Background Human mutations of the coatomer coat protein alpha subunit (COPA) affect retrograde Golgi-to-endoplasmic reticulum (ER) protein transport, resulting in endoplasmic reticulum (ER) stress and a clinical syndrome consisting of polyarthritis and diffuse alveolar hemorrhage (DAH) with autoimmune features. Some SLE patients also develop DAH and C57BL/6 (B6) mice with pristane-induced lupus develop monocyte-dependent DAH closely resembling the human disorder. In contrast, BALB/c mice are resistant to DAH. We examined the role of COPA and ER stress in lupus mice with DAH.

Methods B6 and BALB/c mice were treated with pristane. Expression of Copa and markers of ER stress and vascular injury were assessed by quantitative PCR and immunohistochemistry.

Lung tissue was disrupted by Gentle MACS and ER stress was assessed in CD45+CD146- bone marrow-derived cells and CD45-CD146+ lung microvascular endothelial cells by flow cytometry. COPA transcripts were quantified in peripheral blood from 54 SLE patients and 22 controls.

Results DAH in B6 mice was associated with impaired Copa mRNA and protein expression and evidence of ER stress (increased Ddit3 and CHOP protein, Hspa5 and BiP protein, and other markers). Although DAH did not develop in BALB/c mice treated with either pristane or the ER stress inducer thapsigargin, DAH with impaired Copa expression and evidence of ER stress was induced when BALB/c mice were treated with pristane plus low dose thapsigargin. (BALB/c X B6)F1 mice did not develop DAH or ER stress, suggesting that susceptibility was recessively inherited. Flow cytometry of single cell suspensions of lung tissue revealed increased expression of the ER stress protein BiP in CD45-CD146+ pulmonary endothelial cells and CD45+CD146- myeloid cells from pristane-treated B6 mice and also in CD45- cells from BALB/c mice treated with pristane + thapsigargin. von Willebrand factor (Vwf), a marker of endothelial injury, and the monocyte-attractive chemokine Ccl2 increased in lung from B6 mice with DAH, but not in lung from BALB/c mice treated with pristane (without thapsigargin). These data suggest that pristane- induced ER stress and impaired Copa expression promote lung microvascular injury (indicated by increased Vwf expression) and the production of monocyte-attractive chemokines, such as Ccl2. COPA expression also was low in SLE patients with interstitial lung disease or nephritis, suggesting that increased susceptibility to ER stress and impaired retrograde Golgi-to-ER transport also may be associated with a subset of human lupus.

Conclusion DAH in a mouse lupus model appears to be initiated by genetically-regulated susceptibility of the lung microvasculature endothelium to pristane-induced injury, resulting in an ER stress response and culminating in a monocyte-dependent inflammatory response. Lupus- associated DAH in mice and possibly humans may represent an acquired form of COPA syndrome.

Acknowledgments This work was supported by the National Institutes of Health (NIAMS) grant number R01-AR44731.

Lay summary Lung hemorrhage is a severe complication of SLE. Patients with mutations affecting intracellular protein transport (“COPA syndrome”) also develop lung hemorrhage along with other clinical features of reminiscent of lupus (arthritis, autoantibodies, and abnormal regulation of interferon production). Using a mouse model, we found that lung hemorrhage in lupus replicates some of the clinical features of COPA syndrome, including low expression levels of COPA mRNA and protein and evidence of a stress response in lung microvascular endothelial cells. Additionally, we found that COPA expression was low in a subset of SLE patients, raising the possibility that these individuals might be at increased risk to develop lung hemorrhage.