Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Ragin, M. J. Articles by August, A. Search for Related Content PubMed PubMed Citation Articles by Ragin, M. J. Articles by August, A.

 Previous Article  |  Next Article 

Infection and Immunity, January 2006, p. 282-288, Vol. 74, No. 1
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.1.282-288.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Differential Regulation of Cytokine Production by CD1d-Restricted NKT Cells in Response to Superantigen Staphylococcal Enterotoxin B Exposure

Melanie J. Ragin,^1,2 Nisebita Sahu,^1,3 and Avery August^1*

Center for Molecular Immunology & Infectious Disease and Department of Veterinary and Biomedical Sciences,¹ Pathobiology Graduate Program,² Department of Biochemistry & Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802³

Received 20 September 2005/ Returned for modification 8 October 2005/ Accepted 13 October 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
NKT cells are a heterogeneous population characterized by the ability to rapidly produce cytokines, such as interleukin 2 (IL-2), IL-4, and gamma interferon (IFN-) in response to infections by viruses, bacteria, and parasites. The bacterial superantigen staphylococcal enterotoxin B (SEB) interacts with T cells bearing the Vß3, -7, or -8 T-cell receptors, inducing their expansion and cytokine secretion, leading to death in some cases due to cytokine poisoning. The majority of NKT cells bear the Vß7 or -8 T-cell receptor, suggesting that they may play a role in regulating this response. Using mice lacking NKT cells (CD1d^–/– and J18^–/– mice), we set out to identify the role of these cells in T-cell expansion, cytokine secretion, and toxicity induced by exposure to SEB. We find that Vß8⁺ CD4⁺ T-cell populations similarly expand in wild-type (WT) and NKT cell-null mice and that NKT cells did not regulate the secretion of IL-2. By contrast, these cells positively regulated the secretion of IL-4 and IFN- production and negatively regulated the secretion of tumor necrosis factor alpha (TNF-). However, this negative regulation of TNF- secretion by NKT cells provides only a minor protective effect on SEB-mediated shock in WT mice compared to mice lacking NKT cells. These data suggest that NKT cells may regulate the nature of the cytokine response to exposure to the superantigen SEB and may act as regulatory T cells during exposure to this superantigen.

Top Abstract Introduction Materials and Methods Results Discussion References

 
CD1d-restricted invariant NKT (iNKT) cells, characterized by the presence of an invariant T-cell receptor (TCR) chain composed of V14 and J18 segments in mice, are a unique subset of cells that have characteristics of both NK cells and T cells and constitute only 1% of the lymphocyte population (23, 31). The majority of these cells express a TCR with a specific TCR chain rearrangement, V14/J18, which is associated with Vß chains, mainly Vß8.2 (and Vß7 and -2 to a lesser degree) (23). They also express markers specific for iNKT cells, CD161 (NK1.1) or NKR-P1C, a member of the C-type lectin family. NKT cells are also characterized by an activated and/or memory phenotype (CD69⁺, CD62L^low, and CD44^high). NKT cells recognize and seem to be most potently activated by the glycolipid -galactosylceramide (-GalCer), derived from marine sponge, which has been identified as a specific ligand for both mouse and human iNKT cells, instead of peptide antigens, presented by the ß₂ microglobulin-associated nonclassical major histocompatibility complex class I (MHC-I)-like molecule CD1d (28). More recently, a number of other ligands have been reported that can activate NKT cells, including endogenous isoglobotrihexosylceramide and glycosylphosphatidylinositol, and lipids derived from bacteria including Sphingomonas and Mycobacteria (12, 20, 30, 45, 52, 54). Activation of NKT cells by -GalCer results in a vigorous response marked by proliferation, expression of activation molecules, and secretion of both Th1 and Th2 cytokines, specifically interleukin 4 (IL-4) and gamma interferon (IFN-), suggesting an early regulatory function for NKT cells in immune responses (23).

The bacterial superantigen staphylococcal enterotoxin B (SEB) can cause lethal toxic shock in humans and in sensitized mice (32). This protein acts as a superantigen by cross-linking antigen-presenting cells and T cells via simultaneous interaction with MHC-II and the TCR, resulting in large-scale activation and cytokine production (37). SEB exposure is characterized by robust T-cell activation, specifically of those cells carrying the Vß8 region of the TCR (as well as Vß3 and -7), resulting in expansion of these cells, and massive release of cytokines, such as IL-2, IFN-, and tumor necrosis factor alpha (TNF-) (32). This T-cell activation and cytokine release caused by SEB exposure, particularly of TNF-, can contribute to the development of lethal toxic shock (32).

As a large majority of NKT cells carry the Vß7 or -8 TCR, both of which are reactive to SEB, we reasoned that they may play a role in modulating the cytokine and toxicity responses observed during exposure to this superantigen-toxin. Using mice lacking CD1d or J18, both of which have been reported to lack NKT cells (23), we tested the hypothesis that NKT cells play a role in SEB-mediated toxicity. We report here that while NKT cells differentially regulate cytokine secretion, particularly TNF-, in response to SEB, they play only a minor role if any in SEB-induced toxicity.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice. The following strains of mice were used: BALB/C (wild-type [WT]), CD1d^–/– (Jackson Laboratory, Maine) (44), and J18^–/– (a kind gift of Masaru Taniguchi, Chiba University Graduate School of Medicine, Chiba, Japan, via Moriya Tsuji, New York University School of Medicine, New York, N.Y.) (9). Mice between 8 and 10 weeks of age were used in all experiments. All mice were on a BALB/c background, and the experiments were approved by the Institutional Animal Care and Use Committee at The Pennsylvania State University.

Analysis of T-cell expansion and anergy induction in vivo in response to SEB. WT, CD1d^–/–, and J18^–/– mice were injected intraperitoneally with 50 µg SEB (Sigma-Aldrich, St. Louis, MO) and eye bled at 0, 2, 4, and 5 days. Peripheral blood lymphocytes were stained with antibodies specific for Vß8 (SEB-reactive T cells) or Vß6 (nonreactive T cells) and CD4 fluorescein isothiocyanate, phycoerythrin, or CyChrome (BD Pharmingen, San Diego, CA). Cells were then analyzed by fluorescence-activated cell sorter analysis, with postanalysis of the data using WinMDI. Anergy was induced by injection of 10 µg SEB or phosphate-buffered saline (PBS) on days 0, 2, and 4; mice were sacrificed 5 days following the last SEB injection. Splenocytes were then stimulated with 0, 1, or 10 µg of SEB/ml or phorbol myristate acetate-ionomycin as a control for 72 h. Proliferation was assessed by pulsing with 0.5 µCi [³H]thymidine for the last 18 h of the culture, followed by harvesting onto glass fiber mats and counting.

Analysis of in vivo cytokine secretion. WT, CD1d^–/–, and J18^–/– mice were injected with 50 µg SEB intraperitoneally, and serum was isolated from cardiac blood at 0, 1, 2, 4, 8, 12, and 24 h and analyzed for IL-2 by enzyme-linked immunosorbent assay, following the manufacturer's instructions (BDPharmingen, San Diego, CA). IL-4, IFN-, and TNF- cytokine analyses were performed with a Bio-Plex cytokine assay system (Bio-Rad Hercules, California).

RT-PCR analysis of cytokine expression. WT, CD1d^–/–, and J18^–/– mice were injected with 50 µg SEB intraperitoneally, and lymph nodes (brachial, cervical, inguinal, and axillary), spleen, and liver were isolated after 2 h (IL-2, IL-4, and TNF-) or 10 h (IFN-) for mRNA isolation. Tissues were homogenized in Trizol for isolation of RNA, which was used to generate cDNA using a kit from Amersham Biosciences (Piscataway, NJ), following the manufacturer's instructions. Reverse transcription-PCR (RT-PCR) was then performed using specific primers for the IL-2, IL-4, IFN-, and TNF- genes, with ß-actin used as a control. Primer sequences are available upon request. PCRs were generally run for 35 cycles, except for the ß-actin PCRs, which were run for 26 cycles.

Analysis of in vivo toxicity. WT, CD1d^–/–, and J18^–/– mice were sensitized with an intraperitoneal injection of 20 mg D-galactosamine (D-GalN) (Sigma, St. Louis, MO); 2 h later, mice were injected intraperitoneally with 100 µg SEB. Mice were monitored for 24 h for severity of sickness. Surviving mice were then sacrificed at the 24-h time point.

Data analysis. Statistical evaluation was conducted using Student's t test or the normal theory test, with P values of <0.05 considered statistically significant (38).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Similar expansion of Vß8⁺ CD4⁺ population in NKT-null and WT animals in response to SEB exposure in vivo. SEB has been shown to cause severe illness, due to activation of T cells and the resultant cytokine production (27). T cells are known to be required for this response (27). A majority of NKT cells carry the Vß7 or Vß8 regions of the TCR, and SEB preferentially interacts with T cells bearing these V regions (in addition to Vß3) (23, 51). A hallmark of SEB exposure is the expansion of T cells bearing the Vß8 TCR. Exposure of WT mice to SEB results in an expansion of the SEB reactive T-cell population with a peak at around 2 days postexposure, followed by a decrease that may reflect cell death of this expanded population (26).

We therefore used two models of mice lacking NKT cells, CD1d-null mice, which lack CD1d and thus lack the selective MHC molecules for the development of these cells, and J18-null mice, which lack the specific TCR J region used by the TCR of NKT cells and so lack NKT cells, to determine the role of these cells in SEB-mediated responses. To determine if the NKT cell-null mice exhibited differential T-cell responses, we examined the expansion of the SEB-reactive T cells in these mice. WT, CD1d-null, or J18-null mice were eye bled at day 0 to get a baseline level of their Vß8⁺ CD4⁺ T cells, as well as that of an SEB-nonresponsive population (Vß6⁺ CD4⁺ T cells) as a control. Mice were then injected with SEB and bled on days 2, 4, and 5 following exposure. Peripheral blood Vß8⁺ CD4⁺ and Vß6⁺ CD4⁺ T-cell populations were analyzed by flow cytometry at 2, 4, and 5 days as a measure of T-cell expansion. Figure 1A illustrates that similar expansion of Vß8⁺ CD4⁺, but not Vß6⁺ CD4⁺ T cells (Fig. 1B) in blood from WT, CD1d-null, and J18-null animals was observed following SEB exposure, with expansion peaking around day 2 (26). Therefore, we conclude NKT cells do not regulate the expansion of SEB-reactive T cells following SEB exposure.

View larger version (22K): [in this window] [in a new window]

FIG. 1. NKT cells do not regulate T-cell expansion upon SEB exposure in vivo. WT, CD1d–/–, or J18–/– mice were exposed to SEB, and percentages of CD4+ Vß8+ T cells were analyzed over the indicated time periods. (A) Vß8+ CD4+ T cells; (B) Vß6+ CD4+ T cells (the results of two experiments are shown for each group).

Differential cytokine regulation by J18- and CD1d-null mice in vivo. Cytokine production is a hallmark of SEB exposure (27). In particular, IL-2, IFN-, and TNF- are secreted during exposure to SEB, with toxicity proposed to be due to TNF- and perhaps IFN- production via a T-cell-dependent process (32). NKT cells rapidly secreted predominantly IL-4 and IFN-, as well as IL-2, upon activation (23). Although it is unlikely that NKT cells would be responsible for all of the cytokines observed following SEB exposure, it is possible that they contribute to or modulate their levels. We therefore examined whether NKT cells regulate the level of cytokine secretion in response to SEB exposure in vivo. Mice (WT, CD1d-null, or J18-null) were injected with SEB, and serum samples from these mice were analyzed at various time periods following SEB exposure. Examination of IL-4 secretion revealed that both CD1d- and J18-null mice secreted significantly less IL-4 than WT mice, indicating that CD1d-restricted NKT cells are responsible for most of the IL-4 secretion observed in response to SEB exposure (Fig. 2A). However, we did detect some serum IL-4 in these animals, indicating that conventional T cells responding to SEB stimulation can also secrete this cytokine, although NKT cells may be responsible for the secretion of the majority of this cytokine in WT mice. Surprisingly, examination of IFN- secretion over the same time period indicated that CD1d-null mice produced significantly more of this cytokine than WT or J18-null mice, indicating a role for CD1d-restricted T cells in negatively regulating the secretion of IFN- in response to SEB exposure (Fig. 2B). By contrast, J18-null mice secreted significantly less IFN- than WT mice in response to SEB exposure, suggesting that similar to IL-4, NKT cells may be responsible for the secretion of this cytokine in WT mice (Fig. 2B). Significantly, we found that NKT cells played little role in SEB-induced IL-2 secretion, as there were few differences in the serum levels of this cytokine over the time course of exposure (Fig. 2C). These data suggest that conventional T cells are primarily responsible for IL-2 secretion in response to SEB exposure. Examination of serum TNF- levels indicated that both CD1d- and J18-null mice secreted significantly more of this cytokine than WT mice, although J18-null mice secreted significantly more TNF- than CD1d-null mice (Fig. 3). These data suggest that NKT cells negatively regulate the secretion of TNF- in response to SEB.

View larger version (22K): [in this window] [in a new window]

FIG. 2. Differential regulation of SEB-induced cytokine secretion by CD1d-restricted and J18-bearing T cells in vivo. WT, CD1d–/–, or J18–/– mice were exposed to SEB and sacrificed at the indicated time periods; serum samples were analyzed for IL-4 (A), IFN- (B), or IL-2 (C). •, WT mice; , CD1d–/– mice; , J18–/– mice (the results of two experiments are shown for each group).

View larger version (16K): [in this window] [in a new window]

FIG. 3. NKT cells negatively regulate SEB-induced TNF- secretion in vivo. WT, CD1d–/–, or J18–/– mice were exposed to SEB and sacrificed at the indicated time periods; serum samples were analyzed for TNF-. •, WT mice; , CD1d–/– mice; , J18–/– mice (the results of two experiments are shown for each group).

To determine which tissues were responsible for cytokine production following SEB exposure in these mice, lymph nodes, spleens, and livers were collected from animals exposed to SEB for 2 h, which was near the peak of cytokine secretion in serum for IL-2, IL-4, and TNF-, and at 10 h, which was near the peak of IFN- secretion in serum (Fig. 3). Semiquantitative RT-PCR was performed on RNA from these tissues to determine the source of the observed cytokines (Fig. 4). The data showed that the major source of all the observed cytokines was the lymph nodes and spleen, with much less message observed in the liver. We note that this analysis was not sensitive enough to detect major differences in cytokine message between the different mice, particularly TNF-. Alternatively, the secretion of these cytokines may also be controlled at the level of translation or secretion. Nevertheless, these data show that the main sources of IL-2, IL-4, and TNF- message are the lymph nodes and spleen, with the liver contributing to IFN- production.

View larger version (29K): [in this window] [in a new window]

FIG. 4. Lymph node and spleen are the main source of cytokine message in SEB-exposed animals. WT, CD1d–/–, or J18–/– mice were exposed to 50 µg SEB and sacrificed after 2 h (IL-2, IL-4, and TNF-) or 10 h (IFN-), and RNA was isolated from lymph, node, spleen, and liver. RT-PCR was performed for the indicated cytokine message, as well as ß-actin as a control. Results for lymph node (A), spleen (B), and liver (C) are shown. Note that the ß-actin RT-PCRs shown are for the 2-h time point, with similar results seen for the 10-h time point.

NKT cells do not regulate anergy induced by SEB. A well-recognized response to superantigen exposure in mice is the development of superantigen-specific anergy, where superantigen-reactive T cells exhibit reduced responses upon reexposure to superantigen. NKT cells have been suggested to play a role in modulating the proliferation of CD4⁺ CD25⁺ Treg cells (17). These CD4⁺ CD25⁺ Treg cells have been suggested to play a role in the induction of anergy by superantigens (11). Indeed, the fact that J18- and CD1d-null mice exhibit altered cytokine production in response to SEB in vivo could lead to altered induction of anergy in SEB-reactive T-cell populations. We therefore exposed WT, J18-null, and CD1d-null mice to SEB or PBS and then analyzed their T-cell response to SEB in vitro. Figure 5 demonstrates that while T cells from WT mice exposed to PBS responded to SEB stimulation in vitro, those taken from mice previously exposed to SEB had much-reduced responses. This induction of anergy was also observed in cells taken from mice lacking J18 and CD1d. These data indicate that NKT cell populations are not required for the induction of anergy to superantigen exposure.

View larger version (17K): [in this window] [in a new window]

FIG. 5. NKT cells do not regulate SEB-induced anergy. WT, CD1d–/–, or J18–/– mice were exposed to SEB and then sacrificed, and splenocytes were incubated in vitro with the indicated concentrations of SEB to measure T-cell proliferative responses. (A) WT mice; (B) CD1d–/– mice; (C) J18–/– mice. , PBS injected, •, SEB injected. (D) Splenocytes stimulated with phorbol myristate acetate and ionomycin. Filled bars, WT mice; open bars, CD1d–/– mice; hatched bars, J18–/– mice (the results of two of experiments are shown for each group).

Role of NKT cells in SEB induced toxicity. Our data suggested that CD1d-restricted NKT cells may negatively regulate TNF- and/or IFN- production in response to SEB exposure. As TNF- has been suggested to play a significant role in death due to SEB toxicity (32), we wanted to determine if NKT cells would play a role in SEB-induced toxicity. WT, CD1d^–/–, and J18^–/– mice were sensitized with D-GalN, injected with SEB, and monitored for signs of illness (48). Our experiments demonstrated that >60% of the WT mice died within 8 h after SEB exposure, while <43% of the CD1d^–/– and the J18^–/– mice died during this period (Table 1). The remaining mice all exhibited symptoms but did not die over the 24-h period. This difference in death between the strains was not statistically significant, suggesting that the presence of NKT cells in WT mice does not provide a significant protective effect against SEB induced toxicity, despite the large difference in TNF- secretion in these mice.

View this table: [in this window] [in a new window]

TABLE 1. Equivalent SEB-induced toxicity in NKT cell-null mice

Top Abstract Introduction Materials and Methods Results Discussion References

 
The ability of SEB to bind and activate T cells that carry the Vß8 and -7 TCRs and the fact that a majority of NKT cells carry the Vß7 or -8 TCR prompted us to determine whether this population of T cells would play a role in the response to SEB exposure in vivo. Our studies determined that in the absence of CD1d or NKT cells, the SEB induced expansion of reactive T cells and consequent induction of anergy was unchanged, although Ho et al. suggested that a CD1d-restricted subpopulation of NKT cells could regulate T-cell expansion in response to specific antigen (15). While NKT cells have been suggested to play a role in modulating CD4⁺ CD25⁺ Treg cells and these cells may play a role in superantigen-induced anergy, our findings would suggest that this response is intact in mice lacking NKT cell populations (11, 17). Analysis of cytokine secretion indicated that NKT cells are primarily responsible for the secretion of IL-4 in response to SEB. Furthermore, CD1d-restricted T cells negatively regulate the secretion of IFN-, while NKT cells may be involved in the secretion of this cytokine. By contrast, both CD1d-restricted T-cell populations and NKT cells negatively regulated the secretion of TNF-, while neither population significantly modulated IL-2 secretion. However, these differences in cytokine secretion did not translate into significant differences in modulating SEB induced toxicity in vivo.

Although NKT cells are normally restricted by CD1d, it is very likely that they can be directly activated by SEB. Indeed, CD8⁺ T cells can be directly activated by SEB, and they are normally MHC class I restricted (14). Note that some NKT cells are CD4⁺, similar to CD4⁺ T cells; thus, SEB may interact with MHC class II-positive antigen-presenting cells to present SEB to NKT cells, resulting in their activation (31). It is also possible that they secrete cytokine upon receiving signals produced by other cells activated by SEB. Our findings of differences in cytokine secretion between mice lacking CD1d and those lacking J18 suggest that these mice may lack slightly different populations of cells. Indeed, other workers have been able to detect populations of T cells in CD1d-null mice that carry both NK1.1 and the TCR, hallmarks of NKT cells, although they lack cells that can bind to -GalCer/CD1d tetramers (3, 6). Both CD1d-null mice and J18-null mice lack a major population of NKT cells, those bearing the V14 TCR (9, 44). However, mice lacking CD1d may also lack other T cells that are restricted by CD1d, which may be present in mice lacking J18 (4, 16, 36, 50). In addition, it is also possible that the J18-null mice have other populations of invariant NKT cells that bear the Vß8 TCR region and can respond to SEB. Indeed, recent identification of invariant NKT cells bearing other V regions such as V10, -11, -15, -17, or -19.1 may support this view (5, 43). These other invariant NKT cells may also carry SEB-reactive Vß8, -3, or -7 and may be able to respond to SEB by secreting or modulating cytokine secretion.

It is possible that IFN- and IL-4 may be secreted by different populations of NKT cells, an idea that has been proposed by others as one explanation for the ability of different NKT cell ligands to induce differential cytokine secretion (19, 46). Thus, IL-4-secreting NKT cells may be uniformly absent or reduced in CD1d- and J18-null mice, resulting in reduced IL-4 secretion in response to SEB, as we observed. Although reduced, IL-4 was secreted in response to SEB in these mice, and it has previously been reported that low levels of IL-4 can be detected being secreted by conventional T cells in response to SEB (8, 40). It should be noted that both strains of mice carry conventional T cells capable of secreting wild-type levels of IL-4 (49). By contrast, IFN--secreting NKT cells may also be missing or reduced in J18-null mice, while still present in CD1d-null mice, thus accounting for the increased levels of this cytokine observed in the latter mice.

Dobashi et al. previously examined NK and NKT cells in cytokine secretion induced by SEB exposure and found that Kupffer cells in the liver were important for inducing cytokine secretion. However, they only examined IFN- secretion, and most of their experiments examined in vitro cultures (up to 48 h) following exposure (10). They did find, however, that whether examined in vitro or in serum following exposure, depletion of NK and NKT cells with anti-NK1.1 or anti-asialo GM1 antibodies (expressed on both populations) prior to exposure to SEB resulted in reduced IFN- secretion. This finding would be similar to our findings with the J18-null mice. However, we should note that this reagent depletes both NK and NKT cells, and so a definitive conclusion could not be made regarding the role of NKT cells in IFN- secretion in vivo upon SEB exposure. Indeed, a number of studies using this depletion protocol have come to different conclusions compared to the knockout mice. In particular, Korsgren et al. used this reagent to suggest that NK cells but not NKT cells are required for the development of allergic asthma, while more recent studies using the knockout mice have shown that, indeed, NKT cells are required for the development of this disease (1, 22, 24). Our data definitively show that in the absence of J18-bearing NKT cells, IFN- secretion is reduced. We have also examined the secretion of IL-2, IL-4, and TNF-, as well as T-cell expansion, the ability to induce anergy, and toxicity, none of which were addressed by Dobashi et al. It is possible that since IFN- secretion is observed rather late after SEB exposure (unlike the case for IL-2, IL-4, and TNF-), other cells contribute to its appearance in serum, and these are differently affected by the presence of CD1d versus J18, and perhaps NK cells as well.

Of course, we also cannot rule out indirect effects of CD1d-restricted or J18-bearing T cells on conventional T-cell populations for IL-4 and IFN- secretion. Indeed, this may be the case with regards to TNF- secretion, as in the absence of these cells, mice secreted much more of this cytokine in response to SEB. Of interest is the level of TNF- secretion by the two strains of mice; J18-null mice secreted significantly more TNF- than those lacking CD1d. As discussed above, it is possible that different populations of NKT cells exist, which are differentially reduced or missing in the two strains of mice. These different populations may have differential effects on the ability of conventional T cells to secrete TNF-. These cells represent a very small population of total T cells that may be difficult to detect. Our data suggest that these cell populations may negatively regulate the ability of conventional T cells to secrete TNF-. This may be due to reduced IL-4 secretion and its effects on effector cell function or to other properties of NKT cells. Our analysis of cytokine message suggests that the majority of the cytokine is being made by cells in the lymph node and spleen, with cells in the liver making smaller contributions, as has been previously reported (25, 29). Indeed, Schumann et al. have shown that depletion of hepatic Kupffer cells does not affect serum levels of TNF- in mice exposed to SEB, although these cells make message for this cytokine upon SEB exposure (42). We could detect lower levels of IL-2, IL-4, and IFN- in the livers of the mice, a site where a large majority of the lymphocytes are NKT cells. However, it should be noted that the number of these cells in the liver is still much less than the number of T cells in the spleen and lymph nodes, making it much more likely that cytokine message will be detected in the latter tissues. Experiments aimed at sorting NKT cells and analyzing their cytokine message profile or the use of fluorescent protein knockin into these cytokine loci may be able to better address the issue of NKT cell cytokine production in more detail (47). Our semiquantitative RT-PCR assay was not sensitive enough to detect differences in cytokine message between the different strains of mice, as we observed very little difference in message for the different cytokines, except in the liver, where lower levels of IFN- message were detected in CD1d-null mice and lower levels of IL-4 message were detected in the livers of J18-null mice than in the other mice. These data suggest that it is likely that NKT cells play a modulatory role in cytokine secretion, perhaps contributing to signals that may enhance cytokine message or translation and secretion. In addition, NKT cells have a modulatory role in macrophage and/or dendritic cell activation and maturation, which may alter the signals that T cells receive during SEB exposure (21, 33, 53).

Surprisingly and in contrast to what we observed for TNF- secretion, neither CD1d-restricted nor J18-bearing T cells play a significant role in toxicity induced by SEB. While there was slightly less death in mice lacking J18 or CD1d, the differences were not statistically significant. Thus, while TNF- plays a critical role in the toxicity induced by SEB, increased levels of this cytokine do not seem to increase toxicity of SEB, indicating that the amounts found in WT mice may represent the threshold required for the induction of death upon exposure. Indeed, while TNF- is clearly required for SEB-induced toxicity, it is not sufficient. Several other reports have noted differences in SEB toxicity in the presence of little difference in TNF- levels or have observed that direct delivery of TNF- does not lead to similar toxicity, such as that seen with SEB exposure (2, 41). Thus, TNF- is required but not sufficient for SEB-induced toxicity, which may explain our results, suggesting that other factors may play a role in regulating SEB-induced toxicity. These may include requirements for NKT cells in the liver in expressing Fas ligand and inducing hepatocyte apoptosis, an event that may not be seen in the absence of these cells even in the presence of high levels of TNF- (7, 18, 34).

Nevertheless, the data suggest that NKT cells negatively regulate TNF- secretion and are primarily responsible for IL-4 secretion in response to SEB. These data may also have implications for the role of staphylococcal infections in allergic rhinitis and atopic eczema-dermatitis syndrome, where the presence of bacteria secreting SEB or SEB itself has been implicated in the development of these conditions (13, 35, 39).

 
We thank members of the August laboratory and the Center for Molecular Immunology & Infectious Disease at Penn State for helpful comments and suggestions. We also thank Elaine Kunze and Susan Magargee in the Center for Quantitative Cell Analysis at Penn State for excellent technical help and Margherita Cantorna for helpful discussions. We also thank Masaru Taniguchi (Chiba University Graduate School of Medicine, Chiba, Japan) and Moriya Tsuji (New York University School of Medicine, New York, N.Y.) for providing us with J18-null mice.

This work was supported in part by the American Heart Association (0330036N), and Public Health Service Grant AI-51626 (to A.A.). M.J.R. is a Sloan Scholar and a Ford Foundation Scholar.

 
* Corresponding author. Mailing address: Center for Molecular Immunology & Infectious Disease, Department of Veterinary and Biomedical Sciences, The Pennsylvania State University, 115 Henning Building, University Park, PA 16803. Phone: (814) 863-3539. Fax: (814) 863-6140. E-mail: avery{at}psu.edu.

Editor: J. T. Barbieri

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Akbari, O., P. Stock, E. Meyer, M. Kronenberg, S. Sidobre, T. Nakayama, M. Taniguchi, M. Grusby, R. DeKruyff, and D. Umetsu. 2003. Essential role of NKT cells producing IL-4 and IL-13 in the development of allergen-induced airway hyperreactivity. Nat. Med. 9:582-588.[CrossRef][Medline]
2
2. Anderson., M. R., and M. Tary-Lehmann. 2001. Staphylococcal enterotoxin-B-induced lethal shock in mice is T-cell-dependent, but disease susceptibility is defined by the non-T-cell compartment. Clin. Immunol. 98:85-94.[CrossRef][Medline]
3
3. Arrunategui-Correa, V., L. Lenz, and H. Kim. 2004. CD1d-independent regulation of NKT cell migration and cytokine production upon Listeria monocytogenes infection. Cell Immunol. 232:38-48.[CrossRef][Medline]
4
4. Behar, S., and S. Cardell. 2000. Diverse CD1d-restricted T cells: diverse phenotypes, and diverse functions. Semin. Immunol. 12:551-560.[CrossRef][Medline]
5
5. Behar, S., T. Podrebarac, C. Roy, C. Wang, and M. Brenner. 1999. Diverse TCRs recognize murine CD1. J. Immunol. 162:161-167.[Abstract/Free Full Text]
6
6. Benlagha, K., A. Weiss, A. Beavis, L. Teyton, and A. Bendelac. 2000. In vivo identification of glycolipid antigen-specific T cells using fluorescent CD1d tetramers. J. Exp. Med. 191:1895-1903.[CrossRef][Medline]
7
7. Biburger, M., and G. Tiegs. 2005. -Galactosylceramide-induced liver injury in mice is mediated by TNF- but independent of Kupffer cells. J. Immunol. 175:1540-1550.[Abstract/Free Full Text]
8
8. Cardell, S., I. Hoiden, and G. Moller. 1993. Manipulation of the superantigen-induced lymphokine response. Selective induction of interleukin-10 or interferon-gamma synthesis in small resting CD4+ T cells. Eur. J. Immunol. 23:523-529.[Medline]
9
9. Cui, J., T. Shin, T. Kawano, H. Sato, E. Kondo, I. Toura, Y. Kaneko, H. Koseki, M. Kanno, and M. Taniguchi. 1997. Requirement for V14 NKT cells in IL-12-mediated rejection of tumors. Science 278:1623-1626.[Abstract/Free Full Text]
10
10. Dobashi, H., S. Seki, Y. Habu, T. Ohkawa, S. Takeshita, H. Hiraide, and I. Sekine. 1999. Activation of mouse liver natural killer cells and NK1.1(+) T cells by bacterial superantigen-primed Kupffer cells. Hepatology 30:430-436.[Medline]
11
11. Feunou, P., L. Poulin, C. Habran, A. Le Moine, M. Goldman, and M. Y. Braun. 2003. CD4+CD25+ and CD4+CD25- T cells act respectively as inducer and effector T suppressor cells in superantigen-induced tolerance. J. Immunol. 171:3475-3484.[Abstract/Free Full Text]
12
12. Fischer, K., E. Scotet, M. Niemeyer, H. Koebernick, J. Zerrahn, S. Maillet, R. Hurwitz, M. Kursar, M. Bonneville, S. Kaufmann, and U. Schaible. 2004. Mycobacterial phosphatidylinositol mannoside is a natural antigen for CD1d-restricted T cells. Proc. Natl. Acad. Sci. USA 101:10685-10690.[Abstract/Free Full Text]
13
13. Heaton, T., D. Mallon, T. Venaille, and P. Holt. 2003. Staphylococcal enterotoxin induced IL-5 stimulation as a cofactor in the pathogenesis of atopic disease: the hygiene hypothesis in reverse? Allergy 58:252-256.[CrossRef][Medline]
14
14. Herrmann, T., J. Maryanski, P. Romero, B. Fleischer, and H. MacDonald. 1990. Activation of MHC class I-restricted CD8+ CTL by microbial T cell mitogens. Dependence upon MHC class II expression of the target cells and V beta usage of the responder T cells. J. Immunol. 144:1181-1186.[Abstract]
15
15. Ho, L.-P., B. C. Urban, L. Jones, G. S. Ogg, and A. McMichael. 2004. CD4^–CD8 subset of CD1d-restricted NKT cells controls T cell expansion. J. Immunol. 172:7350-7358.[Abstract/Free Full Text]
16
16. Jahng, A., I. Maricic, C. Aguilera, S. Cardell, R. Halder, and V. Kumar. 2004. Prevention of autoimmunity by targeting a distinct, noninvariant CD1d-reactive T cell population reactive to sulfatide. J. Exp. Med. 199:947-957.[Abstract/Free Full Text]
17
17. Jiang, S., D. Game, D. Davies, G. Lombardi, and R. Lechler. 2005. Activated CD1d-restricted natural killer T cells secrete IL-2: innate help for CD4+CD25+ regulatory T cells? Eur. J. Immunol. 35:1193-1200.[CrossRef][Medline]
18
18. Kaneko, Y., M. Harada, T. Kawano, M. Yamashita, Y. Shibata, F. Gejyo, T. Nakayama, and M. Taniguchi. 2000. Augmentation of V14 NKT cell-mediated cytotoxicity by interleukin 4 in an autocrine mechanism resulting in the development of concanavalin A-induced hepatitis. J. Exp. Med. 191:105-114.[Abstract/Free Full Text]
19
19. Kim, C., E. Butcher, and B. Johnston. 2002. Distinct subsets of human V24-invariant NKT cells: cytokine responses and chemokine receptor expression. Trends Immunol. 23:516-519.[CrossRef][Medline]
20
20. Kinjo, Y., D. Wu, G. Kim, G. Xing, M. Poles, D. Ho, M. Tsuji, K. Kawahara, C. Wong, and M. Kronenberg. 2005. Recognition of bacterial glycosphingolipids by natural killer T cells. Nature 434:520-525.[CrossRef][Medline]
21
21. Kojo, S., K. Seino, M. Harada, H. Watarai, H. Wakao, T. Uchida, T. Nakayama, and M. Taniguchi. 2005. Induction of regulatory properties in dendritic cells by V14 NKT Cells. J. Immunol. 175:3648-3655.[Abstract/Free Full Text]
22
22. Korsgren., M., C. Persson, F. Sundler, T. Bjerke, T. Hansson, B. Chambers, S. Hong, L. V. Kaer, H. Ljunggren, and O. Korsgren. 1999. Natural killer cells determine development of allergen-induced eosinophilic airway inflammation in mice. J. Exp. Med. 189:553-562.[Abstract/Free Full Text]
23
23. Kronenberg, M. 2005. Toward an understanding of NKT cell biology: progress and paradoxes. Annu. Rev. Immunol. 23:877-900.[CrossRef][Medline]
24
24. Lisbonne, M., S. Diem, A. de Castro Keller, J. Lefort, L. Araujo, P. Hachem, J. Fourneau, S. Sidobre, M. Kronenberg, M. Taniguchi, P. Van Endert, M. Dy, P. Askenase, M. Russo, B. Vargaftig, A. Herbelin, and M. Leite-de-Moraes. 2003. Cutting edge: invariant V alpha 14 NKT cells are required for allergen-induced airway inflammation and hyperreactivity in an experimental asthma model. J. Immunol. 171:1637-1641.[Abstract/Free Full Text]
25
25. Litton, M., B. Sander, E. Murphy, A. O'Garra, and J. Abrams. 1994. Early expression of cytokines in lymph nodes after treatment in vivo with Staphylococcus enterotoxin B. J. Immunol. Methods 175:47-58.[CrossRef][Medline]
26
26. MacDonald, H., S. Baschieri, and R. Lees. 1991. Clonal expansion precedes anergy and death of V beta 8+ peripheral T cells responding to staphylococcal enterotoxin B in vivo. Eur. J. Immunol. 21:1963-1966.[Medline]
27
27. Marrack, P., M. Blackman, E. Kushnir, and J. Kappler. 1990. The toxicity of staphylococcal enterotoxin B in mice is mediated by T cells. J. Exp. Med. 171:455-464.[Abstract/Free Full Text]
28
28. Matsuda, J., and M. Kronenberg. 2001. Presentation of self and microbial lipids by CD1 molecules. Curr. Opin. Immunol. 13:19-25.[CrossRef][Medline]
29
29. Matsui, S., M. Terabe, A. Mabuchi, M. Takahashi, M. Saizawa, S. Tanaka, and K. Yokomuro. 1997. A unique response to staphylococcal enterotoxin B by intrahepatic lymphocytes and its relevance to the induction of tolerance in the liver. Scand. J. Immunol. 46:230-234.[CrossRef][Medline]
30
30. Mattner, J., K. Debord, N. Ismail, R. Goff, C. Cantu III, D. Zhou, P. Saint-Mezard, V. Wang, Y. Gao, N. Yin, K. Hoebe, O. Schneewind, D. Walker, B. Beutler, L. Teyton, P. Savage, and A. Bendelac. 2005. Exogenous and endogenous glycolipid antigens activate NKT cells during microbial infections. Nature 434:525-529.[CrossRef][Medline]
31
31. Mercer, J. C., M. Ragin, and A. August. 2005. Natural killer T cells: rapid responders controlling immunity and disease. Int. J. Biochem. Cell Biol. 37:1337-1343.[CrossRef][Medline]
32
32. Miethke, T., C. Wahl, K. Heeg, B. Echtenacher, P. Krammer, and H. Wagner. 1992. T cell-mediated lethal shock triggered in mice by the superantigen staphylococcal enterotoxin B: critical role of tumor necrosis factor. J. Exp. Med. 175:91-98.[Abstract/Free Full Text]
33
33. Munz, C., R. Steinman, and S. Fujii. 2005. Dendritic cell maturation by innate lymphocytes: coordinated stimulation of innate and adaptive immunity. J. Exp. Med. 202:203-207.[Abstract/Free Full Text]
34
34. Nakagawa, R., I. Nagafune, Y. Tazunoki, H. Ehara, H. Tomura, R. Iijima, K. Motoki, M. Kamishohara, and S. Seki. 2001. Mechanisms of the antimetastatic effect in the liver and of the hepatocyte injury induced by alpha-galactosylceramide in mice. J. Immunol. 166:6578-6584.[Abstract/Free Full Text]
35
35. Okano, M., T. Takishita, T. Yamamoto, H. Hattori, Y. Yamashita, S. Nishioka, T. Ogawa, and K. Nishizaki. 2001. Presence and characterization of sensitization to staphylococcal enterotoxins in patients with allergic rhinitis. Am. J. Rhinol. 15:417-421.[Medline]
36
36. Park, S., A. Weiss, K. Benlagha, T. Kyin, L. Teyton, and A. Bendelac. 2001. The mouse CD1d-restricted repertoire is dominated by a few autoreactive T cell receptor families. J. Exp. Med. 193:893-904.[Abstract/Free Full Text]
37
37. Petersson, K., G. Forsberg, and B. Walse. 2004. Interplay between superantigens and immunoreceptors. Scand. J. Immunol. 59:345-355.[CrossRef][Medline]
38
38. Rosner, B. 1999. Fundamentals of biostatistics, 2nd ed. Breton Publishers, Boston, Mass.
39
39. Rossi, R., and G. Monasterolo. 2004. Prevalence of serum IgE antibodies to the Staphylococcus aureus enterotoxins (SAE, SEB, SEC, SED, TSST-1) in patients with persistent allergic rhinitis. Int. Arch. Allergy Immunol. 133:261-266.[CrossRef][Medline]
40
40. Schmitz, J., M. Assenmacher, and A. Radbruch. 1993. Regulation of T helper cell cytokine expression: functional dichotomy of antigen-presenting cells. Eur. J. Immunol. 23:191-199.[Medline]
41
41. Schumann, J., H. Bluethmann, and G. Tiegs. 2000. Synergism of Pseudomonas aeruginosa exotoxin A with endotoxin, superantigen, or TNF results in TNFR1- and TNFR2-dependent liver toxicity in mice. Immunol. Lett. 74:165-172.[CrossRef][Medline]
42
42. Schumann, J., D. Wolf, A. Pahl, K. Brune, T. Papadopoulos, N. van Rooijen, and G. Tiegs. 2000. Importance of Kupffer cells for T-cell-dependent liver injury in mice. Am. J. Pathol. 157:1671-1683.[Abstract/Free Full Text]
43
43. Shimamura, M., and Y. Y. Huang. 2002. Presence of a novel subset of NKT cells bearing an invariant V19.1-J.26 TCR chain. FEBS Lett. 516:97-100.[CrossRef][Medline]
44
44. Smiley, S., M. Kaplan, and M. Grusby. 1997. Immunoglobulin E production in the absence of interleukin-4-secreting CD1-dependent cells. Science 275:977-979.[Abstract/Free Full Text]
45
45. Sriram, V., W. Du, J. Gervay-Hague, and R. Brutkiewicz. 2005. Cell wall glycosphingolipids of Sphingomonas paucimobilis are CD1d-specific ligands for NKT cells. Eur. J. Immunol. 35:1692-1701.[CrossRef][Medline]
46
46. Stenstrom, M., M. Skold, A. Ericsson, L. Beaudoin, S. Sidobre, M. Kronenberg, A. Lehuen, and S. Cardell. 2004. Surface receptors identify mouse NK1.1+ T cell subsets distinguished by function and T cell receptor type. Eur. J. Immunol. 34:56-65.[CrossRef][Medline]
47
47. Stetson, D., M. Mohrs, R. Reinhardt, J. Baron, Z. Wang, L. Gapin, M. Kronenberg, and R. Locksley. 2003. Constitutive cytokine mRNAs mark natural killer (NK) and NK T cells poised for rapid effector function. J. Exp. Med. 198:1069-1076.[Abstract/Free Full Text]
48
48. Stiles, B., Y. Campbell, R. Castle, and S. Grove. 1999. Correlation of temperature and toxicity in murine studies of staphylococcal enterotoxins and toxic shock syndrome toxin 1. Infect. Immun. 67:1521-1525.[Abstract/Free Full Text]
49
49. Van Kaer, L. 2004. Regulation of immune responses by CD1d-restricted natural killer T cells. Immunol. Res. 30:139-153.[CrossRef][Medline]
50
50. Van Rhijn, I., D. Young, J. Im, S. Levery, P. Illarionov, G. Besra, S. Porcelli, J. Gumperz, T. Cheng, and D. Moody. 2004. CD1d-restricted T cell activation by nonlipidic small molecules. Proc. Natl. Acad. Sci. USA 101:13578-13583.[Abstract/Free Full Text]
51
51. White, J., A. Herman, A. Pullen, R. Kubo, J. Kappler, and P. Marrack. 1989. The V beta-specific superantigen staphylococcal enterotoxin B: stimulation of mature T cells and clonal deletion in neonatal mice. Cell 56:27-35.[CrossRef][Medline]
52
52. Wu, D., G. Xing, M. Poles, A. Horowitz, Y. Kinjo, B. Sullivan, V. Bodmer-Narkevitch, O. Plettenburg, M. Kronenberg, M. Tsuji, D. Ho, and C. Wong. 2005. Bacterial glycolipids and analogs as antigens for CD1d-restricted NKT cells. Proc. Natl. Acad. Sci. USA 102:1351-1356.[Abstract/Free Full Text]
53
53. Yue, S., A. Shaulov, R. Wang, S. Balk, and M. Exley. 2005. CD1d ligation on human monocytes directly signals rapid NF-B activation and production of bioactive IL-12. Proc. Natl. Acad. Sci. USA 102:11811-11816.[Abstract/Free Full Text]
54
54. Zhou, D., J. Mattner, C. Cantu III, N. Schrantz, N. Yin, Y. Gao, Y. Sagiv, K. Hudspeth, Y. Wu, et al. 2004. Lysosomal glycosphingolipid recognition by NKT cells. Science 306:1786-1789.[Abstract/Free Full Text]

---

Infection and Immunity, January 2006, p. 282-288, Vol. 74, No. 1
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.1.282-288.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Ragin, M. J. Articles by August, A. Search for Related Content PubMed PubMed Citation Articles by Ragin, M. J. Articles by August, A.