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Ann Thorac Surg 2003;75:1118-1122
© 2003 The Society of Thoracic Surgeons

Original article: general thoracic

Broad-spectrum chemokine inhibition ameliorates experimental obliterative bronchiolitis

Babu V. Naidu, FRCSa, Alexander S. Farivar, MDa, Baiya Krishnadasan, MDa, Steven M. Woolley, MRCSa, David J. Grainger, PhDb, Edward D. Verrier, MDa, Michael S. Mulligan, MDa*

a Division of Cardiothoracic Surgery, Department of Surgery, University of Washington, Seattle, Washington, USA
b Department of Medicine, University of Cambridge, Addenbrooke Hospital, Cambridge, United Kingdom

Accepted for publication November 6, 2002.

* Address reprint requests to Dr Mulligan, Box 356310, University of Washington, 1959 NE Pacific St, Seattle, WA 98195, USA
e-mail: msmmd{at}u.washington.edu


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Comment
 References
 
BACKGROUND: Obliterative bronchiolitis (OB) affects over half of all long-term survivors after lung transplantation. Respiratory epithelial cell injury, peribronchial inflammation, and proliferation of fibrovascular connective tissue causing airway occlusion characterize this lesion. Several chemokines participate in experimental OB, and singular blockade is only partially effective. We hypothesized that a broad-spectrum chemokine inhibitor would be an effective intervention in preventing the progression of OB in an established heterotopic tracheal transplantation model.

METHODS: Tracheas from Brown-Norway or Lewis rats were transplanted subcutaneously into Lewis recipients. Treated, allogeneic recipients received either a broad-spectrum chemokine inhibitor in its active (NR58.3.14.3) or inactive (NR58.3.14.4) form at a dose of 30 mg/kg daily. Luminal obstruction, epithelial loss, leukocytic infiltrates, and inflammatory cytokine mRNA levels were assessed in explanted tracheal samples 14 days after transplantation.

RESULTS: After 14 days, allografts receiving the inactive chemokine inhibitor demonstrated marked peribronchial inflammation, near complete loss of respiratory epithelium, and extensive intraluminal proliferation of fibrovascular connective tissue, with a mean 84% ± 5% reduction in airway lumen cross-sectional area. Isografts showed limited inflammation, with minimal loss of epithelium and luminal occlusion. Allogeneic recipients treated with the active chemokine inhibitor showed a significant preservation of respiratory epithelium, minimal peribronchial inflammation, and a marked decrease in the loss of airway cross-sectional area (23% ± 1%) (p < 0.001).

CONCLUSIONS: These findings further characterize the participation of chemokines in OB, and suggest that broad-spectrum chemokine inhibition may potentially be a useful therapeutic tool in slowing the progression of this disease.


   Introduction
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 Abstract
 Introduction
Material and methods
 Results
 Comment
 References
 
Obliterative bronchiolitis (OB) is a progressive and commonly fatal disorder of small airways that affects more than 60% of lung and heart-lung transplant recipients surviving longer than 3 months [1]. Clinically, OB is characterized by progressive dyspnea and a nonproductive cough, with pulmonary function tests revealing reductions in the forced expiratory volume in 1 second (FEV1) and midexpiratory flow volumes [2]. Although treatment typically consists of intensification of immunosuppressive therapy, this serves only to slow its rate of progression. At present, there is no effective therapy to prevent the development of OB or reverse established lesions.

Our knowledge of the pathophysiology of OB is limited, in part, because it has been difficult to model experimentally. A technically simple model of allograft airway transplantation [3] has been used to investigate the pathogenesis of experimental OB. Though originally described in the mouse, this model has been adapted to the rat [4]. The histopathological features of this model largely parallel the changes seen in human recipients developing OB after lung allotransplantation, except complete airway obstruction typically develops by day 28 after heterotopic transplantation of rat airways into allogeneic recipients.

The chemokines are polypeptide molecules that predominantly have proinflammatory activity, and play an important role in both physiologic and pathologic immune function. The CC chemokines include, among others, MCP-1, MIP-1{alpha}, and RANTES [5]. Their predominant function involves recruiting monocytes and various T-cell populations to sites of inflammation. RANTES (regulated upon activation, normal T-cell expressed and secreted) was found to induce dose-dependent chemotaxis of CD4+ T cells [6]. MCP-1 (monocyte chemoattractant protein-1) is not only a monocyte chemoattractant [7], but also a powerful chemotaxin for both CD4+ and CD8+ cells [8]. MIP-1{alpha} (macrophage inflammatory protein-1{alpha}) is unusual among the CC chemokines in that it is a chemoattractant of CD4+ and CD8+ lymphocytes [9], macrophages [10], and neutrophils [11].

Chemokines play functional roles in the development of OB in rat models. Significant increases in mRNA expression of both MCP-1 and RANTES at days 3, 7, and 21 after allograft transplantation have been shown previously [12], and neutralization of RANTES with antibodies reduced both the infiltration of CD4+ T cells and subsequent airway obstruction in rat tracheal allografts [13]. Recently, we have preliminarily confirmed these observations, demonstrating partial protection against the development of OB with administration of neutralizing antibodies to MCP-1, RANTES, or MIP-1{alpha} (unpublished data).

Because several chemokines appear involved in experimental OB, and blockade of a single chemokine is only partially effective [14], we hypothesized that broad-spectrum chemokine inhibition would more favorably modulate the inflammatory responses known to occur that mimic OB in a rat tracheal allograft model.


   Material and methods
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 Abstract
 Introduction
 Material and methods
Results
 Comment
 References
 
Experimental animals
Inbred male Lewis rats and Brown-Norway rats weighing 250 to 275 g were obtained from Simonsen Labs (Gilroy, CA). These two strains were chosen because they represented a complete major histocompatibility complex (MHC) mismatch. Lewis rats served as donors in the isograft experiments and as recipients in the allograft and isograft groups. All animals were cared for in compliance with the "Principles of Laboratory Animal Care" formulated by the Institute of Laboratory Animal Resources, and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (National Institutes of Health Publication No. 86 to 23, revised 1985).

Experimental surgery
After induction of ketamine anesthesia (200 to 250 mg/kg) and systemic heparinization (50 U IV), donor animals were shaved and prepared. A median sternotomy was performed with extension of the incision into the neck. The trachea and mainstem bronchi were explanted, including the first-order branches to the upper and lower lobes, and subsequently placed in cold physiologic saline, as has been described previously [15].

Under sterile conditions, a dorsolateral subcutaneous pocket was fashioned and the underlying fascia was fenestrated overlying the flank musculature. Meticulous hemostasis was maintained throughout the procedure. The graft was secured into place with two interrupted 6-0 sutures to prevent migration, and the wound was closed with 4-0 absorbable sutures in the deep tissue, and nylon for the skin.

The animals were allowed to recover from the surgery and returned to their cages. The wounds were monitored for infection and dehiscence. Animals then received daily subcutaneous injections of either the active broad-spectrum chemokine inhibitor (NR58.3.14.3) described previously [16] or an inactive control of similar structure (NR58.3.14.4) at a dose of 30 mg/kg subcutaneously after the transplant for 14 days in our treated allograft groups. These inhibitors were a generous gift from David Grainger (Biomeasure Inc., Milford, MA). Our control allografts received only saline, as did the isograft recipients. We chose to administer 30 mg/kg to our treatment groups after performing preliminary experiments utilizing doses that ranged from 15 to 60 mg/kg, because it was the lowest dose able achieving maximal response in terms of limiting the development of tracheal occlusion over a 14-day span. At the time of graft harvest on day 14, the animals were anesthetized with lethal doses of pentobarbital (50 mg/kg), and explanted grafts were placed into an appropriate preservative solution. All groups contained at least 4 animals.

Histologic evaluation
Allograft and isograft airways were explanted at day 14 after transplantation and fixed in 10% phosphate-buffered formalin for subsequent sectioning, hematoxylin and eosin (H&E) staining, and examination by light microscopy. The degree of intraluminal and peritracheal inflammation was graded (by a blinded observer), and the composition of the inflammatory cell infiltrates was noted.

Computerized morphometry
Images of the H&E-stained tracheal sections were taken with a high-resolution video camera attached to the microscope. These images were imported to a computer and analyzed using "NIH Image" software, as has been previously described [17]. The percentage of luminal obstruction in transplanted airways was calculated in two steps. First, the outline of the inner surface of the cartilage was traced. The line representing the membranous trachea was drawn straight by connecting the two ends of the cartilage. In the second step, the cursor was used to trace the inner surface of the actual residual lumen. The cross-sectional area of the actual residual lumen was subtracted from the area contained within the inner circumference of the cartilage. The value obtained was then divided by the area within the cartilage to determine the degree of luminal obstruction. The formula for calculating the percent airway obstruction is: (area within cartilage - area of residual lumen)x100/area within cartilage. In normal, unmanipulated airways, the respiratory epithelium and submucosa lie within the cartilaginous rings. Therefore, using this calculation, normal airways will demonstrate a baseline airway obstruction of approximately 3%.

Multiple sections (n > 10) were taken from the middle 1 cm of each graft, and the mean calculated percent obstruction was determined.

Analysis of epithelial preservation
Using the same computerized imaging program, H&E-stained sections of the transplanted airway were examined. The inner circumference of the original airway was determined, and the cursor was used to trace areas of intact normal respiratory epithelium, variant epithelium, and absent or necrotic epithelium. The percentage of the original circumference was then determined for each of these three possibilities. Once again, numerous sections were taken from the central portion of each graft and mean values were determined.

Ribonuclease protection assay (RPA)
Lung RNA was isolated in guanidine thiocyanate, with two rounds of acid phenol/chloroform extraction and alcohol precipitation. RNA integrity was confirmed by agarose gel electrophoresis and quantitated by optical density measurements (260 nm). RNA from each rat was evaluated using the Riboquant system (Pharmingen, San Diego, CA). Rat template rCK1 was used for detection of cytokines. In vitro transcription was carried out in transcription buffer supplemented with {alpha}-[2P]UTP (3,000 Ci/mmol [Amersham Biosciences, Piscataway, NJ]) and T7 RNA polymerase. After DNase I treatment, the riboprobe was isolated by phenol/chloroform extraction and ammonium acetate/ethanol precipitation, and labeling efficiency was determined by measuring Chernokov activity in a scintillation counter. Each riboprobe was diluted to the optimal activity defined by the manufacturer, added to 20 µg of kidney RNA, heated to 90°C, allowed to cool to 56°C, and annealed overnight. After RNase and proteinase K treatment, protected RNA hybrids were purified by phenol/chloroform extraction and ammonium acetate/ethanol precipitation, and separated by electrophoresis on 5% polyacrylamide/8 mol/L urea gels. Gels were dried and subjected to autoradiography using Biomax MS2 Film (Kodak, Rochester, NY).

Statistical analysis
Standard deviation and standard error of the mean were calculated for each group. Comparison between groups was accomplished using Student’s t test. Differences were considered statistically significant at p less than 0.05.


   Results
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 Abstract
 Introduction
 Material and methods
 Results
Comment
 References
 
Broad-spectrum chemokine inhibition reduces luminal obstruction
In isografts harvested 14 days after transplantation, the cross-sectional area of the tracheal lumen was narrowed by approximately 3%. As mentioned previously, normal mucosal and submucosal thickness accounts for this figure. In contrast, the allografts harvested at day 14 from animals treated with saline vehicle revealed almost total luminal obstruction (84% ± 5%) (Fig 1). However, daily treatment with the active broad-spectrum chemokine inhibitor (NR58.3.14.3) significantly reduced the extent of luminal obstruction to 23% ± 1% (p < 0.001 vs saline controls), whereas treatment with the inactive control (NR58.3.14.4) had no effect (83% ± 1%).



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  		Fig 1. Luminal obstruction on day 14. In animals treated with the active chemokine inhibitor, airway obstruction was significantly reduced by 73% (p < 0.004) compared with time-matched controls treated with the inactive inhibitor. Also shown are standard errors of the mean for each treated group. Solid bar = isograft control; open bar = inactive inhibitor; crosshatched bar = active inhibitor.

 
Broad-spectrum chemokine inhibition preserves airway epithelium
Isografts examined at 14 days demonstrated approximately 99% preservation of the respiratory epithelium, with 96% ± 0.7% of the epithelium appearing normal and 3% ± 0.7% variant (cuboidal columnar). In contrast, allografts at 14 days from saline-treated rats lost 98% ± 6% of the respiratory epithelium, and the 2% of epithelium that remained appeared morphologically variant.

However, in allografts from animals treated with the active chemokine inhibitor (NR58.3.14.3), there was significant preservation of epithelium (38% ± 13%; p = 0.03), of which, 18% ± 6% appeared variant and 20% ± 8% normal (Fig 2).



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  		Fig 2. Epithelial loss on day 14. In animals treated with the active chemokine inhibitor, epithelial loss was significantly reduced (p = 0.03) by 61% compared with time-matched controls treated with the inactive inhibitor.

 
Alteration in the balance of immune cells associated with active chemokine inhibition
Analyzing H&E-stained sections, 14-day saline-treated isografts revealed few inflammatory cells, most of which were lymphocytes and macrophages, with occasional neutrophils. In contrast, saline-treated allografts had dense peritracheal inflammation characterized by the presence of mononuclear cells. Almost the entire airway cross-sectional area was obscured by fibrovascular connective tissue with scattered accumulations of inflammatory cells (Fig 3). However, allografts treated with the active chemokine inhibitor (NR58.3.14.3) had a marked reduction in inflammatory cell accumulation.



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  		Fig 3. Histology (hematoxylin and eosin, x100). (A) Inactive inhibitor. By day 14, there is complete loss of respiratory epithelium and dense peritracheal inflammation. Furthermore, fibrovascular connective tissue and scattered accumulations of inflammatory cells fill the lumen. (B) Active inhibitor. By day 14, sections revealed that most of the epithelial lining was retained, and that there was only a modest degree of airway obstruction.

 
Changes in the expression of inflammatory mediators with the broad-spectrum chemokine inhibition
Having demonstrated that active chemokine inhibition ameliorates the progression of tracheal occlusion, we tested whether this protection corresponded to a change in the expression of inflammatory mediators. This was determined utilizing a ribonuclease protection assay, which analyzes a panel of cytokine mRNA. Densitometric analysis is presented graphically for allografts treated with either the inactive or active chemokine inhibitor. Isografts contained little detectable mRNA for any of the cytokines tested, consistent with the previously noted absence of inflammation or airway obstruction. Allografts treated with inactive inhibitor demonstrated an increased expression of a range of cytokines, including interleukin (IL)-1ß, tumor necrosis factor (TNF)-{alpha}, interferon (IFN)-{gamma} and IL-6, and IL-2. Fourteen days of treatment with the active inhibitor substantially reduced the expression of TNF-{alpha}, IL-6, and IFN-{gamma}.


   Comment
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 Abstract
 Introduction
 Material and methods
 Results
 Comment
References
 
Chronic inflammatory diseases, including idiopathic pulmonary fibrosis [18], asthma [19], and HIV [20], are associated with an inappropriate production of chemokines. Additionally, recent animal studies have implicated chemokines in chronic solid organ allograft rejection. The cells that mediate chronic rejection, namely T-cells and monocytes, are the target cells of the chemokines. Despite a relative wealth of descriptive studies, few interventional studies have demonstrated the effectiveness of chemokine blockade in vivo in the setting of transplantation. Using a model of murine kidney transplantation, methionine-extended RANTES (met-RANTES), an antagonist of the chemokine receptors CCR1 and CCR5, reduced leukocyte infiltration and tubular injury [21]. CCR1-deficient mice demonstrated allograft acceptance with MHC class II mismatch, and a delayed pattern of rejection with MHC class I mismatch in a cardiac transplant model [22]. Given that chemokine antagonism may be helpful in preventing experimental acute and chronic allograft rejection, this strategy may have clinical potential.

The action of approximately 50 members of the chemokine superfamily is mediated through binding to a family of 14 G-protein–coupled receptors. This promiscuity of receptor binding makes targeting any one particular chemokine or its receptor largely ineffective due to redundancy. Furthermore, the chemokine receptors are closely related to a wide range of other heptahelical receptors that mediate a plethora of important physiologic functions [23]. Therefore, the limitation of blocking a single chemokine, particularly when there is a need to block recruitment of at least two of the key immune cells involved in chronic rejection, namely T-cells and monocytes, makes the idea of broad-spectrum inhibition especially attractive.

The active form of the chemokine inhibitor used in this study (NR58.3.14.3) is a D amino acid peptide that is conformationally restrained by intermolecular disulfide linkage, and is derived from a conserved region at the C terminus of monocyte chemoattractant protein (MCP). In vitro, it inhibits migration of both CXC and CC chemokines in human monocytes and THP-1 cells [24]. It has been shown to abolish both TNF-{alpha} secretion and neutrophil sequestration in an in vivo lipopolysaccharide model [25]. Its utility in ameliorating reperfusion injury has also been demonstrated in rat cerebral [16] and lung (unpublished data) ischemia models.

Though the model we employed mirrors the changes seen in humans who develop OB, there are some limitations. As previously mentioned, fibrous obliteration of the lumen typically occurs by day 28. This is a more rapid response than that characterized in human subjects, in whom OB develops over months to years after transplantation. Second, the immune response to donor antigens is assessed in a large rat airway, as opposed to the smaller human bronchioles, which may contain different numbers of antigen-presenting cells such as macrophages and dendritic cells [26]. Nonetheless, this model provides an alternative to whole-lung transplantation in small rodents, and affords insight into potential interventions that may retard the progression to OB in humans.

In the present study, we have shown that active chemokine inhibition effectively reduces injury to respiratory epithelium and the subsequent inflammation that leads to fibrous obliteration of heterotopic tracheal transplants. This protection is associated with both a reduction in inflammatory infiltrates and expression of proinflammatory cytokine mRNA. These effects surpass the protection demonstrated previously by individual chemokine blockade, presumably due to the inhibition of multiple chemokines. Unfortunately, the cost of synthesizing D amino acids, poor oral bioavailability, and a short plasma half-life are prohibitive to its use in clinical trials at this time. However, these results show a positive effect of chemokine inhibition in ameliorating the inflammation associated with the development of experimental OB, and afford insight into possible future interventions that may limit the development of chronic lung transplant rejection.


   References
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 Abstract
 Introduction
 Material and methods
 Results
 Comment
 References
 

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