Chemokine receptors recruit U0126 order leukocytes to the alveolar site of inflammation and orchestrate local immune responses. In the previous studies we demon strated that, among a plethora of chemokine receptors involved in this network, specifically CCR2 on lympho cytes and CXCR1 on neutrophils, modulate pulmonary immunity in human inflammatory lung diseases. Therefore, we examined whether cells expressing SP CI73T stimulated the expression of CCR2 on lymphocytes and CXCR1 on neutrophils by incubating isolated neu trophils or lymphocytes with 7 fold concentrated super natants of MLE 12 cells expressing WT or I73T SP C. As a result, CD8 lymphocytes did not show a difference between WT and I73T mutant, however CD4 lympho cytes showed an increased level of surface receptor CCR2 expression in response to the supernatant of proSP CI73T expressing cells.

We observed the same pat tern with CXCR1, which was increased on CD4 lym phocytes after incubation with the mutant cell supernatant, but was unaltered on CD8 lymphocytes. We further analyzed the surface receptor expression on neutrophils. The supernatant of SP CI73T expressing cells increased the level of CXCR1 expression on neutrophils, but did not affect CD11b levels. Non concentrated supernatants gave the same results, although less pronounced and a clear concentra tion dependency of the effects was observed. This suggests that SP CI73T expressing MLE 12 cells were able to modulate the surface receptor expres sion on the cells of immune system through the secretion of soluble factors into the medium.

Discussion Mutations in the SFTPC gene are a known cause of sur factant deficiency and very variable genetic ILD in chil dren and adults. We investigated the intracellular disturbances and intercellular signaling of MLE 12 cells expressing SP CI73T and the ability of pharmaceutical drugs used in ILD therapy to modulate some of the cel lular consequences of SP C deficiency caused by this mutation. MLE 12 cells were chosen as a model system since they contain structures, which resemble lamellar bodies seen in AECII. The presence of lamellar body like structures in the cells used was confirmed by electron microscopy. Here we named the organelles detectable as LAMP3 positive vesicles, lamellar body like structures. A potential limitation of the study is that our system corresponds rather to a homozygous than to a heterozy gous SFTPC mutation where one WT copy is still pre sent. Although endogenous SP C is expressed in the MLE 12 cells, expression of exogenous SP C from the CMV promoter present on the plasmid vector is likely higher. However, all known patients with SP C mutations are heterozygous, expressing one copy of the wild type gene. GSK-3 Thus, the experimental model reflects the in vivo condition.