Latent TGF-beta1 associated with SP-A: Possible novel mechanism to regulate TGF-beta1 mediated inflammation and fibrosis in the lung

Surfactant protein A (SP-A) and active transforming growth factor beta (TGF-beta) 1 have been shown to modulate the functions of various immune cells in the lung. TGF-beta1 is released by cells as latent, biologically inactive complexes. After an activation step TGF-beta1 signaling from the cell membrane to the nucleus is mediated through Smad2/3 proteins. The signaling pathway of SP-A is however unknown. The aim of the present study was to elucidate if the Smad signaling pathway plays also a role in mediating the immune modulation of SP-A.

Primary human CD4+ T lymphocytes were transfected with a Smad3 signaling specific reporter gene using the nucleofection technology. The transfected cells were then treated with SP-A, obtained from patients with alveolar proteinosis. SP-A had no effect on Smad signaling. After acidification of SP-A samples, changing pH to 2.0, “activated“ SP-A enhanced the reporter gene activity in a dose dependent manner. Maximal reporter gene activation was 3.0±0.3 following addition of acid “activated“ SP-A compared with control. Furthermore western blot analysis showed Smad2 phosphorylation by acid “activated“ SP-A. The effect of acid “activated“ SP-A could be specifically antagonized by neutralising antibodies against TGF-beta1 or a soluble receptor for TGF-beta (sTGF-betaRII). SP-A samples were analyzed for TGF-beta1 by ELISA. Acidification of SP-A samples increased TGF-beta1 concentration 18.5-fold (before acidification: 0.2±0.05 pg TGF-beta1/ug SP-A; after acidification: 3.7±0.3 pg TGF-beta1/ug SP-A). Acid “activated“ SP-A could induce Smad signaling independent of the used purification system for SP-A (mannose affinity and anion exchange chromatography). In order to examine if “activation“ of SP-A had an influence on the inhibition of human CD4+ T lymphocyte proliferation, CD4+ T lymphocytes were stimulated with anti-CD3. The addition of non acid “activated“ SP-A resulted in a small decrease in proliferation (-21±4%), while acid “activated“ SP-A decreased proliferation to -60±6%. The inhibitory effect of acid “activated“ SP-A could be antagonized by anti-TGF-beta1 and sTGF-betaRII.

These results demonstrate that SP-A most likely binds latent TGF-beta1. We speculate that the inhibitory effect of acid “activated“ SP-A on CD4+ T lymphocyte proliferation may be – at least in part – due to TGF-beta1. The possible ability of SP-A to associate with latent TGF-beta1 could permit the storage of this cytokine in the lung. Inflammation associated changes in the homeostasis of the lung could activate latent TGF-beta1 bound to SP-A. The storage capability of SP-A for latent TGF-beta1 could be a novel mechanism to regulate TGF-beta1 mediated inflammation and fibrosis reactions in the lung.