Down-modulation of lung immune responses by interleukin-10 and transforming growth factor beta (TGF-beta) and analysis of TGF-beta receptors I and II in active tuberculosis

Abstract

Immune factors influencing progression to active tuberculosis (TB) remain poorly defined. In this study, we investigated the expression of immunoregulatory cytokines and receptors by using lung bronchoalveolar lavage cells obtained from patients with pulmonary TB, patients with other lung diseases (OLD patients), and healthy volunteers (VOL) by using reverse transcriptase PCR, a transforming growth factor beta (TGF-beta) bioactivity assay, and an enzyme immunoassay. TB patients were significantly more likely than OLD patients to coexpress TGF-beta receptor I (RI) and RII mRNA, as well as interleukin-10 (IL-10) mRNA (thereby indicating the state of active gene transcription in the alveolar cells at harvest). In contrast, gamma interferon (IFN-gamma) and IL-2 mRNA was seen in both TB and OLD patients. Likewise, significantly elevated pulmonary steady-state protein levels of IL-10, IFN-gamma, and bioactive TGF-beta were found in TB patients versus those in OLD patients and VOL. These data suggest that the combined production of the immunosuppressants IL-10 and TGF-beta, as well as coexpression of TGF-beta RI and RII (required for cellular response to TGF-beta), may act to down-modulate host anti-Mycobacterium tuberculosis immunity and thereby allow uncontrolled bacterial replication and overt disease. Delineating the underlying mechanisms of M. tuberculosis-triggered expression of these immune elements may provide a molecular-level understanding of TB immunopathogenesis.

FIG. 1.

The BAL cells of TB patients express transcripts for IL-2, IFN-γ, and IL-10, as well as TGF-β RI and RII. Illustrated are the autoradiographic blot analyses of RT-PCR products for IL-2, IFN-γ, IL-10, TGF-β RI, and TGF-β RII, as well as the ethidium bromide-stained RT-PCR β-actin amplicons, from BAL cells obtained from active-TB patients (TB; n = 16) or control patients with OLD (OLD; n = 6). The amounts of input cDNA from cytokine and cytokine receptor RT-PCR were each normalized to the ratio of the sample's β-actin amplicon density to the mean β-actin amplicon density (see Materials and Methods). Amplicons were electrophoresed, transferred onto a nylon membrane, hybridized with specific ³²P-labeled probes, and exposed to X-ray film. The number above each lane corresponds to a patient code. The expected band sizes are given at right. Water (H₂O) was loaded as the negative control. Thirteen patients were studied for NOS2 by RT-PCR and Southern analysis, and all except three patients (numbers 5, 6, and 34) had NOS2 transcripts detected. NOS2 expression in 11 of these patients (numbers 2, 3, 5, 6, 27, 30, 34, 35, 44, R2, and R3) was previously reported (37). Patients 13 and 18 were also positive for NOS2 (data not shown). Patients R2 and R3 were from New York and all others were from Brazil. Fourteen patients with TB (numbers 2, 3, 5, 6, 13, 18, 23, 27, 30, 34, 35, 44, 48, and 58) and six patients with OLD (numbers 43, 46, 47, 56, 57, and 59) had both RT-PCR assays and TGF-β bioassays performed (see the text). IL-10 was also assayed by both RT-PCR and EIA for two TB patients (numbers 44 and 58) and two patients with OLD (numbers 46 and 59) (see the text). This image was created using Adobe PhotoDeluxe Home Edition 3.1.

FIG. 2.

TGF-β activity is elevated in the BAL fluids of TB patients. Active and neutralizable TGF-β activity from the lavage fluids (concentrated by molecular sieve centrifugation) of TB patients (TB; n = 24), controls with OLD (OLD; n = 25), and VOL (n = 8) was detected by a bioassay that evaluates TGF-β-induced proliferation inhibition of the Mv1Lu mink lung epithelial cell line. The amount of TGF-β was extrapolated from a standard curve by using recombinant human TGF-β1, and the specific TGF-β activity was established in parallel by using antisera with neutralizing activity against TGF-β1, TGF-β2, and TGF-β3. Specific TGF-β activity was calculated as the difference between results for the no-antisera and the neutralizing antisera conditions. The results of statistical analyses of the data by the unpaired two-tailed t test are shown. TGF-β levels determined by bioassay were comparatively examined within the TB group by three separate categorical segregations: cavitary versus noncavitary disease, extensive (two or more lobes affected) versus limited (one lobe affected) disease, and TB diagnosis based upon AFB culture positivity versus diagnosis based upon clinical criteria (as given in Table 1). No differences were seen in protein levels of TGF-β among the segregated TB subgroupings.

FIG. 3.

Levels of IFN-γ and IL-10 are elevated in the BAL fluids from TB patients. Lung lavage fluids from TB patients (TB; n = 10), controls with OLD (OLD; n = 10), and VOL (n = 8) were assayed for both IFN-γ and IL-10 by commercial EIA. The standard-error bars, as well as the results of statistical analyses of the data by the unpaired one-tailed t test, are shown.