL expressing a classical helper phenotype.

The immunobiological function of lymphocytes within the epithelium (IELs) of the small intestine is incompletely understood; however, it has been shown that intestinal IEL T cells exhibit cytotoxicity, produce cytokines, and perform some regulatory roles. In this study, we have focused on CD4+, alphabeta TCR+ IELs, which comprise approximately 15 - 20% of the total population, for helper functions. We have assessed the profile of type 1 or type 2 Th cell cytokines produced in alphabeta TCR bearing CD4+CD8- and CD4+CD8+ (double positive, DP) intestinal IELs by cytokine-specific ELISPOT and by reverse transcription-polymerase chain reaction. Help for B cells taken from Peyer's patches (PP) allowed us to assess IEL function for mucosal antibody responses. Freshly isolated CD4+CD8- IEL T cells contain T(h)2-type cells, e.g. high numbers of IL-5 secreting (spot forming) cells (SFC) followed by IL-4 and IL-6, while DP T cells have IL-5 and IL-6 producing cells, but not IL-4. In addition to T(h)2-like cytokine producing T cells, both CD4+ T cell subsets contain IFN-gamma secreting T(h)1-type cells but neither subset synthesizes IL-2. Stimulation of CD4+CD8- and DP T cells with solid phase mAb anti-CD3 resulted in production of IL-2 in addition to IFN-gamma, IL-5, and IL-6, and this treatment stimulated DP T cells to produce IL-4. When freshly isolated intestinal IEL T cells were separated into CD4+ and CD4- cells, and co-cultured with PP B cells, the former subset supported Ig synthesis including IgA responses, while the later fraction did not. Further, purified alphabeta TCR+, CD4+CD8- T cells and DP T cells from IELs when added to PP B cell cultures induced increased numbers of Ig secreting cells. However, CD4-CD8+ T cells which produced a similar profile of cytokines did not provide helper function for B cells. Our study has shown that alphabeta TCR+ T cells from intestinal IELs exhibit T(h)1- and T(h)2-type cell functions. Of significant interest was the finding that DP T cells exhibit cytokine synthesis and helper function in the epithelium of the gastrointestinal tract in addition to CD3+ IELs expressing a classical helper phenotype (CD4+CD8-).

Antigen-specific B cell responses to mucosally delivered proteins are dependent upon CD4-positive T helper (Th) cells, and the frequency of Th1 and Th2 cell responses after oral immunization may determine the level and isotype of mucosal antibody responses. We have used a protein-based vaccine, tetanus toxoid (TT), together with the mucosal adjuvant cholera toxin (CT), for oral immunization of mice to study the nature of antigen-specific Th cell subsets induced in Peyer's patches (PP) of the gastrointestinal (GI) tract and in the spleen (SP) during peak antibody responses. Mice orally immunized with TT and CT responded with antigen-specific secretory immunoglobulin A (S-IgA) antibodies in the GI tract, and with both IgG and IgA antibody responses in serum. PP and SP CD4+ T cells from mice orally immunized with TT plus CT were cultured with antigen-coated latex microspheres for induction of proliferative responses and for enumeration of cytokine producing CD4+ T cells. Interestingly, both PP and SP CD4+ T cell cultures showed increased numbers of IL-4- and IL-5 (Th2-type)-producing, spot-forming cells (SFCs) after 21 d of immunization, while essentially no interferon-gamma (IFN-gamma) or IL-2 (Th1-type) SFCs were noted. Cytokine-specific Northern blots and RT-PCR also revealed that significant IL-4 and IL-5 mRNA levels, but not IFN-gamma or IL-2 mRNA, were present in CD4+ T cells isolated from antigen-stimulated cultures. However, systemic immunization with TT and CT induced antigen-specific IgG and IgM but not IgA antibodies in serum. Further, both IL-2- and IFN-gamma-producing Th1-type cells as well as IL-4- and IL-5-secreting Th2-type cells were generated in SP. Our results show that oral immunization with TT and the mucosal adjuvant CT selectively induced antigen-specific Th2-type responses which may represent the major helper cell phenotype involved in mucosal IgA responses in the GI tract.

Cholera toxin (CT) is an effective mucosal antigen and acts as an adjuvant when given orally with various antigens; however, few studies have compared the levels of antibody responses to CT and coadministered protein in systemic and mucosal tissues. In this study, we used tetanus toxoid (TT) for assessment of immune responses. Time course and dose-response studies established that 250 mug of TT given orally with 10 mug of CT three times at weekly intervals induced high serum and gastrointestinal tract anti-TT and anti-CT antibody responses. Oral immunization with TT alone induced no detectable mucosal immunoglobulin A (IgA) antibodies in fecal extracts and only weak serum IgG anti-TT responses. The coadministration of CT and TT induced peak serum IgG anti-TT responses following two oral doses that remained constant after the third oral immunization, while optimal mucosal IgA responses were seen after the third oral immunization. The serum anti-TT response obtained with CT and TT proved protective against TT challenge (100 minimum lethal doses), whereas mice orally given CT or TT alone died. Antigen-specific B-cell responses were assessed with an isotype-specific Elispot assay of isolated lymphoid cells from the spleen, Peyer's patches, and the small intestinal lamina propria. Interestingly, approximately fourfold-higher numbers of IgA anti-CT than of anti-TT antibody-producing (spot-forming) cells occurred in lymphocytes from the lamina propria of mice orally immunized with both TT and CT. The adjuvant CT did not induce polyclonal B-cell responses in mice given CT by the oral route, since no significant differences in total numbers of B cells producing IgA, IgG, or IgM were found compared with the numbers in mice given TT alone. The results clearly indicate that serum and mucosal antibody responses develop with different kinetics and that protective TT-specific antibody responses are generated in the systemic compartment when TT is administered with CT via the oral route.

Murine intraepithelial lymphocytes (IEL) respond poorly to T cell mitogens and to monoclonal antibody stimulation of T cell receptor (TCR)- and CD3- associated molecules. In contrast, we found that a soluble extract of Mycobacterium tuberculosis (Mtb), but not purified protein derivative of tuberculin, induced significant proliferative responses in IEL cultures.The active component was apparently a heat shock protein (HSP), since recombinant 7 1-kDa HSP f rom Mtb induced IEL to proliferate, while 65-kDa HSP from M. bovis and M. leprae did not. Both alpha/beta and gamma/delta TCR-enriched IEL gave proliferative responses to 71-kDa HSP. Further, culture supernatants from IEL stimulated with 71-kDa HSP contained elevated levels of interleukin-(IL)-3/granulocyte-macrophage colony-stimulating factor, interferon-gamma and IL-6, but not IL-2, IL-4, IL-5 or transforming growth factor-beta. Finally, several IEL T cell clones have been maintained for up to 6 weeks, when stimulated with 71-kDa HSP, IL-2 and feeder cells. Our results show that the 71-kDa HSP of Mtb induces IELT cells to divide and to secrete cytokines and this may offer a model for cloning and study of IEL T cells in vitro.

LOCALIZED AND CHRONICALLY-INFLAMED GINGIVAL TISSUES of adult periodontitis (AP) are generally characterized as a hyper-responsiveness of B lineage cells where increased numbers of plasma cells occur. It was previously shown that high numbers of IgG subclass antibody-secreting cells (e.g., IgG1 > IgG2 > IgG3 greater-than-or-equal-to IgG4) with significant numbers of IgA subclass antibody-producing cells were seen in enzymatically dissociated gingival mononuclear cells (GMC) from inflamed periodontal tissues. An interesting finding was that the frequency of IgA2 plasma cells was elevated in the severe stage of AP when compared with the moderate stage. IgM plasma cells were essentially not found in these tissues. To understand the cytokine involvement in these increased B cell responses in inflamed gingiva, GMC isolated from inflamed tissues of AP patients were examined for cytokine production, specifically for IL-2, IL-4, IL-5, and IL-6 at both the protein and mRNA levels, since these cytokines have been shown to be essential interleukins for the regulation of the B cell response. Freshly-isolated GMC and peripheral blood mononuclear cells (PBMC) from AP patients were initially examined for IL-6 production because of its essential role for the terminal differentiation of B cells to become Ig-producing plasma cells. High levels of IL-6 were produced by GMC but not by PBMC unless cells were stimulated with T cell mitogen. A similar finding was also obtained when levels of IL-6 specific mRNA were examined in GMC and PBMC. Further, freshly-isolated GMC expressed IL-6 receptors, but PMBC from the same patient did not harbor this receptor on their cell surfaces. When production of the other cytokines including IL-2, IL-4, and IL-5 was examined, it was interesting to note that a lack of IL-2 and IL-4 expression was seen, while IL-5 was produced by GMC in significant amounts. In summary, these findings showed that a unique profile of cytokine synthesis was seen in GMC isolated from the inflamed periodontium. Further, high production of cytokines, namely IL-5 and IL-6, which have the ability to regulate later stages of B cell development, to induce them to become Ig-secreting plasma cells, and to support plasmacytoma growth, are important immunopathological elements for the induction of the elevated B cell response in the inflamed gingiva of patients with AP.

A unique characteristic of the localized inflammatory tissue in the periodontium (eg., adult periodontitis [AP]) is the accumulation of IgG (IgG1 > IgG2 > IgG3 greater-than-or-equal-to IgG4) followed by IgA plasma cells (IgA1 > IgA2). However, the exact molecular mechanisms contributing to these elevated B-cell responses at the local disease site are still unknown. Thus, this study has examined the production of cytokines of importance in B-cell responses, eg., interleukin (IL)-2, IL-4, IL-5, and IL-6 by gingival mononuclear cells (GMC) isolated from patients in severe stages of AP. These cytokines were assessed at the protein and messenger (m)RNA levels to understand their importance for the observed increased B-cell responses present in these tissues. Among the four cytokines tested by respective cytokine-specific, polymerase chain reaction and dot-blot hybridization, high levels of IL-5- and IL-6-specific mRNA were noted in GMC freshly isolated from AP Patients. On the other band, specific message for IL-2 and IL-4 were not present. Further, the analysis of culture supernatants of GMC also revealed that cells from AP patients spontaneously produced IL-5 and IL-6 but not IL-2 and IL-4. In contrast, when peripheral blood mononuclear cells isolated from the same patients were examined for these cytokines, no detectable levels of mRNA or secreted cytokines were noted. These results showed that GMC from localized inflammatory tissues in severe stages of AP possess a distinct cytokine profile represented by high levels of IL-5 and IL-6 mRNA expression and protein synthesis, whereas IL-2 and IL-4 were not detected Further, this study supports the concept that AP is a localized inflammatory disease, because GMC from the inflamed tissue actively produce IL-5 and IL-6, whereas peripheral blood mononuclear cells from the same patients do not.

In order to assess cytokine-producing cells at the single cell level, the cytokine-specific ELISPOT assay has proven to be an important and sensitive method. The purpose of this study was to adapt this method to elucidate individual cells producing murine IL-2, IL-4 or IL-6. In order to establish these cytokine-specific ELISPOT assays, IL-2-, IL-4- and IL-6-specific cDNA transfected myeloma cell lines, e.g., X63-Ag8-653 X2, X63-Ag8-653 X4 and X63-Ag8-653 X6, respectively, were used as specific cytokine-producing cells. In the IL-2 ELISPOT assay, the coating reagent, monoclonal antibody (mAb) rat IgG2a anti-mouse IL-2 (CR #40014) was used while rabbit IgG polyclonal anti-mouse IL-2 was employed for detection of IL-2 spot forming cells (SFC). The mAbs anti-mouse IL-4, BVD4-1D11 and BVD6-24G2 were selected as capture and detection antibodies for enumeration of IL-4 SFC. For the IL-6 ELISPOT assay, anti-mouse IL-6 (MP5-20F3) mAb was used for coating and MP5-32C11 mAb was used for detection of IL-6 SFC. When IL-2 producing X63-Ag8-653 X2 cells were subjected to these three different ELISPOT assays, IL-2-specific SFC were only noted with the IL-2 ELISPOT system. In the case of IL-4 SFC, only X63-Ag8-653 X4 cells formed specific spots using the tandem of BVD4-1D11 and BVD6-24G2 mAbs. IL-6-specific spots developed in MP5-20F3 mAb pre-coated wells containing X63-Ag8-653 X6 cells, when developed with mAb anti-IL-6 (MP5-32C11). Addition of cycloheximide (50 Ag/ml) inhibited formation of IL-2, IL-4 and IL-6 SFC by approximately 90%. When an unrelated mAb was used as detection antibody in these three different cytokine-specific ELISPOT assays, IL-2-, IL-4- and IL-6-specific SFC were not detected. Further, when concanavalin A stimulated T cells from Peyer's patch of normal mice were subjected to the respective cytokine-specific ELISPOT assay, IL-2, IL-4 and IL-6 SFC were enumerated. These results have shown that cytokine-specific IL-2, IL-4 and IL-6 ELISPOT assays have now been established and will allow analysis of the frequency of cytokine-secreting cells at the single cell level.

Murine intestinal intraepithelial lymphocytes (IEL) have been shown to contain subsets of alpha/beta TCR+ and gamma/delta TCR+ T cells that spontaneously produce cytokines such as IFN-gamma and IL-5. We have now determined the nature and cell cycle stage of these cytokine-producing T lymphocytes in IEL by using IFN-gamma and IL-5-specific ELISPOT assay, cytokine-specific mRNA-cDNA dot-blot hybridization and polymerase chain reaction, and flow cytometry (FACS) for DNA analysis. When CD3+ T cells from IEL of normal C3H/HeN mice were separated into low and high density fractions by discontinuous Percoll gradients, IFN-gamma and IL-5 spot-forming cells were only found in the former population. Analysis of MRNA for these cytokines by both IFN-gamma and IL-5-specific dot-blot hybridization and polymerase chain reaction revealed that higher levels of message for IFN-gamma and IL-5 were also seen in the low density fraction. However, cell cycle analysis of these two fractions by FACS using propidium iodide showed a similar pattern of cell cycle stages in both low and high density populations (G0 + G1 approximately 96 to 98% and S/G2 + M approximately 2 to 4%). Finally, mRNA from gamma/delta TCR+ and alpha/beta TCR+ T cells in both low and high density fractions of IEL were analyzed for IFN-gamma and IL-5 message by polymerase chain reaction. After 35 cycles of amplification, both gamma/delta TCR+ and alpha/beta TCR+ T cells in the low density fraction expressed higher levels of message for these two cytokines when compared with the high density population. These results have now shown that both gamma/delta and alpha/beta TCR+ IEL can be separated into low and high density subsets and both fractions possess a similar stage of cell cycle. However, only the low density cells (in G1 phase) of both gamma/delta and alpha/beta TCR types possess increased cytokine-specific mRNA and produce the cytokines IFN-gamma and IL-5. Our results suggest that alpha/beta TCR+ and gamma/delta TCR+ IEL can produce cytokines without cell proliferation.

MRL mice, which develop a lymphoproliferative disease characterized by increased numbers of alpha/beta T cell receptor+ (TCR+) B220/6B2+CD4-CD8- T cells [lymphoproliferation (Ipr) T cells], were studied for the effect of the Ipr/Ipr mutation on the mucosal immune system in the gastrointestinal (GI) tract. We analyzed the effect of the Ipr gene mutation on T and B cell populations in the Peyer's patches (PP) and the lamina propria lymphocytes (LPLs), as examples of major IgA inductive and effector tissues in the GI tract respectively. Normal mouse PP contain B cells committed to IgA (surface IgA+) but only low numbers of B cells producing IgA. However, enhanced spontaneous IgA and IgG synthesis occurs in the PP of MRL mice. Further, we have now shown that PP of MRL mice are populated by Ipr T cells. Interestingly, Ipr T cells were not present in significant numbers in LPLs of MRL mice, even in older animals. Of interest was the finding that the ratio of CD4+ to CD8+ T cells in the lamina propria was lower in MRL when compared with control mice, and the CD8+ T cell subset actually predominates in LPLs of autoimmune mice. In addition, the number of gamma/delta TCR+ T cells in LPL of MRL Ipr/Ipr mice was significantly increased, especially in MRL Ipr/Ipr mice at 6 and 12 weeks of age. When the isotype distribution of B cells in LPLs was analyzed, no changes were noted in MRL Ipr/Ipr mice in comparison with MRL +/+ or normal control mice, and the pattern was IgA > > IgM >IgG. These results show that although increased numbers of CD8+ T cells and gamma/delta TCR+ cells occur in the LPLs of MRL mice, a normal distribution of plasma cell isotypes (IgA > > IgM > IgG) is found in this mucosal compartment. Further, Ipr T cells do not develop in the lamina propria compartment of the GI tract.

Past work has shown that a subset of effector T cells with unique characteristics could abrogate hapten- or antigen-induced tolerance, and the reconstitution of this immune response has been termed contrasuppression. We have studied contrasuppression in a model of oral tolerance (OT) in which adoptively transferred antigen-specific T contrasuppressor (Tcs) cells reverse OT and result in antibody responses to the eliciting antigen. In the present study, we show that murine intraepithelial lymphocytes (IELs) from mice orally immunized with sheep red blood cells (SRBC) contain T cells that exhibit Tcs cell activity. This effect was mediated by CD3+ gamma/delta T cell receptor-positive (TCR+), but not alpha/beta-TCR+ T cells, and gamma/delta TCR+ Tcs cells were associated with both the CD4-,CD8+ and CD4-,CD8- (double-negative) IEL fractions. The CD4-,CD8+ gamma/delta-TCR+ IELs were further separated into Vicia villosa-adherent and -nonadherent fractions. Adoptive transfer of V villosa-adherent gamma/delta-TCR+ T cells to mice with OT to SRBC resulted in splenic IgA, IgM, and IgG subclass anti-SR-BC responses, while V. villosa-nonadherent gamma/delta-TCR+ T cells were without activity. The gamma/delta-TCR+ IELs did not support in vitro antibody responses in B cell cultures, while alpha/beta-TCR+ IELs were effective T helper cells. Further, cytokine production by the gamma/delta-TCR+ IELs was examined, and the gamma/delta-TCR+ V villosa-adherent fraction, which possessed contrasuppressor function, contained low levels of IL-5 mRNA and small numbers of IL-5-producing cells when compared with alpha/beta-TCR+ IELs and V villosa-nonadherent gamma/delta-TCR+ IELs. Our results now show that mouse IELs contain two distinct types of T cells that function in the immune response, e.g., alpha/beta-TCR+ T cells that produce IL-5 and function as helper cells, and gamma/delta-TCR+ T cells that restore antibody responses in mice that had been orally tolerized with antigen.

The mucosal immune system of the gastrointestinal (GI) tract consists of Peyer's patches (PP), which are IgA inductive sites, and more diffuse effector regions which include cells in the intraepithelial lymphocyte (IEL) compartment. Since autoimmune MRL lpr/lpr (MRL/lpr) mice develop a proliferating CD3+, CD4-, CD8- (double negative; DN), B220+ T cell subset in systemic lymphoid tissue, we have initiated studies to determine the distribution of CD3+, DN, B220+ T cells (B220+ T cells or lpr/lpr T cells) in the GI immune system. Specifically, we examined T cell subsets separated according to expression of CD4, CD8, Thy-1, B220, alpha/beta T cell receptor (TcR) and gamma/delta TcR in PP and IEL of MRL/lpr mice at 6, 12 and 21 weeks of age. Increased numbers of CD3+ T cells were noted in both PP and spleen of 12- and 21-week-old mice in which the development of autoimmune disorders were also evident. However, normal numbers of CD3+ IEL T cells were seen in MRL/lpr mice in all three age groups tested. When the presence of T cell lymphadenopathy was examined in both IgA inductive and effector tissues, the PP followed the B220+ T cell pattern seen in the spleen, where approximately 30%-50% of CD3+ T cells in the PP of 12- and 21-week-old MRL/lpr mice expressed the phenotype of lpr/lpr T cells and > 90% were alpha/beta TcR+. On the other hand, B220+ T cells had not developed in PP or spleen of 6-week-old MRL/lpr mice. Of interest was the finding that IEL from lpr/lpr homozygous mice did not contain B220+ T cells in any age group tested. In this regard, the IEL of MRL/lpr mice comprised an identical pattern and frequency of CD4-/CD8+, CD4+/CD8-, DN and CD4+/CD8+ (double positive, DP) T cell subsets as their normal counterparts (i.e. MRL +/+, BALB/c and C3H/HeN mice) which consisted of approximately 75%, approximately 7.5%, approximately 7.5% and approximately 10%, respectively. Further, Thy-1, gamma/delta TcR and alpha/beta TcR expression in these four subsets of MRL/lpr IEL were very similar to normal mice. These results suggest that the intestinal IEL compartment is minimally affected by the lpr/lpr mutation which induces T cell abnormalities and indicate that B220+ T cells do not preferentially home to IEL. Further, our results support the concept that IEL T cells develop as a separate T cell lineage from thymus-derived cells.

Intestinal intraepithelial lymphocytes (IEL) from mice are > 80% CD3+ T cells and could be separated into four subsets according to expression of CD4 and CD8. In our studies designed to assess the functions of IEL, namely, cytokine production, it was important to initially characterize the various subsets of T cells that reside in IEL. The major subset was CD4-, CD8+ (75% of CD3+ T cells), which contained approximately 45 to 65% gamma/delta TCR+ and 35 to 45% alpha/beta TCR+ T cells. Approximately 7.5% of IEL T cells were CD4-, CD8- (double negative) and gamma/delta+ population. On the other hand, CD4+, CD8+ (double positive) and CD4+, CD8- fractions represented 10% and 7.5% of CD3+ T cells, respectively, which were all alpha/beta TCR+. Inasmuch as CD3+, CD4-, CD8+ T cells are a major subset of IEL which contain both gamma/delta TCR or alpha/beta TCR-bearing cells, the present study was focused on the capability of this subset of IEL T cells to produce the cytokines IFN-gamma and IL-5. Both gamma/delta TCR+ and alpha/beta TCR+ IEL spontaneously produced IFN-gamma and IL-5, although higher frequencies of cytokine spot-forming cells were associated with the alpha/beta TCR+ subset. Approximately 30% of CD8+, gamma/delta TCR+ cells produced both cytokines, whereas approximately 90% of alpha/beta TCR+ T cells produced either IFN-gamma or IL-5. Both gamma/delta TCR+ and alpha/beta TCR+ IEL possessed large quantities of cytokine-specific mRNA, clearly showing that these IEL were programmed for cytokine production. When IEL were activated with anti-gamma/delta or anti-CD8 antibodies, higher numbers of IFN-gamma and IL-5 spot-forming cells were noted. The present study has provided direct evidence that a major function of IEL involves cytokine production, and this is the first evidence that gamma/delta TCR+ cells in IEL possess the capability of producing both IL-5 and IFN-gamma.

Past studies have shown that freshly isolated human B cells from peripheral blood and tonsils do not express IL-6 receptors (IL-6R); however, mitogen or antigen activation of these B cells induces IL-6R and responsiveness to IL-6. In this study, we have shown that a high proportion of B cells enzymatically dissociated from human appendix, a gut-associated lymphoreticular tissue (GALT), expresses the IL-6R, and that recombinant human IL-6 induces significant increases in the number of Ig-producing cells. The recombinant human IL-6-induced increase in Ig-producing cells is restricted to the IgA isotype. Further, IgA2 is the major subclass; however, significant numbers of IgA1 producing cells are also seen. In contrast, human tonsillar and peripheral blood B cells express low levels of IL-6R, and exogenous IL-6 does not increase numbers of Ig-producing cells. When PBMC or tonsillar cells are stimulated with PWM, the former display an equal distribution of IgA1 and IgA2 secreting cells, while tonsillar B cells are mainly of the IgA1 subclass. The distribution of surface Ig-positive (sIg+) B cells in the appendix B cell population is sIgA+ > sIgG+ > sIgM+, and the sIgA+ B cells express higher levels of IL-6R when compared with sIgG+ or sIgM+ B cells. These studies show that human appendix contains B cell subsets that constitutively express IL-6R, and that a high proportion of these cells are committed to the IgA isotype. Furthermore, higher numbers of IL-6 responsive IgA2 B cells are present in the human appendix as compared to tonsils or PBMC.

It is well established that increased numbers of plasma cells occur in the localized tissues of chronic inflammatory diseases such as adult periodontitis, and enzymatic isolation has shown that most B lineage cells produce IgG-subclass with some IgA-subclass responses. It would be of importance to determine if excess production of cytokines in the localized lesion account for these responses and in the present study we have assessed gingival mononuclear cell (GMC) supernatants for cytokines that activate B cells including IL-6R expression and for levels of IL-6 present. Inasmuch as limited numbers (approximately 1 to 3 x 10(6) cells) of GMC were obtained from surgically removed tissues (approximately 400 mg), we have focused on the analysis of IL-6 production by GMC in this study. Further, initial evidence of additional cytokines that are produced by GMC and induce expression of IL-6R on resting B cells has been obtained. The GMC and PBMC from individual patients were cultured in the presence (or absence) of Con A. Higher levels of IL-6 were produced spontaneously by GMC when compared with Con A-stimulated PBMC. When PBMC cultures were supplemented with GMC supernatants obtained from the same patient, high numbers of spot-forming cells (SFC), mainly of IgG followed by IgA isotype, were seen. The induction of SFC by GMC supernatants was inhibited by incubation with a goat anti-human IL-6 antibody. When the effect of GMC supernatants on subclasses of PBMC SFC was determined, the response was IgG1 > IgG2 > IgG3 = IgG4 and IgA1 > IgA2, a pattern remarkably similar to the distribution of plasma cells in the GMC itself. To assess for cytokines in GMC supernatants that mediated B cell activation, supernatants containing anti-IL-6 were cultured with PBMC or purified B cells for 72 h. This treatment induced small proliferative B cell responses and elevated expression of IL-6R on B cells, but did not induce SFC responses. Further, incubation of B cells with GMC supernatants induced resting B cells (G0/G1) to enter the cell cycle (S and G2/M). Addition of human rIL-6 to these cultures on day 3 restored IgG- and IgA-subclass SFC responses by day 7. Cytokine-induced IL-6R expression also occurred in vivo because freshly isolated GMC expressed high levels of this receptor. These results show that GMC produce cytokines that induce B cell activation including IL-6R expression and secrete IL-6 that regulates B cell terminal differentiation into plasma cells of IgG- and IgA-subclasses. We are currently assessing the cytokines produced by GMC that induce the expression of IL-6R on B cells.