Acute Systemic Immune Activation following Conjunctival Exposure to Staphylococcal Enterotoxin B

Abstract

Conjunctival exposure to the Staphylococcus aureus superantigen staphylococcal enterotoxin B (SEB) may occur accidentally, as a result of bioterrorism, or during colonization or infection of the external eye. Using human leukocyte antigen class II transgenic mice, we show for the first time that conjunctival exposure to SEB can cause robust systemic immune activation.

Staphylococcal enterotoxin B (SEB) is a superantigenic exotoxin produced by Staphylococcus aureus that is capable of inducing strong polyclonal activation of CD4⁺ and CD8⁺ T lymphocytes even at extremely low concentrations (23). The potent immunostimulatory property of SEB has led to its development as a biological weapon (4, 19), delivered either through contaminated food or water or in an aerosolized form. While ingested SEB causes acute food poisoning (2), airway exposure to SEB aerosol can cause robust systemic immune activation and a clinical condition resembling toxic shock (3, 20, 26, 29).

In the event of exposure to aerosolized SEB, the eyes, in addition to the airways, would also be readily exposed to SEB. In such a case, the conjunctival mucous membranes may serve as a portal of absorption of SEB. However, it is not known whether conjunctival exposure to SEB can have systemic effects. Isolated cases of accidental conjunctival SEB exposure do suggest systemic effects (30). This has potential clinical relevance in addition to its biodefense significance for the following reasons. Conjunctival exposure to SEB can occur during extraocular staphylococcal infections such as keratitis, conjunctivitis, or blepharitis, albeit in smaller amounts (10, 11, 14, 15, 18, 31, 32). Patients with atopic dermatitis particularly tend to have high levels of S. aureus in the conjunctival sacs and eyelid margins; these isolates are capable of elaborating superantigen exotoxins (12, 16, 21, 22, 36). The hemolytic alpha-, beta-, delta-, and gamma-toxins of S. aureus are produced during such extraocular infections and play a role in the etiopathogenesis of staphylococcal eye diseases (6, 7, 9, 13). Staphylococcal superantigen exotoxins are also produced during such infections (1, 33-35, 38), but their pathogenic role is unknown. Given these observations that conjunctival exposure to SEB can occur accidentally, as a result of bioterrorism, or during colonization or infection of the external eye, it is imperative to understand its systemic effects. Therefore, in the present study, we evaluated the systemic immune effects of conjunctival exposure to SEB. It should be noted that conventional mice mount a weak immune response to SEB due poor binding of SEB to endogenous murine major histocompatibility complex class II molecules (5, 8, 28, 29, 37). Nonetheless, transgenic mice expressing human leukocyte antigen-DR3 (HLA-DR3) or -DQ8 or the interleukin-10 (IL-10)-deficient HLA-DQ8 transgenic mice are extremely sensitive to immune activation by SEB (24-27) and hence were used in our study.

For conjunctival challenge, 5 μl of phosphate-buffered saline (PBS) alone or SEB (5 μg/μl, highly purified, endotoxin-reduced, <1 endotoxin unit/mg; Toxin Laboratories, Sarasota, FL) was instilled onto each eye of anesthetized mice, and the animals were sacrificed at specific time points. Conjunctival application of SEB caused a time-dependent alteration in the expression patterns of selected activation markers on CD4⁺ and CD8⁺ T cells bearing SEB-reactive TCR Vβ8 in the draining cervical lymph nodes (Fig. 1). Although the expression of CD62L decreased by 24 h in SEB-treated mice, the level returned to that seen in PBS-treated mice by 72 h. Consistent with the activated phenotype, the expression of CD69 increased by 24 h, peaked by 48 h, and returned to low levels by 72 h. CD44, another activation marker, showed a similar profile (data not shown). The taller histogram peaks at 72 h in SEB-treated mice were due to the expansion of TCR Vβ8-bearing T cells at this time point, resulting in higher cell counts (see below).

FIG. 1.

Systemic T-cell activation after conjunctival exposure to SEB. Age-matched HLA-DQ8.IL-10^−/− mice were anesthetized, and 5 μl (25 μg) of SEB was applied onto each eye. The expression of activation markers CD62L (A) and CD69 (B) on SEB-reactive TCR Vβ8⁺ CD4⁺ and CD8⁺ T cells in the cervical lymph nodes at the indicated time points was determined by flow cytometry. Representative histograms from one of four similar experiments are shown.

Conjunctival application of SEB also resulted in an expansion of CD4⁺ and CD8⁺ T cells bearing the SEB-reactive TCR Vβ8 but not of cells bearing the SEB-nonreactive TCR Vβ6, in the draining cervical lymph nodes as well as in the spleen (Fig. 2), indicating a systemic response to SEB. It had been shown earlier that after systemic administration of superantigen (by intraperitoneal or intravenous routes), while peripheral T cells undergo expansion, CD4 CD8 double-positive thymocytes undergo massive deletion (17). As depicted in Fig. 3, there was a time-dependent reduction in the percentage of CD4 CD8 double-positive thymocytes (upper right quadrants) consistent with apoptosis of immature double-positive thymocytes, with a proportional increase in the percentages of CD4 and CD8 single-positive thymocytes (lower right and upper left quadrants, respectively). The absolute number of thymocytes was reduced by 15-fold by 72 h compared to PBS-treated mice.

FIG. 2.

Peripheral T-cell expansion after conjunctival exposure to SEB. Age-matched HLA-DQ8.IL-10^−/− mice were anesthetized, and 5 μl (25 μg) of SEB was applied onto each eye. Mice were sacrificed at the indicated time points, and the distribution of CD4⁺ and CD8⁺ T cells expressing TCR Vβ6 (SEB-nonreactive) or Vβ8 (SEB-reactive) in the cervical lymph nodes (A) and spleens (B) was analyzed by flow cytometry. Bars represent mean + the standard error from four to five mice/group.

FIG. 3.

Thymocyte apoptosis after conjunctival exposure to SEB. Age-matched HLA-DQ8.IL-10^−/− mice were anesthetized, and 5 μl (25 μg) of SEB was applied onto each eye. Mice were sacrificed at the indicated time points, and the distributions of thymocyte subsets were analyzed by flow cytometry. Representative dot plots from one of four similar experiments are shown.

Systemic immune activation after conjunctival exposure to SEB was also evident in HLA-DR3 transgenic mice as assessed by similar parameters (data not shown). In addition, HLA-DR3 transgenic mice were challenged conjunctivally with SEB and given bromodeoxyuridine (BrdU; Sigma, St. Louis, MO) daily in their drinking water at a concentration of 0.8 mg/ml. BrdU incorporated into newly synthesized DNA was detected by using a BrdU flow kit (Becton Dickinson, San Jose, CA). As depicted in Fig. 4, ocular instillation of SEB led to the proliferation of CD4⁺ and CD8⁺ T cells in the draining lymph nodes (Fig. 4), as well as in the spleen (data not shown), resulting in BrdU incorporation only in T cells bearing TCR Vβ8 but not in T cells bearing TCR Vβ6, further supporting a systemic effect of SEB delivered through the conjunctival mucosa. On the other hand, BrdU incorporation was indistinguishable between T cells bearing Vβ6 and Vβ8 in mice instilled with PBS. Thus, we have shown for the first time that conjunctival exposure to SEB can cause systemic immune activation.

FIG. 4.

Peripheral T-cell expansion after conjunctival exposure to SEB. Age-matched HLA-DR3 transgenic mice were anesthetized, and 5 μl (25 μg) of SEB was applied onto each eye. Mice were treated with BrdU in drinking water daily and sacrificed at 72 h. BrdU incorporated in CD4⁺ and CD8⁺ T cells expressing TCR Vβ6 (SEB-nonreactive) or Vβ8 (SEB-reactive) in cervical lymph nodes in gated population (indicated by the circles) was analyzed by flow cytometry. Representative plots are shown. In histograms, shaded and clear areas represent BrdU incorporated in Vβ6- and Vβ8-gated cells, respectively.

The amount of SEB ultimately absorbed after conjunctival application in mice would be much lower than what was applied because of grooming activity. In spite of this fact, we were still able to show systemic immune activation. High levels of human exposure to SEB can occur during accidental laboratory exposure or during intentional bioterrorism. Nonetheless, it should also be noted that humans are far more sensitive to SEB than are HLA class II transgenic mice. For example, the estimated 50% lethal dose of aerosolized SEB is 0.02 μg/kg in humans (30), whereas that for HLA-DQ8 transgenic mice is 70 μg/kg (29). Thus, conjunctival exposure to smaller amounts of SEB as would occur during infections could probably have systemic effects in humans. Acute or chronic conjunctival exposure to SEB or even other superantigens after a staphylococcal eye colonization or infection or after exposure to aerosols likely has other implications, including the activation of autoreactive T cells and other immunological consequences similar to what we have we shown recently after exposure through the nasal route (24, 26).