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The surfactant protein C mutation A116D alters cellular processing, stress tolerance, surfactant lipid composition, and immune cell activation.

Zarbock R, Woischnik M, Sparr C, Thurm T, Kern S, Kaltenborn E, Hector A, Hartl D, Liebisch G, Schmitz G, Griese M - BMC Pulm Med (2012)

Bottom Line: Furthermore, SP-CA116D cells secreted soluble factors into the medium that modulated surface expression of CCR2 or CXCR1 receptors on CD4+ lymphocytes and neutrophils, suggesting a direct paracrine effect of SP-CA116D on neighboring cells in the alveolar space.In addition, we show that some of the effects of the mutation on cellular homeostasis can be antagonized by application of pharmaceuticals commonly applied in ILD therapy.Our findings shed new light on the pathomechanisms underlying SP-C deficiency associated ILD and provide insight into the mechanisms by which drugs currently used in ILD therapy act.

View Article: PubMed Central - HTML - PubMed

Affiliation: Childrens' Hospital of the Ludwig-Maximilians-University, Lindwurmstr, 4, 80337 Munich, Germany.

ABSTRACT

Background: Surfactant protein C (SP-C) is important for the function of pulmonary surfactant. Heterozygous mutations in SFTPC, the gene encoding SP-C, cause sporadic and familial interstitial lung disease (ILD) in children and adults. Mutations mapping to the BRICHOS domain located within the SP-C proprotein result in perinuclear aggregation of the proprotein. In this study, we investigated the effects of the mutation A116D in the BRICHOS domain of SP-C on cellular homeostasis. We also evaluated the ability of drugs currently used in ILD therapy to counteract these effects.

Methods: SP-CA116D was expressed in MLE-12 alveolar epithelial cells. We assessed in vitro the consequences for cellular homeostasis, immune response and effects of azathioprine, hydroxychloroquine, methylprednisolone and cyclophosphamide.

Results: Stable expression of SP-CA116D in MLE-12 alveolar epithelial cells resulted in increased intracellular accumulation of proSP-C processing intermediates. SP-CA116D expression further led to reduced cell viability and increased levels of the chaperones Hsp90, Hsp70, calreticulin and calnexin. Lipid analysis revealed decreased intracellular levels of phosphatidylcholine (PC) and increased lyso-PC levels. Treatment with methylprednisolone or hydroxychloroquine partially restored these lipid alterations. Furthermore, SP-CA116D cells secreted soluble factors into the medium that modulated surface expression of CCR2 or CXCR1 receptors on CD4+ lymphocytes and neutrophils, suggesting a direct paracrine effect of SP-CA116D on neighboring cells in the alveolar space.

Conclusions: We show that the A116D mutation leads to impaired processing of proSP-C in alveolar epithelial cells, alters cell viability and lipid composition, and also activates cells of the immune system. In addition, we show that some of the effects of the mutation on cellular homeostasis can be antagonized by application of pharmaceuticals commonly applied in ILD therapy. Our findings shed new light on the pathomechanisms underlying SP-C deficiency associated ILD and provide insight into the mechanisms by which drugs currently used in ILD therapy act.

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Surface receptor expression on human lymphocytes and neutrophils. Neutrophils and lymphocytes were isolated from the whole blood of different human donors and incubated with 7-fold concentrated supernatants obtained from MLE-12 cells expressing SP-CWT or SP-CA116D prior to flow cytometry analysis. Non-concentrated supernatants gave the same results, although less pronounced with a clear concentration dependency of the effects (data not shown). The receptor levels on the surface of lymphocytes after incubation with antibodies directed against (A) CCR2 or (B) CXCR1 are shown and expressed as mean fluorescence intensity (MFI). Another second marker-specific antibody was applied to distinguish between CD4+ and CD8+ lymphocytes. (C) The levels of CXCR1 and CD11b on isolated neutrophils. Significant changes are depicted with the corresponding p-values.
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Figure 6: Surface receptor expression on human lymphocytes and neutrophils. Neutrophils and lymphocytes were isolated from the whole blood of different human donors and incubated with 7-fold concentrated supernatants obtained from MLE-12 cells expressing SP-CWT or SP-CA116D prior to flow cytometry analysis. Non-concentrated supernatants gave the same results, although less pronounced with a clear concentration dependency of the effects (data not shown). The receptor levels on the surface of lymphocytes after incubation with antibodies directed against (A) CCR2 or (B) CXCR1 are shown and expressed as mean fluorescence intensity (MFI). Another second marker-specific antibody was applied to distinguish between CD4+ and CD8+ lymphocytes. (C) The levels of CXCR1 and CD11b on isolated neutrophils. Significant changes are depicted with the corresponding p-values.

Mentions: We hypothesized that accumulation of misfolded SP-C might somehow lead to an enhanced accumulation of leukocytes and thus boost the inflammatory reaction. To test this hypothesis, we examined whether cells expressing SP-CA116D stimulated the expression of CCR2 on lymphocytes and CXCR1 on neutrophils by incubating isolated neutrophils or lymphocytes with 7-fold concentrated supernatants of MLE-12 cells expressing either WT or A116D SP-C. While no difference in surface receptor expression between WT and mutant was observed in CD8+ lymphocytes, CD4+ lymphocytes showed a highly significant increase in the level of surface receptor CCR2 expression in response to the supernatant of SP-CA116D expressing cells (Figure 6A). The same was true in the case of CXCR1, which was increased on CD4+ lymphocytes after incubation with the mutant cell supernatant, but remained unaltered on CD8+ lymphocytes (Figure 6B). We further analyzed the surface receptor expression on neutrophils. The supernatant of cells expressing SP-CA116D increased CXCR1 expression on neutrophils, but did not affect CD11b levels (Figure 6C). Non-concentrated supernatants gave the same results by tendency, although less pronounced. A clear concentration dependency of the effects was observed (data not shown). This suggests that SP-CA116D-expressing MLE-12 cells were able to modulate the surface receptor expression on the cells of immune system through the secretion of soluble factors into the medium.


The surfactant protein C mutation A116D alters cellular processing, stress tolerance, surfactant lipid composition, and immune cell activation.

Zarbock R, Woischnik M, Sparr C, Thurm T, Kern S, Kaltenborn E, Hector A, Hartl D, Liebisch G, Schmitz G, Griese M - BMC Pulm Med (2012)

Surface receptor expression on human lymphocytes and neutrophils. Neutrophils and lymphocytes were isolated from the whole blood of different human donors and incubated with 7-fold concentrated supernatants obtained from MLE-12 cells expressing SP-CWT or SP-CA116D prior to flow cytometry analysis. Non-concentrated supernatants gave the same results, although less pronounced with a clear concentration dependency of the effects (data not shown). The receptor levels on the surface of lymphocytes after incubation with antibodies directed against (A) CCR2 or (B) CXCR1 are shown and expressed as mean fluorescence intensity (MFI). Another second marker-specific antibody was applied to distinguish between CD4+ and CD8+ lymphocytes. (C) The levels of CXCR1 and CD11b on isolated neutrophils. Significant changes are depicted with the corresponding p-values.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3376036&req=5

Figure 6: Surface receptor expression on human lymphocytes and neutrophils. Neutrophils and lymphocytes were isolated from the whole blood of different human donors and incubated with 7-fold concentrated supernatants obtained from MLE-12 cells expressing SP-CWT or SP-CA116D prior to flow cytometry analysis. Non-concentrated supernatants gave the same results, although less pronounced with a clear concentration dependency of the effects (data not shown). The receptor levels on the surface of lymphocytes after incubation with antibodies directed against (A) CCR2 or (B) CXCR1 are shown and expressed as mean fluorescence intensity (MFI). Another second marker-specific antibody was applied to distinguish between CD4+ and CD8+ lymphocytes. (C) The levels of CXCR1 and CD11b on isolated neutrophils. Significant changes are depicted with the corresponding p-values.
Mentions: We hypothesized that accumulation of misfolded SP-C might somehow lead to an enhanced accumulation of leukocytes and thus boost the inflammatory reaction. To test this hypothesis, we examined whether cells expressing SP-CA116D stimulated the expression of CCR2 on lymphocytes and CXCR1 on neutrophils by incubating isolated neutrophils or lymphocytes with 7-fold concentrated supernatants of MLE-12 cells expressing either WT or A116D SP-C. While no difference in surface receptor expression between WT and mutant was observed in CD8+ lymphocytes, CD4+ lymphocytes showed a highly significant increase in the level of surface receptor CCR2 expression in response to the supernatant of SP-CA116D expressing cells (Figure 6A). The same was true in the case of CXCR1, which was increased on CD4+ lymphocytes after incubation with the mutant cell supernatant, but remained unaltered on CD8+ lymphocytes (Figure 6B). We further analyzed the surface receptor expression on neutrophils. The supernatant of cells expressing SP-CA116D increased CXCR1 expression on neutrophils, but did not affect CD11b levels (Figure 6C). Non-concentrated supernatants gave the same results by tendency, although less pronounced. A clear concentration dependency of the effects was observed (data not shown). This suggests that SP-CA116D-expressing MLE-12 cells were able to modulate the surface receptor expression on the cells of immune system through the secretion of soluble factors into the medium.

Bottom Line: Furthermore, SP-CA116D cells secreted soluble factors into the medium that modulated surface expression of CCR2 or CXCR1 receptors on CD4+ lymphocytes and neutrophils, suggesting a direct paracrine effect of SP-CA116D on neighboring cells in the alveolar space.In addition, we show that some of the effects of the mutation on cellular homeostasis can be antagonized by application of pharmaceuticals commonly applied in ILD therapy.Our findings shed new light on the pathomechanisms underlying SP-C deficiency associated ILD and provide insight into the mechanisms by which drugs currently used in ILD therapy act.

View Article: PubMed Central - HTML - PubMed

Affiliation: Childrens' Hospital of the Ludwig-Maximilians-University, Lindwurmstr, 4, 80337 Munich, Germany.

ABSTRACT

Background: Surfactant protein C (SP-C) is important for the function of pulmonary surfactant. Heterozygous mutations in SFTPC, the gene encoding SP-C, cause sporadic and familial interstitial lung disease (ILD) in children and adults. Mutations mapping to the BRICHOS domain located within the SP-C proprotein result in perinuclear aggregation of the proprotein. In this study, we investigated the effects of the mutation A116D in the BRICHOS domain of SP-C on cellular homeostasis. We also evaluated the ability of drugs currently used in ILD therapy to counteract these effects.

Methods: SP-CA116D was expressed in MLE-12 alveolar epithelial cells. We assessed in vitro the consequences for cellular homeostasis, immune response and effects of azathioprine, hydroxychloroquine, methylprednisolone and cyclophosphamide.

Results: Stable expression of SP-CA116D in MLE-12 alveolar epithelial cells resulted in increased intracellular accumulation of proSP-C processing intermediates. SP-CA116D expression further led to reduced cell viability and increased levels of the chaperones Hsp90, Hsp70, calreticulin and calnexin. Lipid analysis revealed decreased intracellular levels of phosphatidylcholine (PC) and increased lyso-PC levels. Treatment with methylprednisolone or hydroxychloroquine partially restored these lipid alterations. Furthermore, SP-CA116D cells secreted soluble factors into the medium that modulated surface expression of CCR2 or CXCR1 receptors on CD4+ lymphocytes and neutrophils, suggesting a direct paracrine effect of SP-CA116D on neighboring cells in the alveolar space.

Conclusions: We show that the A116D mutation leads to impaired processing of proSP-C in alveolar epithelial cells, alters cell viability and lipid composition, and also activates cells of the immune system. In addition, we show that some of the effects of the mutation on cellular homeostasis can be antagonized by application of pharmaceuticals commonly applied in ILD therapy. Our findings shed new light on the pathomechanisms underlying SP-C deficiency associated ILD and provide insight into the mechanisms by which drugs currently used in ILD therapy act.

Show MeSH
Related in: MedlinePlus