Th-17, monokines, collagen type V, and primary graft dysfunction in lung transplantation

Abstract

Rationale: The pathogenesis of primary graft dysfunction (PGD), a serious complication of lung transplantation, is poorly understood. Human studies and rodent models have shown that collagen type V (col[V]), stimulates IL-17-dependent cellular immunity after lung transplantation.

Objectives: To determine whether patients with end-stage lung disease develop pretransplant col(V)-specific cellular immunity, and if so, the impact of this response on PGD.

Methods: Trans-vivo delayed-type hypersensitivity (TV-DTH) assays were used to evaluate memory T-cell responses to col(V) in 55 patients awaiting lung transplantation. Pa(O(2))/Fi(O(2)) index data were used to assess PGD. Univariate risk factor analysis was performed to identify variables associated with PGD. Rats immunized with col(V) or irrelevant antigen underwent lung isografting to determine if prior anti-col(V) immunity triggers PGD in the absence of alloreactivity.

Measurements and main results: We found that 58.8% (10/17) of patients with idiopathic pulmonary fibrosis, and 15.8% (6/38) of patients without idiopathic pulmonary fibrosis tested while on the wait list for a lung transplant were col(V) DTH positive. Col(V) reactivity was CD4(+) T-cell and monocyte mediated, and dependent on IL-17, IL-1beta, and tumor necrosis factor (TNF)-alpha. Pa(O(2))/Fi(O(2)) indices were impaired significantly 6-72 hours after transplantation in col(V)-reactive versus nonreactive patients. Univariate risk factor analysis identified only preoperative TV-DTH to col(V) and ischemic time as predictors of PGD. Finally, in a rat lung isograft model, col(V) sensitization resulted in significantly lower Pa(O(2))/Fi(O(2)), increased local TNF-alpha and IL-1beta production, and a moderate-to-severe bronchiolitis/vasculitis when compared with control isografts.

Conclusions: The data suggest that activation of innate immunity by col(V)-specific Th-17 memory cells represents a novel pathway to PGD after lung transplantation.

Figures

Figure 1.

Consistency and correlation between peripheral and intrathoracic-derived mononuclear cells for collagen V (col[V]) trans-vivo delayed-type hypersensitivity (TV-DTH). (A) Mean recall response from Epstein-Barr virus (EBV) over all time points was 37. 5 × 10−4 inches, with overall col(V) responses ranging from 22.5 to 40 × 10−4 inches of net swelling. Mean col(V) swelling response over all time points was 30 × 10−4 inches. Collagen type II (col[II]) did not elicit a TV-DTH swelling response. Triangles, EBV; circles, col(V); squares, col(II) (B) Differences in TV-DTH response from peripheral and hilar lymph node–derived mononuclear cells (PBMC and LN) are presented in five col(V) responders and five col(V) nonresponders. Dotted lines denote paired observations for each patient and solid lines represent group means. There were three patients with borderline responses (20 × 10−4 inches) who could not be characterized based on PBMC samples alone, but with LN sampling clearly demarcated their respective TV-DTH status. Closed circles and squares, PBMC; open circles and squares, LN.

Figure 2.

Evidence for differences in pretransplant collagen V (col[V]) trans-vivo delayed-type hypersensitivity (TV-DTH) status by lung disease group. Pretransplant TV-DTH responses to collagen II (col[II]) (circles) or col(V) (squares) in 55 patients with end-stage lung disease and 8 healthy control subjects. The diagnostic category “other” includes those patients defined in Table 1. Responses were averaged from duplicate determinations for each patient including peripheral blood leukocyte and lymph node preparations. Shaded area represents negative TV-DTH values (<25 × 10−4 inches). AAD = α1-antitrypsin disease; CF = cystic fibrosis; COPD = chronic obstructive pulmonary disease IPF = idiopathic pulmonary fibrosis.

Figure 3.

Cytokine and cellular requirements of the trans-vivo delayed-type hypersensitivity (TV-DTH) response to collagen V (col[V]). (A) Cytokine neutralization was conducted by coinjection of blocking antibodies at the time of TV-DTH injection. Isotype control injections had no effect on tetanus toxoid (TT) recall or col(V) TV-DTH responses. Classic TT recall swelling responses were blocked by IFN-γ (P = 0.008); however, this antibody had no effect on col(V) swelling responses. Blockade of either IL-17 (P = 0.004), IL-1β (P = 0.003), or tumor necrosis factor-α (P = 0.001) completely abrogated swelling responses to col(V), but not recall antigen. Solid bars, IgG, control; dark shaded bars, IFN-γ; light shaded bars, α-IL-17; open bars, IL-1β; dotted bars, TNF-α. (B) Cellular depletion studies were performed in response to TT recall antigen and col(V). TT responses were abolished by depletion of CD4+ cells, but were not affected by depletion of CD14+ or CD8+ cells. Col(V) responses were equally abrogated by depletion of either CD4+ or CD14+ cells. Solid bars, peripheral blood leukocytes; dark shaded bars, CD8 depleted; light shaded bars, CD4 depleted; open bars, CD14 depleted; dotted bars, sham depleted.

Figure 4.

PaO2/FiO2 as a function of trans-vivo delayed-type hypersensitivity (TV-DTH) response and disease type. Subgroup analyses based on least squares means with associated SEM. Dotted line represents an index value of 200, the threshold for primary graft dysfunction diagnosis. Functional data presented from collagen V TV-DTH–reactive versus nonreactive patients (n = 33). Statistical analyses were performed via a least squares mean repeated-measures analysis of variance; *P ⩽ 0.05; **P ⩽ 0.01. Open squares, DTH negative (n = 24); solid squares, DTH positive (n = 9).

Figure 5.

Functional, histologic, and biochemical variation among orthotopic rat lung transplant cohorts. (A) Comparison of PaO2/FiO2 index among treatment rat groups, showing severe (P = 0.008) functional decline after collagen V (col[V]) immunization (n = 3) compared with hen egg lysozyme (HEL)–immunized (n = 4) control rats at 72 hours post-transplantation. *P-value significant compared with HEL. (B) Seventy-two-hour histology is also shown. Col(V)-immunized animals have significant mononuclear cell infiltration around small airways and vascular structures consistent with acute rejection (grade 2). Data are representative of three to four rats in each group (×10 original magnification). Some perivascular and peribronchiolar infiltration is seen in HEL-immunized animals, likely due to complete Freund's adjuvant exposure. (C) Bronchoalveolar lavage (BAL) of native and isograft lungs at 72 hours post-transplantation shows disruption of the normally macrophage-dominated cellular differential. A trend (P > 0.05) toward more polymorphonuclear leukocytes (PMNs) was seen in the col(V)-immunized isograft, but not in the native lung or HEL-immunized animals. (D) Cytokine analysis in BAL fluid from col(V)- and HEL-immunized animals, 72 hours post-transplantation, was also performed. Col(V)-immunized isograft BAL fluid contained significantly more tumor necrosis factor (TNF)-α (P = 0.04) and IL-1β (P = 0.04) when compared with HEL-immunized isograft counterparts (Mann-Whitney U test for nonparametric data). *P value significant compared with HEL. (C and D) Solid bars, col(v); open bars, HEL.