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. 2008 Oct 15;181(8):5738-47.
doi: 10.4049/jimmunol.181.8.5738.

Anti-type V collagen humoral immunity in lung transplant primary graft dysfunction

Takekazu Iwata  1 , Alexander Philipovskiy, Amanda J Fisher, Robert G Presson Jr, Masako Chiyo, Jae Lee, Elizabeth Mickler, Gerald N Smith, Irina Petrache, David B Brand, William J Burlingham, Bagavathi Gopalakrishnan, Daniel S Greenspan, Jason D Christie, David S Wilkes
Affiliations

Anti-type V collagen humoral immunity in lung transplant primary graft dysfunction

Takekazu Iwata et al. J Immunol. .

Abstract

Primary graft dysfunction (PGD) is a major complication following lung transplantation. We reported that anti-type V collagen (col(V)) T cell immunity was strongly associated with PGD. However, the role of preformed anti-col(V) Abs and their potential target in PGD are unknown. Col(V) immune serum, purified IgG or B cells from col(V) immune rats were transferred to WKY rat lung isograft recipients followed by assessments of lung pathology, cytokines, and PaO(2)/FiO(2), an index of lung dysfunction in PGD. Immune serum, purified IgG, and B cells all induced pathology consistent with PGD within 4 days posttransfer; up-regulated IFN-gamma, TNF-alpha, and IL-1beta locally; and induced significant reductions in PaO(2)/FiO(2). Depleting anti-col(V) Abs before transfer demonstrated that IgG2c was a major subtype mediating injury. Confocal microscopy revealed strong apical col(V) expression on lung epithelial, but not endothelial cells; which was consistent with the ability of col(V) immune serum to induce complement-dependent cytotoxicity only in the epithelial cells. Examination of plasma from patients with or without PGD revealed that higher levels of preformed anti-col(V) Abs were strongly associated with PGD development. This study demonstrates a major role for anti-col(V) humoral immunity in PGD, and identifies the airway epithelium as a target in PGD.

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Figures

FIGURE 1
FIGURE 1
Detection of systemic anti-col(V) or anti-HEL Abs. Serum was collected from rats there were immunized with col(V) or HEL and incubated with Sepharose beads coated with col(V) or HEL. Flow cytometry was used to detect IgG Abs bound to col(V) or HEL-coated beads. Data shown are derived from serum collected from an individual rat immunized with col(V) or HEL and are representative of n = 5–6 rats immunized with each Ag.
FIGURE 2
FIGURE 2
Passive transfer of col(V) immune serum or purified IgG, or adoptive transfer of purified B cells from to isograft recipients induces rejection-like pathology in isograft lungs. Col(V) or HEL immune serum or purified IgG or B cells from col(V)-immunized rats were isolated and transferred to lung isograft recipients and lung tissues collected posttransfer as described in Materials and Methods. A, Passive transfer of col(V) immune serum. B, Passive transfer of IgG purified from col(V) immune serum. C, Adoptive transfer of B cells from col(V) immunized rats. D, Passive transfer of HEL immune serum. E, Passive transfer of IgG purified from HEL immune serum. No pathologic lesions are observed in isograft lungs of rats that received HEL immune sera or IgG purified from HEL-immunized rats (A and B, respectively). Arrows indicate perivascular and peribronchiolar infiltrates (A–C). Data are representative of n = 4–6 rats in each group. Magnification of H&E stained lung sections is ×20.
FIGURE 3
FIGURE 3
Quantitation of IgG subtypes col(V) immune serum. IgG subtypes were quantified in serum from col(V)-immunized rats by ELISA as reported in Materials and Methods. A, IgG subtypes in immune serum. Data representative of mean ± SEM of IgG subtypes present in serum isolated from n = 4 immunized rats. B, IgG subtypes present in IgG-purified from immune serum then incubated with anti-col(V) beads to deplete anti-col(V) Abs. Data derived from serum pooled from n = 4 col(V)-immunized rats. N.D., Not detected.
FIGURE 4
FIGURE 4
PaO2/FIO2 as a function of treatment groups. A, Ratio determined as reported in Materials and Methods for untreated (normal) WKY rats and WKY isograft recipients posttransfer of serum or purified IgG. Data represent mean ± SEM of n = 3–4 rats in each group. *, p < 0.01 and **, p < 0.001. B, Histology of isograft lungs posttransfer of purified IgG depleted of anti-col(V) Abs. Data are representative of n = 3 rats per group. magnification with H&E stain is ×20.
FIGURE 5
FIGURE 5
Cytokine profiles in BAL fluid. Cytokine IFN-γ (A), TNF-α (B), and IL-1β (C) levels in BAL fluid in untreated isograft recipients and isograft recipients passive posttransfer of serum or IgG preparations. Data represent mean ± SEM of n = 3 rats in each group. *, p < 0.001; **, p < 0.01; and ***, p < 0.05. N.d., Not detected.
FIGURE 6
FIGURE 6
Immunohistochemical staining for col(V) in native and isograft lungs. Native and isograft lungs were harvested from WKY rats 4 days posttransplantation and stained for col(V) performed as described in Materials and Methods. Arrows indicate airway epithelium or vascular endothelium. Col(V) staining identified by brown. Data are representative of n = 3 rats per group. Magnification is ×40.
FIGURE 7
FIGURE 7
Confocal microscopy expression of col(V) on apical but not basal surface of airway epithelial cells. Primary rat airway epithelial cells were stained for col(V) (Rhodamine red), type IV collagen (col(IV) FITC, green), and nuclei identified by DAPI (blue) staining. Confocal imaging was used to scan sections from apical (left) to basal (right) surface as shown. Data are representative of three separate experiments with similar results.
FIGURE 8
FIGURE 8
Col(V) immune serum induction of complement-dependent cytotoxicity in airway epithelial cells. After growing to confluence, rat airway epithelial cells were incubated with HEL or col(V) immune serum in the presence or absence complement. After an incubation period, cells were stained with propidium iodide (PI) and analyzed by examined by flow cytometry to detect dead cells (propidium iodide-positive) by side scatter light (SS) analysis. Data are representative of two experiments with similar results.
FIGURE 9
FIGURE 9
Circulating Ab levels in patients with PGD and in non-PGD controls during the first 3 days after lung transplant. Box plots represent medians with intraquartile ranges, with whiskers representing full ranges. *, p ≤ 0.05 for the comparison at each time point between PGD and non-PGD. p < 0.001 for comparisons across all time points.
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