Bronchial epithelial injury in the context of alloimmunity promotes lymphocytic bronchiolitis through hyaluronan expression

Abstract

Epithelial injury is often detected in lung allografts, however, its relation to rejection pathogenesis is unknown. We hypothesized that sterile epithelial injury can lead to alloimmune activation in the lung. We performed adoptive transfer of mismatched splenocytes into recombinant activating gene 1 (Rag1)-deficient mice to induce an alloimmune status and then exposed these mice to naphthalene to induce sterile epithelial injury. We evaluated lungs for presence of alloimmune lung injury, endoplasmic reticulum (ER) stress, and hyaluronan expression, examined the effect of ER stress induction on hyaluronan expression and lymphocyte trapping by bronchial epithelia in vitro, and examined airways from patients with bronchiolitis obliterans syndrome and normal controls histologically. We found that Rag1-deficient mice that received mismatched splenocytes and naphthalene injection displayed bronchial epithelial ER stress, peribronchial hyaluronan expression, and lymphocytic bronchitis. Bronchial epithelial ER stress led to the expression of lymphocyte-trapping hyaluronan cables in vitro. Blockade of hyaluronan binding ameliorated naphthalene-induced lymphocytic bronchitis. ER stress was present histologically in >40% of bronchial epithelia of BOS patients and associated with subepithelial hyaluronan deposition. We conclude that sterile bronchial epithelial injury in the context of alloimmunity can lead to sustained ER stress and promote allograft rejection through hyaluronan expression.

Keywords: endoplasmic reticulum stress; lung rejection.

Fig. 1.

Histological evaluation of alloimmune adoptive transfer (allo-AT) lungs after naphthalene exposures. A and B: one week after exposure, there is evidence of epithelial injury in naphthalene-treated mice (A, inset: higher-magnification image of boxed-in area) but not corn oil-treated mice (B). C and D: 3 wk after exposure, naphthalene-treated mice show peribronchial infiltration (C), and corn oil-treated mice show infiltration around vessels (D). Magnification: A and B, ×4 (insets: ×20); C and D, ×2, HE staining.

Fig. 2.

Histological evaluation of allo-AT lungs after naphthalene exposures. A and B: 3 wk after exposure, corn oil-treated mice show infiltration around vessels (A), and naphthalene-treated mice show peribronchial infiltration (B). C: higher-power magnification of a naphthalene-treated allo-AT mouse lung, demonstrating infiltration of epithelia by immune cells, which morphologically appear lymphocytic. D: peribronchial fibrosis in a mouse that received naphthalene two times after adoptive transfer. Immunohistochemically, there is little or no peribronchial staining for CD3 in corn oil-treated mice (E), but clearly visible CD3⁺ cells in naphthalene-treated mice (F). Panels on bottom show higher-magnification images of boxed-in areas in E and F. G: semiquantitative evaluation of infiltration score over time after exposure. There is similar and not progressive perivascular infiltration between naphthalene- and corn oil-treated mice (left), but statistically significant and sustained increase in peribronchial CD3 infiltration in naphthalene-treated mice only (right). N = 12–20 mice/group. Magnification: A and B, ×10; C, ×40, HE staining; D, ×20, Masson-Trichrome staining; E and F, ×4 (top) and ×20 (bottom), CD3 staining.

Fig. 3.

α-Smooth muscle actin (α-SMA) staining around airways is sparse and discontinuous in corn-oil-treated mice (A, arrows) but much more pronounced and expanded in naphthalene-treated mice (B, arrowheads). Magnification: ×4.

Fig. 4.

A: immunohistochemical staining of allo-AT lungs for endoplasmic reticulum (ER) stress with C/EBP homology protein (CHOP, green) and activating transcription factor (ATF)-6 (red). There is significant (but not universal) positive staining in naphthalene-treated mice that is statistically more than in corn oil-treated AT-mice and non-AT naphthalene-treated mice. N = 12–20 mice/group. Magnification: ×20. B: quantitative RT-PCR shows statistically significant upregulation of ER stress genes ATF-4 and CHOP. N = 15–20 mice/group. C: hyaluronan expression in allo-AT mouse lungs. Naphthalene-treated allo-AT lungs demonstrate significant deposition of hyaluronan and its binding partner IαI (top). Note the nuclear density in DAPI staining that colocalizes with the hyaluronan area. There is no increase of hyaluronan expression over baseline in corn oil-treated lungs (bottom). Magnification: ×10. D: allo-AT mice demonstrate a sustained increased in mRNA expression of all three hyaluronan synthases (HAS) over 3 wk (top), whereas iso-AT mouse lungs demonstrate an increase in HAS expression after naphthalene expression, but only in the 1st wk after exposure (bottom). N = 4/group.

Fig. 5.

A: in vitro ER stress induction in BEAS-2B cells leads to upregulation of HAS1 and HAS2 (top), and deposition of hyaluronan (in green, bottom), which appears to partly form cable-like structures. *P < 0.05 compared with control-treated cells. Eight experimental repeats per exposure. B: coculture with ER-stressed BEAS-2B cells (previously exposed to tunicamycin for 24 h) leads to upregulation of HAS enzymes in MRC-5 cells. C: hyaluronan cables (green) trap lymphocytes (visible from blue nuclear DAPI staining). The lymphocytes can be found on net-like hyaluronan structures (top) or form beads-on-a-string structures. D: quantification of trapped lymphocytes shows the positive effect of tunicamycin, which is abolished by treatment with hyaluronidase. Eight experimental repeats per exposure.

Fig. 6.

A: hyaluronan binding blockade ameliorated lymphocytic bronchitis in the naphthalene model. Pep-1-treated mice had significant reduction in peribronchial inflammation (left) compared with scrambled peptide-treated mice (right). B: quantification of inflammatory score. There is no difference in perivascular immune infiltrates by treatment, but pep-1 reduces the peribronchial infiltration score. N = 6–8/group.

Fig. 7.

A: evidence for ER stress in an airway that is affected by bronchiolitis obliterans. The immunofluorescent staining depicts the boxed-in area of the airway highlighted in Masson-Trichrome staining. B: quantification of ER stress in bronchiolitis obliterans-afflicted airways. A: some airways demonstrate almost continuous positive staining for CHOP (top left, in red). Other airways show skip-pattern lesions (top right). Negative control (rejected transplant lung) shows no staining (bottom left). Magnification: ×40. Quantification shows a significant difference in CHOP/ATF6-positive staining (bottom right). N = 4–6/group. C: consecutive sections of a scarified airway (Masson-trichrome and hematoxylin-eosin, top) stain strongly positive for hyaluronan (bottom left, brown) and IαI (bottom right, orange) in the subepithelial area and scar tissue. Magnification: ×10.