Enhanced Monocyte Chemoattractant Protein-3/CC Chemokine Ligand-7 in Usual Interstitial Pneumonia

Abstract

Section:

Chemokines are increased and may exert effects on both inflammatory and remodeling events in idiopathic pulmonary pneumonia (IIP). Accordingly, we examined the concomitant expression of inflammatory CC chemotactic cytokines or chemokines and their corresponding receptors in surgical lung biopsies obtained at the time of disease diagnosis and pulmonary fibroblasts grown from these biopsies. By gene array analysis, upper and lower lobe biopsies and primary fibroblast lines from patients with usual interstitial pneumonia (UIP), nonspecific interstitial pneumonia, and respiratory bronchiolitis-interstitial lung disease, but not patients without IIP, exhibited CCL7 gene expression. TAQMAN, immunohistochemical, and ELISA analyses confirmed that CCL7 was expressed at significantly higher levels in UIP lung biopsies compared with biopsies from patients with nonspecific interstitial pneumonia, respiratory bronchiolitis-interstitial lung disease, and from patients without IIP. Higher levels of CCL7 were present in cultures of IIP fibroblasts compared with non-IIP fibroblasts, and CCL5, a CCR5 agonist, significantly increased the synthesis of CCL7 by UIP fibroblasts. Together, these data suggest that CCL7 is highly expressed in biopsies and pulmonary fibroblast lines obtained from patients with UIP relative to patients with other IIP and patients without IIP, and that this CC chemokine may have a major role in the progression of fibrosis in this IIP patient group.

Keywords:

chemokine; chemokine receptor; idiopathic interstitial pneumonia; idiopathic pulmonary fibrosis

Fibrotic lung diseases grouped in the category of idiopathic interstitial pneumonias (IIP) remain difficult to diagnose and a tremendous challenge to treat (1). Surgical lung biopsies (SLBs) are often taken for diagnostic purposes with the primary goal directed toward differentiating usual interstitial pneumonia (UIP) from IIPs with a more favorable prognosis such as nonspecific interstitial pneumonia (NSIP) and respiratory bronchiolitis-interstitial lung disease (RBILD), among others (2). The most common histologic pattern is UIP (also referred to as idiopathic pulmonary fibrosis [IPF] if the disease is idiopathic), for which treatment options are unsatisfactory (1), and patients with this form of IIP have a median survival of less than 3 years after diagnosis (1). Although considerable histopathologic data is gleaned from SLBs pertaining to the degree of cellularity and fibrosis (3), these biopsies may also provide important molecular clues as to the etiopathogensis of IIP. Because fibroblasts are readily grown from IIP SLBs, the abnormal growth and synthetic properties of these cells have also been examined (4).

The pathogenesis of IIP is believed to result, in part, from a miscommunication between inflammatory and structural cells, which is driven by the presence of several soluble factors including cytokines, chemokines, and growth factors (5). The list of chemokines that have been recognized in IIP is large, but little is known about the relative contribution of these mediators in the fibrotic process within the human lung (6). Approximately 94% of all known chemokines fall into two main families that are differentiated by their sequence homology and the position of two conserved cysteine residues in the N-terminus of these small (∼ 8- to 14-kD) proteins. Presently, there are 16 CXC ligands (CXCL1–16) and 28 CC ligands (CCL1–28) that have been identified and recently ordered using a new classification scheme (7). All chemokine ligands bind to G protein–coupled 7 transmembrane or serpentine receptors: 6 are known CXC chemokine receptors and 10 known functional CC chemokine receptors.

Thus, the aim of the present study was to examine SLBs and primary fibroblast lines from patients suspected of having IIP with SLBs and primary fibroblast lines obtained from patients for diseases other than IIP using gene array, quantitative TAQMAN polymerase chain reaction, and ELISA analyses to compare CC chemokine and chemokine receptor expression. Given the recent prior focus on characterizing CXC chemokines in SLBs from patients with IIP and the importance of these factors in angiogenesis during pulmonary fibrosis (8), this study focused instead on the concomitant gene and/or protein expression of inflammatory CC chemokines (CCL1–CCL18) and their respective receptors (CCR1–5 and 8). Many of these CC ligands have been examined individually or in combination with CXC ligands in biologic samples from patients with IIP. The present study expands the findings of previous studies by revealing, for the first time, that monocyte chemoattractant protein-3 or CCL7 (according to its new designation) is increased in the context of UIP and the synthesis of this profibrotic CC chemokine (9) is augmented via CCR5 on UIP fibroblasts. In addition, the data presented herein suggest that the examination of concomitant inflammatory chemokine and chemokine receptor profiles in SLBs by specific gene array, TAQMAN, and specific protein ELISA may provide further valuable information of diagnostic significance in IIP.

METHODS

Section:

Patients

The Institutional Review Board at the University of Michigan Medical School approved this study and informed consent was obtained from each patient. The clinical characteristics of the forty-four patients studied are summarized in Table 1

TABLE 1. Clinical characteristics of patients studied
	UIP (n = 21)	NSIP (n = 6)	RB (n = 8)	Non-IIP (n = 9)
Gender (M:F)	14:7	4:2	2:6	3:6
Age, yr*	59 ± 7	61 ± 7	45 ± 11	50 ± 15
Duration of symptoms, yr	2.1 ± 2.4	0.6 ± 0.5	4.4 ± 4.9	1.0 ± 0.6
Smoking, % current or ex	71	50	83	63
FVC, %pred^†	61 ± 19	68 ± 21	72 ± 20	97 ± 19
TLC, %pred^‡	66 ± 18	77 ± 23	85 ± 21	107 ± 26
DL_CO, % pred	44 ± 19	38 ± 17	58 ± 12	54 ± 18
CT-fib	1.57 ± 0.68	1.53 ± 1.60	0.55 ± 0.07	0.90 ± 1.15
CT-alv	1.44 ± 0.89	1.13 ± 1.71	1.55 ± 1.20	2.03 ± 0.45
Mean FF^§	1.41 ± 0.82	0.58 ± 0.51	0.17 ± 0.41	0.05 ± 0.12
Max FF^¶	1.59 ± 0.85	0.63 ± 0.48	0 ± 0	0.08 ± 0.20

^* p = 0.002, UIP and NSIP greater than RB.

^† p = 0.002, UIP lower than non-IIP.

^‡ p = 0.001, UIP lower than non-IIP.

^§ p = 0.002, UIP greater than RB and non-IIP.

^¶ p < 0.0001, UIP greater than RB and non-IIP.

Definition of abbreviations: CT-alv = HRCT alveolar abnormality; CT-fib = HRCT fibrotic abnormality; DL_CO = diffusing capacity for carbon monoxide; FF = fibroblastic foci profusion; TLC = total lung capacity.

. These patients were referred by members of the Michigan End-Stage Lung Disease network with a suspicion of IIP based on a compilation of symptoms, physiologic symptoms, and radiographic findings. Additional detail regarding patient inclusion in this study is provided in an online data supplement.

Isolation and Culture of Primary Pulmonary Fibroblast Lines

Pulmonary fibroblasts were grown from upper- and lower-lobe IIP and non-IIP SLBs and each primary fibroblast line was serially passaged five times to yield pure populations as previously described elsewhere (10).

Preparation of RNA and cDNA from SLBs and Pulmonary Fibroblast Lines

The portion of each SLB used for molecular analysis was snap-frozen in liquid nitrogen and stored at −80°C. For RNA isolation, these samples were thawed, and mechanically homogenized in TRIzol Reagent (Invitrogen Life Technologies, Carlsbad, CA) and total RNA was then prepared according to the manufacturer's instructions. Purified fibroblast lines from each patient were added to 24-well tissue culture plates at a cell density of 1 × 10⁵ cells/well. Twenty-four hours after plating, fibroblasts were exposed to fresh DMEM-15 or DMEM-15 to which 10 ng/ml of CCL5 or CCL22 had been added. All chemokines were purchased from R&D Systems (Minneapolis, MN). After 24 hours, TRIzol Reagent was added to each well for RNA isolation. Purified RNA from SLBs and pulmonary fibroblast lines was subsequently reverse transcribed into cDNA using a BRL reverse transcription kit and oligo (dT) 12–18 primers. The amplification buffer contained 50 mM KCl, 10 mM Tris-HCl (pH 8.3, and 2.5 mM MgCl₂).

Gene Array Membranes

Non-Rad GEArray gene array membranes from SuperArray Inc (Bethesda, MD) were used to analyze changes in gene profiles for human chemokines and chemokine receptors as described in the online supplement.

Real-Time TAQMAN Polymerase Chain Reaction Analysis

Human CCL2, CCL3, CCL5, CCL7, CCL11, and CCL22 gene expression in IIP and non-IIP SLBs was analyzed by real-time quantitative reverse transcriptase–polymerase chain reaction procedure using an ABI PRISM 7,700 Sequence Detection System (Applied Biosystems, Foster City, CA) as previously described (11).

ELISA Analysis

Human CCL2, CCL3, CCL5, CCL7, CCL11, and CCL22 protein levels were measured in 50-μl cell-free supernatants from homogenized IIP and non-IIP SLBs, and from human fibroblast cultures using a sandwich ELISA technique (R&D Systems) (12). Recombinant human chemokines were used to generate the standard curves from which sample concentrations were derived. The limit of detection for each chemokine was consistently above 1 pg/ml. Chemokine levels in each sample was normalized to total protein levels.

Immunochemistry

Routine immunochemistry techniques were used to detect CCL7 in SLBs and CCR5 on primary fibroblast lines as described in the online supplement.

Statistical Analysis

All results are expressed as mean ± SEM. ANOVA analysis and the Kruskal-Wallis or Tukey-Kramer Multiple Comparisons tests were used to reveal statistical differences between patient groups. p < 0.05 was considered statistically significant.

RESULTS

Section:

Pathologic and Clinical Features of Patients Studied

Forty-four patients were enrolled in the present study and the baseline pathologic and clinical characteristics of this group are summarized in Table 1. Twenty-one of the patients exhibited histologic features consistent with UIP; 20 of these patients had concordant UIP, whereas 1 had NSIP in one lobe and UIP in another lobe (3). The cases of NSIP included two patients with a cellular pattern and four with a fibrotic variant. The non-IIP cases included five patients that were classified as nondiagnostic (i.e., histologically normal), three patients that were diagnosed with hypersensitivity pneumonitis, and one patient that was diagnosed with sarcoid.

Inflammatory CC Chemokine Receptor Gene Expression in SLBs from Patients with IIP and Patients without IIP: Gene Array Analysis

The SuperArray gene analysis for CC chemokine receptors in SLBs from UIP, NSIP, RBILD, and non-IIP (i.e., normal) patient groups is summarized in Figure 1

Figure 1. SuperArray gene analysis of inflammatory CC chemokine receptor gene expression in upper and lower lobe surgical lung biopsies (SLBS) from UIP (n = 5 upper lobe [solid bars] SLBs, n = 5 lower lobe [darkly shaded bars] SLBs), NSIP (n = 3 upper lobes, n = 3 lower lobe), RBILD (n = 5 upper lobes, n = 3 lower lobes) and non-IIP groups (n = 3 upper lobes, n = 3 lower lobes). Also shown is SuperArray gene analysis of inflammatory CC chemokine receptor gene expression in primary pulmonary fibroblast lines grown from upper and lower lobe SLBS from UIP (n = 4 upper lobe fibroblast lines [open bars], n = 4 lower lobe fibroblast lines [lightly shaded bars]), NSIP (n = 5 upper lobe fibroblast lines, n = 6 lower lobe fibroblast lines), RBILD (n = 3 upper lobe fibroblast lines, n = 4 lower lobe fibroblast lines) and non-IIP disease control groups (n = 3 upper lobe fibroblast lines, n = 4 lower lobe fibroblast lines). The data shown are mean ± SEM.

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. For this initial gene array analysis, representative upper and lower SLBs were selected at random from the UIP, NSIP, RBILD, and non-IIP patient groups. In the non-IIP group, only biopsies that were diagnostically normal were selected for this initial gene analysis. All inflammatory CC chemokine receptors were detected by SuperArray analysis in upper- and lower-lobe SLBs from the UIP and NSIP patient groups (Figure 1). In addition, all inflammatory CC chemokine receptors were detected in fibroblasts grown from upper- and lower-lobe biopsies (same panels). However, although all were detected in SLBS from the RBILD patient group, inflammatory CC chemokine receptor transcripts were rarely detected in primary pulmonary fibroblast lines grown from this patient group (Figure 1). All inflammatory chemokine receptors were detected in upper- and lower-lobe SLBs from the non-IIP patient group. Again, the expression of CC chemokine receptor transcripts was not always consistently observed in upper and lower primary fibroblasts grown from non-IIP SLBs. For example, CCR3 was not detected in lower-lobe fibroblasts, whereas upper-lobe fibroblasts did not express CCR8 (Figure 1). Thus, using a SuperArray gene analysis for the detection of inflammatory CC chemokine receptors, it was apparent that these receptors were more consistently detected in SLBs and primary fibroblast lines from patients with severer forms of IIP relative to RBILD and non-IIP biopsies and fibroblasts lines.

Inflammatory CC Chemokine Ligand Gene Expression in SLBs from Patients with IIP and Patients without IIP: Gene Array Analysis

Several inflammatory CC ligands were present on each SuperArray gene array, thereby facilitating a concomitant analysis of these ligands with their corresponding receptors (Figure 2)

Figure 2. SuperArray gene analysis of CC chemokine gene expression in upper- and lower-lobe surgical lung biopsies (SLBS) from UIP (n = 5 upper-lobe [solid bars] SLBs, n = 5 lower-lobe [darkly shaded bars] SLBs), NSIP (n = 3 upper lobes, n = 3 lower lobe), RBILD (n = 5 upper lobes, n = 3 lower lobes) and non-IIP groups (n = 3 upper lobes, n = 3 lower lobes). Also shown is SuperArray gene analysis of inflammatory CC chemokine receptor gene expression in primary pulmonary fibroblast lines grown from upper- and lower-lobe SLBS from UIP (n = 3 upper-lobe [open bars] fibroblast lines, n = 4 lower-lobe [lightly shaded bars] fibroblast lines), NSIP (n = 5 upper-lobe fibroblast lines, n = 6 lower-lobe fibroblast lines), RBILD (n = 3 upper-lobe fibroblast lines, n = 4 lower-lobe fibroblast lines) and non-IIP disease control groups (n = 3 upper-lobe fibroblast lines, n = 4 lower lobe). The data shown are mean ± SEM.

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. The following inflammatory CC chemokine gene transcripts were present on the SuperArray: CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, and CCL22. The majority of these inflammatory CC chemokine ligand transcripts were present in both SLBs and primary fibroblast lines from all four patient groups. However, one notable exception was CCL7, which was detected in SLBs and primary fibroblast lines from UIP and NSIP patient groups and in SLBs alone from the RBILD patient group. However, gene transcripts for this inflammatory CC chemokine were absent in upper- and lower-lobe SLBs and primary fibroblast lines from the non-IIP group. Thus, this SuperArray analysis suggested that differences in CCL7 expression could differentiate IIP from non-IIP patient groups.

Inflammatory CC Chemokine Ligand Gene Expression in SLBs from Patients with IIP and Patients without IIP: Quantitative TAQMAN Polymerase Chain Reaction Analysis

Given the suggestion that CCL7 gene expression differed between IIP and non-IIP SLBs, we turned to quantitative real-time TAQMAN polymerase chain reaction (PCR) and used this technique to explore whether CCL7 was indeed altered in IIP relative to non-IIP SLBs. A group of five other inflammatory CC chemokines (CCL2, CCL3, CCL5, CCL11, and CCL22) that were consistently detected in the SuperArray analysis was also examined in IIP and non-IIP SLBs for comparison. Quantitative TAQMAN PCR analysis was performed on upper and lower SLBs from all patients enrolled in the present study (Figure 3)

Figure 3. Quantitative TAQMAN PCR analysis of CCL2, CCL3, CCL5, CCL7, CCL11, and CCL22 gene expression in upper lobe (solid bars) and lower lobe (shaded bars) surgical lung biopsies (SLBS) from UIP (n = 21 patients), NSIP (n = 6 patients), RBILD (n = 8 patients), and non-IIP (n = 9 patients). The data shown are mean ± SEM. *p ⩽ 0.05 compared with appropriate lobe from the non-IIP patient group.

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. Transcript levels of the CC chemokines shown were consistently increased twofold or greater in the IIP group relative to the non-IIP group, whereas the majority of other CC chemokine transcripts present on the SuperArray did not show this consistent fold-increase in expression or were not detected using real-time TAQMAN PCR. Higher fold increases in transcript expression (above non-IIP levels) for CCL2, CCL3, CCL5, CCL7, CCL11, and CCL22 were typically observed in SLBs from the UIP and NSIP patient groups compared with biopsies from the RBILD group. In addition, CCL7 and CCL22 gene levels were significantly increased in upper- and lower-lobe SLBs from the UIP patient group compared with the corresponding SLB from the non-IIP group. Thus, the quantitative real-time TAQMAN PCR analysis provided evidence that CCL7 and CCL22 transcript levels were significantly altered in IIP and that the greatest expression of both chemokines was present in UIP SLBs.

Inflammatory CC Chemokine Ligand Protein Expression in SLBs from Patients with IIP and Patients without IIP: ELISA Analysis

We next examined whether the changes in gene expression for the CC ligands detected by TAQMAN PCR correlated with changes in protein levels for the same CC ligands. As shown in Figure 4

Figure 4. ELISA analysis of CCL2, CCL5, CCL7, and CCL22 in upper lobe (solid bars) and lower lobe (shaded bars) surgical lung biopsies (SLBS) from UIP (n = 21 patients), NSIP (n = 6 patients), RBILD (n = 8 patients), and non-IIP (n = 9 patients). The data shown are mean ± SEM. *p ⩽ 0.05 compared with lower lobe SLBs from non-IIP patient group.

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, the expression of CCL2, CCL5, CCL7, and CCL22 in upper- and lower-lung biopsies varied among the IIP and non-IIP patient groups examined. Among the IIP patient groups, CCL2 was more abundantly expressed in SLBs from the patients with UIP and patients with NSIP compared with the RBILD and non-IIP groups, but these differences did not reach statistical significance. However, significant elevations in CCL5 were observed in upper- and lower-lobe SLBs from the NSIP group (compared with the appropriate non-IIP SLB). In UIP SLBs, both upper- and lower-lobe SLBs contained significantly greater levels of CCL7 and CCL22 protein compared with upper and lower SLBs from the non-IIP patient group (Figure 4). Upper- and lower-lobe SLBs from the UIP patient group contained 13.2 ± 5.1 and 12.0 ± 3.0 pg of CCL7/mg of protein, respectively, whereas upper- and lower-lobe SLBs from the non-IIP patient group contained 0.9 ± 0.9 and 1.3 ± 1.3 pg of CCL7/mg of protein. Upper- and lower-lobe SLBs from the UIP patient group contained 166.1 ± 34.6 and 157.7 ± 33.4 pg of CCL22/mg of protein, respectively, whereas upper- and lower-lobe SLBs from the non-IIP patient group contained 56.9 ± 28.5 and 48.9 ± 32.1 pg of CCL22/mg of protein. Accordingly, whole lung levels of CCL7 in UIP SLBs were increased approximately 12- to 13-fold above levels measured in non-IIP SLBs, in contrast to an approximate 3-fold increase in whole-lung levels of CCL22 in UIP SLBs relative to their non-IIP counterparts. Whole-lung levels of immunoreactive CCL3 and CCL11 were consistently below the limits of ELISA detection (data not shown). Thus, these data agreed with the TAQMAN quantitative PCR data showing that SLBs from the UIP patient group are characterized by a markedly increased expression of CCL7 and CCL22.

Immunohistochemical Detection of CCL7 in SLBs from Patients with IIP and Patients without IIP

Although further examination of CCL22 was hampered by the lack of a suitable antibody for immunohistochemical analysis of this chemokine, the presence of CCL7 in SLBs was determined using routine immunohistochemical techniques (Figure 5)

Figure 5. Immunohistochemical analysis of CCL7 in SLBs from UIP upper (A and B) and lower (C and D), NSIP upper (E and F), and RBILD upper (G and H). Control staining is shown in A, C, E, and G. CCL7 immunoreactivity (red staining) was observed in focal areas of upper (B) and lower (D) SLBs from patients with UIP. More diffuse CCL7 staining was observed in SLBs from patients with NSIP (F), whereas CCL7 was predominately detected around blood vessels in SLBs from patients with RBILD (H). Original magnification: ×200.

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. ELISA analysis suggested that CCL7 was present in greater abundance in UIP and NSIP SLBs compared with other IIP and non-IIP SLBs. The aim of this immunohistochemical analysis was to localize CCL7 protein in IIP and non-IIP biopsies. As shown in Figure 5, CCL7 was localized to interstitial areas of lung samples from UIP (Figure 5B, upper lobe, and Figure 5D, lower lobe) and NSIP (Figure 5F) patient groups, but CCL7 expression appeared to localize more frequently in distinct foci within upper- and lower-lobe samples from patients with UIP (Figures 5B and 5D). Immunoreactive CCL7 was also localized in vascular smooth muscle cells associated with vessels in NSIP (Figure 5F) and RBILD (Figure 5H) biopsies, but CCL7 was not detected in non-IIP control biopsies (not shown). Although both our TAQMAN and ELISA analyses indicated that CCL22 was significantly increased in SLBs from patients with UIP relative to biopsies from patients without IIP, we were unable to localize the expression of this chemokine using immunohistochemical analysis in any of the biopsies we examined. This technical conundrum prevented us from examining in greater detail the identity of the cells that may be sources of CCL22 in UIP. Thus, this data showed that CCL7 was localized to interstitial areas of the lung, particularly in UIP and NSIP SLB biopsies.

Immunocytochemical Analysis of CCR5 Expression by IIP and Non-IIP Pulmonary Fibroblasts

SuperArray analysis gene analysis of primary fibroblast lines grown from upper- and lower-lobe biopsies from the RBILD patient group indicated that inflammatory CC chemokine receptor gene expression was nearly entirely absent in these cells. In contrast, all other primary fibroblast lines showed gene expression for CCR1–5 and 8. We therefore next examined whether pulmonary fibroblast lines grown from both IIP and non-IIP biopsies expressed inflammatory CC chemokine receptors at the protein level. Given that CCR5 was present in upper- and lower-lobe fibroblasts in the UIP, NSIP, and non-IIP patient groups and a highly specific and sensitive antibody was available for immunocytochemical detection, we focused on this CC chemokine receptor. Primary pulmonary fibroblast lines from patients with UIP (Figure 6)

Figure 6. Immunocytochemical analysis of CCR5 expression by upper lobe (A and B) and lower lobe (C and D) pulmonary fibroblasts grown from surgical lung biopsies obtained from patients with UIP. Control staining is shown in A and C, whereas CCR5 is stained red in B and D. Original magnification: ×200.

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and from patients with other IIP and without IIP (Figure 7)

Figure 7. Immunocytochemical analysis of CCR5 expression by upper lobe pulmonary fibroblasts grown from surgical biopsies obtained from patients with NSIP (A and B), patients with RBILD (C and D), and patients without IIP (E and F). Control staining is shown in A, C, and E, whereas CCR5 is stained red in B, D, and F. Original magnification: ×200.

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were examined using routine immunocytochemical techniques to detect the presence of CCR5. Upper- and lower-lobe fibroblasts from patients with UIP exhibited very strong expression of CCR5, and this receptor was observed on the majority of the fibroblasts in culture (Figure 6). Although CCR5 expression was observed in cultures of NSIP (Figure 7B), RBILD (Figure 7D), and non-IIP (Figure 7F), this expression was less dramatic than that observed in cultures of UIP fibroblasts. Taken together, this analysis showed that human fibroblasts from all four patient groups expressed immunoreactive CCR5, thereby highlighting an as yet unexplained disconnect between transcript and protein expression for this chemokine receptor (and possibly other CC receptors) in primary human fibroblast lines. These data also emphasize the importance of concomitant analyses of the CC chemokines and their respective receptors at the transcript and protein levels.

ELISA Analysis of CCL7 Synthesis by Primary Pulmonary Fibroblasts

We next determined whether fibroblasts were a source of CCL7, given that the immunohistochemical analysis of SLBs suggested that this chemokine was localized to interstitial areas. The synthesis of CCL7 by primary pulmonary fibroblast lines from upper- and lower-lobe IIP and non-IIP biopsies was analyzed, and the data from this analysis is summarized in Figure 8

Figure 8. CCL7 synthesis by primary pulmonary fibroblasts grown from upper and lower lobe biopsies obtains from patients with UIP (n = 21), patients with NSIP (n = 4), patients with RBILD (n = 8), and patients without IIP (n = 9). ELISA analysis was performed on whole biopsy homogenates from each patient group. The data shown are mean ± SEM. *p ⩽ 0.05 compared with CCL7 levels measured in control cultures of lower lobe UIP fibroblasts.

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. The constitutive synthesis of CCL7 by upper- and lower-lobe IIP fibroblasts was consistently greater than that observed in cultures of non-IIP fibroblasts (Figure 8), but no differences were detected among the IIP fibroblast lines examined. When CCL22 at 10 ng/ml was added to cultures of IIP and non-IIP fibroblasts for 24 h, no changes in CCL7 synthesis by these cells was observed (Figure 8). However, the addition of 10 ng/ml of CCL5 significantly increased CCL7 generation by lower lobe UIP fibroblasts (Figure 8) compared with CCL7 generation observed in control cultures of these fibroblasts. The presence of CCL5 in cultures of upper lobe NSIP fibroblasts increased CCL7 generation twofold, but this increase did not reach statistical significance. We saw no evidence that pulmonary fibroblasts from patients with IIP and patients without IIP synthesized CCL5 or CCL22 (data not shown). These data suggest that pulmonary fibroblasts are a prominent source of CCL7, but CCL7 was found in greater abundance in cultures of IIP fibroblasts compared with non-IIP fibroblasts, and the expression of this chemokine by UIP and NSIP fibroblasts was augmented by the presence of CCL5.

DISCUSSION

Section:

IIPs are a heterogeneous group of disorders consisting of several clinicopathologic entities with differing histopathologic patterns, clinical course, response to therapy, and prognosis (2, 13, 14). It is now recognized that accurate diagnosis is required in IIP, particularly in distinguishing various clinicopathologic entities of this disease from UIP, the most common histologic pattern in IIP. With this recognition, an expert, international panel of pulmonologists, radiologists, and pathologists has recently suggested that examination of the SLB in isolation is no longer considered “gold standard” for diagnosis (2). However, SLBs have provided researchers with important clues as to identity of soluble mediators that may account for the subtype differences in IIP. Given that the CC ligands and CC receptors appear to be the altered the greatest during the development of experimental fibrosis (15), the present study focused on this chemokine family in clinical fibrosis. SuperArray molecular analysis showed that CCR1–5 and CCR8 were present in upper- and lower-lobe biopsy samples from the IIP and non-IIP patient groups, but gene expression for these receptors was absent in primary fibroblast lines grown from biopsies of patients with RBILD. Upon further molecular and protein analysis it was observed that CCL7 was significantly increased in SLBs from the UIP patient group. CCL7 was present in interstitial areas of UIP and NSIP SLBs, and primary fibroblast lines from patients with IIP were an excellent source of this chemokine. More importantly, the synthesis of CCL7 was enhanced by CCL5, another chemokine that is prominently expressed UIP SLBs. First identified as a fibroblast-derived chemokine (or fibroblast-induced cytokine [16]), the protein sequence of CCL7 shows 74% identity with CCL2, but secreted CCL7 protein differs from CCL2 in being N-glycosylated (17) and is a more potent activator of basophils (16). Similar to CCL2, CCL7 specifically stimulates the directional migration of T cells as well as monocytes via CCR2 (18). More recently, CCL7 was shown to be a major CC chemokine associated with experimental fibrosis (9). Thus, through the use of gene and protein analyses, the findings from the present study suggest that assessment of CCL7 levels in SLBs and primary fibroblast lines grown from these biopsies may aid in the differential diagnosis of IIP subsets.

The observation that CCL5 protein was significantly elevated in NSIP SLBs was a novel and surprising finding in the present study because this CC chemokine and its major receptor, CCR5, have been predominantly associated with sarcoidosis (19). For the ELISA analysis in the present study, the non-IIP SLB protein data was derived from both upper and lower biopsies from nondiagnostic, sarcoid, and hypersensitivity pneumonitis patient groups, but we did not observe any differences in CCL5 levels amongst these three non-IIP groups. Our data may differ from previous studies due to the fact that we analyzed fewer patients with sarcoid or hypersensitivity pneumonitis. NSIP remains an IIP entity that requires better characterization, and encompasses a broad spectrum of features including alveolar wall inflammation (cellular), interstitial fibrosis (fibrotic), and mixed cellular and fibrotic (2). In general, the diagnosis of NSIP is associated with a more favorable prognosis than that of UIP. Differences in levels of cytokines, matrix metalloproteinases, and adhesion molecules have been observed between SLBs from NSIP and UIP patient groups, but the importance of these differences has not been explored. The observation that CCL5 is significantly elevated in NSIP SLBs may provide additional evidence that pulmonary events leading to NSIP differ from that of UIP and other forms of IIP. Further analysis of chemokine and chemokine receptors in SLBs from patients with NSIP is certainly warranted.

Given that the immunostaining of whole-lung biopsies from patients with UIP and patients with NSIP showed that CCL7 was predominately located within interstitial areas of lung, the present study also addressed whether cultured primary pulmonary fibroblasts were a putative cellular source of CCL7. All cultured primary fibroblast lines examined in the present study synthesized CCL7, but it was interesting to note that the presence of CCL5 in cultures of lower-lobe UIP fibroblasts significantly augmented the generation of this CC chemokine. This finding is particularly interesting given the marked increase in CCR5 expression that was observed on UIP fibroblasts; however, it is possible that CCL5 may have augmented CCL7 synthesis via CCR1 and/or CCR3 expression by these cells. CCR5 has been thoroughly characterized as a receptor for CCL3, CCL4, and CCL5; however, it has been shown that CCL7 could bind CCR5 with high affinity without eliciting a functional response (20). However, the latter effect of CCL7 was observed in the context of transfected Chinese Hamster Ovary cells and supraphysiologic concentrations (i.e., ⩾ 200 nM) of CCL7 were required to displace CCR5 agonists (i.e., CCL4) or prevent the binding of R5 strains of HIV (20). Clearly, further investigation is required to determine the impact of CCL7, and the CC chemokine receptors it activates, on the proliferative and synthetic properties of pulmonary fibroblasts grown from IIP and non-IIP biopsies.

Research into the etiopathogensis of IIP has largely focused on elucidating the inflammatory mechanisms that initiate and/or maintain the fibrotic response in the lung, and strong arguments have been presented in support of this research direction (21). However, it is well documented that potent antiinflammatory or immunomodulatory therapies are largely ineffective in the treatment of severe forms of IIP, namely UIP and NSIP (22, 23). These failures have lead to the suggestion that these forms of IIP may be associated more with abnormal wound healing than with inflammation (23). Support for this emerging concept was observed in the present study in that it was shown that CCL7 was elevated in IIP and the pulmonary fibroblast appeared to be a major cellular source of this profibrotic chemokine. We provide evidence to support the diagnostic importance of examining this CC ligand and possibly others (i.e., CCL22) in biopsy samples from patients suspected of having IIP. Thus, further characterization of the patterns of CCL7 expression in IIP may aid in the development of promising anti–chemokine-based therapies in IIP.

The authors thank Robin Kunkel for her artistic assistance during the preparation of this manuscript.

References

Section:

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Correspondence and requests for reprints should be addressed to Cory M. Hogaboam, Ph.D., Associate Professor, Department of Pathology, University of Michigan Medical School, Rm. 5216B, Med Sci I, 1301 Catherine Rd., Ann Arbor, MI 48109-0602. E-mail: [email protected]

American Journal of Respiratory and Critical Care Medicine

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