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Virtually all cystic fibrosis (CF) patients are colonized by Pseudomonas aeruginosa, and 90% of those that are colonized die as a result of lung damage (11). One of the hallmarks of the lung damage in CF is an ineffective inflammatory response that results in severe neutrophil-mediated pulmonary damage and an inability to clear the organisms. P. aeruginosa contributes to the lung damage by the production of virulence factors; one of the most important virulence factors, exoenzyme S, has been shown to induce pulmonary damage in animal models (17, 29, 35), and increased levels of exoenzyme S correlate with human disease (18, 36). The cytotoxicity of exoenzyme S for epithelial cells follows both contact-dependent type III translocation into eukaryotic target cells and contact-independent type III secretion, suggesting two mechanisms of cellular activation: intracellular and extracellular (15, 34, 35). We have recently described an additional activity: extracellular exoenzyme S is mitogenic for T cells, inducing tremendous T-cell activation (5-7). Exoenzyme-S-induced activation of T cells is neither dependent upon nor inhibited by ADP-ribosyltransferase activity, and this activity is present in exoenzyme S from both purified and recombinant sources (7). Further, we have found that activation by exoenzyme S induces T-cell apoptosis (6). The current studies were performed to determine whether this T-cell activation culminates in the induction of cytokines that have the potential of influencing immunoinflammatory responses or whether apoptosis precludes cytokine transcription.

Immunoinflammatory responses are orchestrated by cytokines, and T-cell mitogens and superantigens are potent stimuli for cytokine production from T cells, monocytes, and macrophages (2, 3). In CF there is a misdirected or dysregulated response, since there is a chronic and exuberant immunoinflammatory response without clearance of the pathogen. This response may be a result of altered cytokine induction that includes decreased secretion of the anti-inflammatory cytokine interleukin-10 (IL-10) (13, 28) with concordant increases in proinflammatory cytokine production, including tumor necrosis factor alpha (TNF-), IL-1, IL-1, IL-6, and IL-8 (4, 11, 22). T-cell mitogens induce the production of many cytokines and are capable of altering or polarizing the Th1 and Th2 cytokine profile, which can have a potential pathogenic role during infection (8, 9, 14, 16, 31). We hypothesized that exoenzyme S could contribute to a dysregulated immune response by the exaggerated production of both Th1 and Th2 cytokines and proinflammatory cytokines.

Cytokine effects and interactions are complex, with many cytokines influencing the production of others throughout the time course of the response. Because of this, multiparameter technology has been applied to cytokine work (24) with the goal of determining the kinetics of induction and the nature of the most important cytokine response. For this purpose, the RNase protection assay (RPA) has significant advantages; it is a sensitive and quantitative measure of cytokine and chemokine mRNA induction that allows for the simultaneous determination of multiple genes. Three gene panels were used to examine transcriptional expression of Th1 and Th2 cytokines, proinflammatory and immunoregulatory cytokines, and chemokines.

Exoenzyme S purification. ExoS/DG1 from P. aeruginosa strain DG1 was isolated as previously described using (NH₄)₂SO₄ precipitation of culture supernatants, ion-exchange chromatography, and acetone precipitation, followed by gel filtration, and migrated as a 50-kDa band without ADP-ribosyltransferase activity (35). rExoS was isolated from Escherichia coli BL21(DE3) bearing a plasmid encoding histidine-tagged exoenzyme S cloned from P. aeruginosa 388(pETrHisExoS). rExoS was purified by Ni²⁺ affinity chromatography from cellular lysates and migrated as a 52-kDa band possessing ADP-ribosyltransferase activity (7, 21). Neutralizing antibodies generated against ExoS/DG1 were shown to neutralize T-cell activation induced by rExoS, indicating that both preparations share the mitogenic epitope (7).

Cell culture and RPA. Peripheral blood mononuclear cell(s) (PBMC) were isolated from healthy adults by Ficoll-Hypaque density centrifugation (27). Cells (2 × 10⁵ cells/well) were cultured in AIM V serum free medium (Gibco BRL, Burlington, Ontario, Canada) in 24-well nonadherent plates (Costar) in the presence of 10 µg of polymyxin B per ml in order to exclude cellular activation from lipopolysaccharides (LPS) (30). Resting PBMC were stimulated with 1 µg of either ExoS/DG1 or rExoS per ml.

Th1 and Th2 cytokines. T-cell mitogens and superantigens are capable of altering or polarizing the Th1 or Th2 cytokine profile which can have a significant role in the pathogenesis of infection (23, 26, 31). Since exoenzyme S is a T-cell mitogen, we investigated whether T-cell cytokines were induced. A positive response was defined as a >2-fold increase in the intensity compared to unstimulated PBMC ([i.e., stimulated cytokine band intensity/stimulated L32 band intensity]/[unstimulated cytokine band intensity/unstimulated L32 band intensity]). Only those genes that were detected are displayed in tabular form (Tables 1 and 2). Neither ExoS/DG1 nor rExoS induced transcription of IL-4 or IL-5 (Fig. 1 and Table 1). Modest induction of Th1 type cytokines (IL-2 and gamma interferon [IFN-]) was seen (Fig. 1 and Table 1), which was confirmed by reverse transcription-PCR (RT-PCR) (data not shown). We were somewhat surprised by the relative lack of induction, since exoenzyme S causes rapid expression of high levels of CD69 on a large percentage of T cells, which reflects substantial activation (5, 7). By contrast, both ExoS/DG1 and rExoS strongly induced transcription of the proinflammatory cytokines IL-1 and IL-1 that peaked between 5 and 24 h and declined after 5 days. Since strong induction of proinflammatory cytokines and a vigorous inflammatory response characterize the response in CF patients infected with P. aeruginosa (11), this led us to hypothesize that exoenzyme S preferentially induces proinflammatory cytokines. Thus, we examined the ability of exoenzyme S to induce proinflammatory cytokines and chemokines.

View larger version (113K): [in this window] [in a new window]   FIG. 1.   Preferential induction of Th1 cytokines from PBMC by stimulation with exoenzyme S. Resting PBMC were either unstimulated (U), stimulated with 1 µg of ExoS/DG1 per ml (D), or stimulated with 1 µg of rExoS per ml (R) for various times. Undigested fragments were used as markers to determine the identity of protected fragments. Densitometric values are given in Table 1. The experiment was repeated twice with similar results.

Proinflammatory cytokine induction by exoenzyme S. Together with IL-1 and IL-1, ExoS/DG1 and rExoS induced rapid transcription of additional proinflammatory cytokines including TNF- and IL-6 (Fig. 2A and Table 1). While both TNF- and IL-6 peaked at 5 h, transcription of IL-6 decreased significantly by day 1 and transcription of TNF- decreased gradually until day 5. The ExoS/DG1- and rExoS-induced cytokine profile was indicative of a proinflammatory stimulus more so than a polarized Th1-Th2 response with the induction of proinflammatory cytokines such as TNF-, IL-1, IL-1, and IL-6. These cytokines mediate inflammatory responses through a variety of mechanisms, including the activation and recruitment of numerous cell types (9, 19).

View larger version (95K): [in this window] [in a new window]   FIG. 2.   Exoenzyme S induces transcription of proinflammatory cytokine (A) and chemokine (B) mRNA. Resting PBMC were either unstimulated (U), stimulated with 1 µg of ExoS/DG1 per ml (D), or with 1 µg of rExoS per ml (R) for various times. Undigested fragments were used as markers to determine the identity of protected fragments. Densitometric values are given in Tables 1 and 2. The experiment was repeated twice with similar results.

View larger version (24K): [in this window] [in a new window]   FIG. 3.   LPS contamination is not responsible for cytokine upregulation. PBMC were stimulated in RPMI culture media for 3 h. PBMC were either unstimulated (Unst), stimulated with 10 µg of P. aeruginosa per ml (LPS), or stimulated with 1 µg of ExoS/DG1 per ml (DG1) with or without 10 µg of polymyxin B per ml. PBMC were collected and lysed, and the RNA was extracted. RT was performed using random hexamer primers. PCR was performed for 26 cycles using TNF--specific and GAPDH-specific primers. The experiment was repeated twice with similar results.