Type V Collagen–induced Tolerance Prevents Airway Hyperresponsiveness
Authors: Jeremy M. Lott, Sarita Sehra, Purvi Mehrotra, Elizabeth A. Mickler, Amanda J. Fisher, Wenwu Zhang, Robert G. Presson, Michael F. Busk, Shreevrat Goenka, Susan J. Gunst, Mark H. Kaplan, David S. Wilkes, Sally E. Wenzel
Journal: American Journal of Respiratory and Critical Care Medicine (2013)

To the Editor:

Asthma, a leading cause of obstructive lung disease, is characterized by inflammation, mucus secretion, and remodeling of peribronchiolar connective tissue. These changes plus goblet cell metaplasia, smooth muscle hyperplasia, airway thickening, and eosinophil recruitment are regulated by or are a consequence of various cytokines, including IL-4, -13, -5, -9, and more recently -17 (1–8).

Type V collagen (col[V]), a minor collagen, is intercalated within fibrils of type I collagen, the major lung collagen (9). Due to its location, col(V) is considered a sequestered antigen not normally exposed to the immune system. However, interstitial remodeling leads to col(V) exposure, and results in a cryptic antigen becoming a target of the immune response. Indeed, we reported that anti-col(V) immunity developed in the pathogenesis of lung transplant–associated obliterative bronchiolitis and idiopathic pulmonary fibrosis, which are two disorders characterized by extensive interstitial remodeling (10, 11). Studies showing that immune responses to col(V) are involved in disease pathogenesis suggest that immune tolerance to col(V) may be protective of tissue injury. Indeed, col(V)-induced tolerance prevented acute lung allograft rejection and obliterative bronchiolitis (12–14), as well as bleomycin-induced pulmonary fibrosis (15).

Because asthma is a disease that may involve interstitial remodeling, we hypothesized that clinical asthma is associated with anti-col(V) immunity and that col(V)-induced tolerance may limit the asthmatic response. Examination of serum from normal subjects and patients with asthma in the current study show higher levels of systemic anti-col(V) antibodies in individuals with asthma, and overexpression of interstitial col(V) in fatal asthma. col(V)-induced tolerance down-regulated ovalbumin (OVA)-induced airway hyperresponsiveness (AHR) by reduction of airway smooth muscle contraction.

Methods
Methods are available in the online supplement.


Results
Anti-col(V) antibodies and col(V) expression in asthma.
We first determined if clinical asthma was associated with the presence of anti-col(V) antibodies. The study population is described in Table 1. Compared with healthy normal volunteers, individuals with asthma had significantly higher levels of serum anti-col(V) antibodies (P < 0.001) (Figure 1A). To determine if col(V) is highly expressed in the asthmatic lung, col(V) expression was assessed in a lung section from a patient who succumbed to an asthmatic episode. As expected in the normal lung, some col(V) staining was present in the subepithelial tissues and basement membrane, and stained at low levels in adjacent peribronchiolar tissues. In contrast, col(V) staining was increased in the thickened basement membrane and adjacent tissues in fatal asthma (Figure 1B).



TABLE 1.

PATIENT DEMOGRAPHIC DATA





Figure 1.

Detection of systemic anti–type V collagen (col[V]) antibodies in clinical asthma and allergic airway disease. (A) Serum was collected from 20 clinically defined individuals with asthma and 10 nonsmoking individuals without asthma and assayed ...




Induction of col(V) tolerance.
To determine if allergic airway disease (AAD) is also associated with anti-col(V) antibodies, we measured anti-col(V) antibodies in untreated mice and mice after OVA-induced AAD. In parallel studies, col(V) tolerance was induced by a single intravenous injection (100 μg) prior to OVA/alum as reported in the Methods in the online supplement and shown in Figure 1C. Compared with untreated mice or phosphate-buffered saline (PBS)-treated controls, AAD resulted in development of anti-col(V) antibodies (Figure 1D). To determine if OVA-induced AAD resulted in anti-col(V) cellular immune responses, mediastinal lymph node cells were isolated and cultured in the presence of OVA or col(V). As expected, OVA sensitization alone resulted in brisk OVA-induced proliferation (P < 0.01) (Figure 1E). Consistent with detection of anti-col(V) antibodies, OVA-sensitized mice developed a trend toward higher proliferation in response to col(V) (Figure 1E). Notably, col(V) treatment significantly suppressed OVA-induced proliferation, indicating that col(V) administration induced tolerance (P < 0.01) (Figure 1E). Local and systemic humoral responses were studied; the levels of total and OVA-specific IgE were assessed in serum and bronchoalveolar lavage (BAL) of treated animals. Compared with PBS-treated controls, total and OVA-specific IgE levels were unchanged in the serum of col(V)-tolerized mice (Figure 1F). In contrast, col(V)-induced tolerance resulted in significant reductions in total and OVA-specific IgE antibodies in BAL fluid (P < 0.05) (Figure 1G). Collectively, these data indicate that OVA-induced AHR results in anti-col(V) immunity, and col(V)-induced tolerance abrogates local OVA-induced cellular and humoral immune responses.


col(V)-induced tolerance down-regulated AHR.
To study the effects of col(V)-induced tolerance on AHR, we assessed lung function both by noninvasive (enhanced pause [Penh]) and invasive methods of measuring airway resistance and lung compliance. As expected, OVA sensitization and challenge resulted in dose-dependent increases in Penh in response to methacholine in PBS-treated (control) mice (Figure 2A). In contrast, Penh gradually increased in col(V)-tolerized mice at low methacholine concentrations, then plateaued at higher concentrations, which were significantly lower than PBS-treated controls (P < 0.001) (Figure 2A). Resistance, a measure of airway obstruction, was also significantly reduced in col(V)-tolerized mice compared with controls at the highest methacholine concentrations (P < 0.01) (Figure 2B). There was no change in compliance in the col(V)-tolerized group compared with the PBS group (Figure 2C). Consistent with data showing less airway resistance, airway smooth muscle contractile force in response to acetylcholine stimulation was decreased in mice tolerized with col(V) when compared with PBS-treated controls (Figure 2D).



Figure 2.

The effect of type V collagen (col[V])-induced tolerance on allergen-induced airway hyperresponsiveness (AHR). AHR was measured 24 hours after the final intranasal challenge. (A) Twenty-four hours after the final challenge, AHR to aerosolized methacholine ...