Berylliosis

Rationale: Genetic factors are likely to influence the clinical course and pattern of sarcoidosis, a granulomatous disease of unknown origin. Objectives: We tested this hypothesis for C-C chemokine receptor 5 (CCR5), a molecule involved in recruitment and activation of mononuclear cells. Methods: In addition to the known CCR5 Delta 32 insertion/deletion, we evaluated a further eight single-nucleotide polymorphisms in 106 British patients and 142 British unaffected subjects, and second-setted the results in 112 Dutch patients and 169 healthy Dutch control subjects. Measurements and Main results: In the British population, the frequency of one of the identified haplotypes (HHC) was strongly associated with the presence of parenchymal disease (radiographie stage ≥ II versus stages 0 and I) at presentation (odds ratio [OR], 5.2; 95% confidence interval [CI], 1.96-13.7; corrected p = 0.02), at 2 (OR, 6.6; 95% CI, 2.5-17.6; corrected p = 0.006), and at 4 years follow-up (OR, 6.8; 95% CI, 2.5-18.0; corrected p = 0.0045). In the Dutch population, the same association was seen at 2 (OR, 6.7; 95% CI, 2.8-16.4; corrected p = 0.002), and 4 years follow-up (OR, 9.0; 95% CI, 3.5-23.1; corrected p = 0.0009). Conclusions: No association between the CCR5 haplotype HHC and susceptibility to sarcoidosis was observed, indicating that this relevant gene only operates after disease induction. In summary, we report a strong association between CCR5 haplotype HHC and persistent lung involvement in sarcoidosis.

Keywords: cytokines; genetic polymorphisms; sarcoidosis

The accumulated lifetime risk of sarcoidosis is as much as 1.3% for women and almost 1% for men (1). The lungs and thoracic lymph nodes are most commonly affected by this Th1-driven disease. The presentation and the clinical course of pulmonary sarcoidosis vary widely, from self-limited to chronic lung disease. Knowledge of the risk factors for persistent lung impairment in sarcoidosis is limited.

Ample evidence of a strong genetic influence in sarcoidosis exists, as reflected by the ATS/ERS/WASOG conclusions (2). Associations between major histocompatibility complex (MHC) genotypes and sarcoidosis susceptibility/phenotypes are strikingly consistent across ethnic boundaries (2, 3). Nevertheless, roughly half of the patients do not have evidence for a human leukocyte antigen (HLA) contribution to pathogenesis (4), highlighting the importance of studying other genes, either MHC-associated or located in other chromosomal areas, that could drive the disease toward different patterns. It is increasingly clear that the sarcoidosis "family" of diseases comprises distinct phenotypical entities, including Löfgren's syndrome, persistent/progressive lung disease, granulomatous uveitis, and berylliosis, each with potentially distinct genetic associations (4-9).

Chemokines are small peptides that mediate monocyte, lymphocyte, and neutrophil chemotactic activity, by binding to specific G-protein-coupled receptors (10-13). The C-C chemokine receptor 5 (CCR5) gene has been mapped to the short arm of chromosome 3 among a group of genes that encode multiple chemokine receptors including the CCR2 gene (14). Ligands for CCR5, including CCL3, CCL4, CCL5, and CCL8 chemokines (15, 16), play a major role in the recruitment and activation of lymphocytes and monocytes in sarcoidosis (17, 18). CCR5 expression is upregulated in bronchoalveolar lavage (BAL) macrophages and lymphocytes in sarcoidosis (19) and BAL levels of CCL3 and CCL5 correlate with risk of sarcoid progression (20-23). Thus, chemokinenreceptor interactions are likely to regulate the composition and/or the persistence of cellular infiltrates.

The aim of this study was to identify associations between CCR5 haplotypes (including additional promoter polymorphisms) and the presence and nature of sarcoidosis lung involvement. We evaluated the distribution of CCR5 haplotypes in white British patients and control subjects; a second sample of white Dutch patients with sarcoidosis and control subjects was tested for replication.

The results of part of this study have been previously presented in the form of an abstract (24).

METHODS

British Patients and Control Subjects

One hundred six unrelated white British patients were investigated. In all patients the diagnosis of sarcoidosis was histopathologic, and in accordance with the diagnostic criteria defined in the American Thoracic Society/European Respiratory Society International Statement on Sarcoidosis (2). British and Dutch patients presenting with classical Löfgren's syndrome were excluded from the study. Written patient consent was obtained from all subjects; the Ethics Committees of the Royal Bromplon Hospital, London gave authorization for the study. This hospital is a tertiary referral center taking patients mainly from the southeast of the United Kingdom.

The British control population comprised 142 unaffected white subjects.

Dutch Patients and Control Subjects

One hundred twelve unrelated white Dutch patients were included in the study. The diagnosis of sarcoidosis was established as described above (2). Written consent was obtained from all subjects and authorization was given by the Ethics Committee of the Sint Antonius Hospital, Nieuwegein, The Netherlands. This hospital is a secondary referral center.

The Dutch control group comprised 169 healthy white subjects. Both British and Dutch control subjects were anonymized. However, all were healthy as judged by a self-administered questionnaire and by the routine laboratory investigations performed on blood donors.

Evaluation of Pulmonary Disease Severity

Radiography. Chest radiographs for each patient were examined and compared to determine disease severity and course. Staging of disease was classified according to the joint ATS/ERS/WASOG Consensus Statement on Sarcoidosis (2) into stage 0 (normal chest radiograph), stage I (bilateral hilar lymphadenopathy, BHL), stage II (BHL and pulmonary infiltrates), stage III (pulmonary infiltrates without BHL), and stage IV (pulmonary fibrosis).

In the United Kingdom, chest radiographs at presentation, 2, and 4 years were evaluated independently by two experienced pulmonary radiologists (see also expanded Methods section in the online supplement). In Holland, one experienced chest radiologist performed the same procedure. Presentation chest radiographie data were available for 215 patients (104 British, 111 Dutch). Chest radiographs at 2 years after initial presentation were available for 198 patients (99 British, 99 Dutch). A total of 183 patients (91 British, 92 Dutch) also had radiographic follow-up at 4 years. For three patients (2 British, 1 Dutch), no chest radiograph was available for evaluation at presentation, 2, or 4 years. The difference in patient numbers at presentation compared with 2 and 4 years is mainly due to the inclusion in our study of a small number of patients in whom a diagnosis has been made recently. These patients have not been followed up long enough to be included in the 2- and 4-year analysis.

Pulmonary function testing. Both groups of patients had pulmonary function tests performed at the referral center. The same normal values for calculations of lung functions were used in the United Kingdom and in Holland. Pulmonary function tests included FEV^sub 1^ and FVC by using spirometry with carbon monoxide diffusion capacity (DL^sub CO^) as measured by the single-breath technique and expressed as percentage of predicted. The calculations are based on the ATS recommendations (25, 26). Lung function data were available for 106 British and 107 Dutch patients.

BAL. In Holland and the United Kingdom, bronchoscopy was performed and BAL fluid processed as described previously (27) (see also expanded Methods in the online supplement). BAL data were available for 44 British and 78 Dutch patients.

Clinical features of British and Dutch patients are summarized in Table E1 in the online supplement.

Sequence-Specific Primers and Polymerase Chain Reaction

Polymorphisms were determined using sequence-specific primers (SSPs) and polymerase chain reaction (PCR). All PCR reactions were run under identical conditions as previously described (28).

To identify the same CCR5 promoter haplogroups described by Gonzalez and coworkers (29), the CCR2 SNP 190 (GIA) was also genotyped.

Data Analysis

The genotype, allele, and carriage frequencies (i.e., number of individuals carrying the allele either in both [homozygous] or in only one [heterozygous] chromosome), were determined by direct counting. Haplotypes were identified by the computer program PHASE, version 2 (30). PHASE uses a Bayesian approach with a coalescent theory to estimate haplotype structure. It is similar to the expectation maximization (EM) algorithm but predicts the structure of the next sampled haplotype by comparing it to haplotypes already assigned rather than to genotypes. PHASE is suited to tightly linked loci as in the CCR5 gene region; in this regard pairwise D' values were calculated before implementing the PHASE algorithm using the computer program Arlequin (31), PHASE is freely available from http://www.stat.Washington, edu/stephens/software.html (see also expanded Methods section in the online supplement).

Subsequently, the carriage frequency of each haplotype was determined by direct counting. Proportions were compared using chi-square statistics or Fisher's exact test as appropriate. Adjustment for multiple tests was made using the formula p^sub c^ = p × n, where p^sub c^ is the corrected value, p the uncorrected value, and n the number of tests performed (Bonferroni method). A value of p

RESULTS

We investigated eight biallelic CCR5 single nucleotide polymorphisms (SNPs). To better define the CCR5 promoter region, in addition to the well known polymorphisms at positions -2459 A/G (Promoter), -2135 T/C (Exon), -2086 A/G (Exon), -1835 T/C (Exon/Intron boundary), and Delta 32 wt/Δ32 insertion/ deletion (Exon, coding sequence), we genotyped our samples for the SNPs at position -5663, -3900, and -3458 (reported in the publicly available SNP database) (32), which had not been previously included in any of the known haplogroups. We first provided data on genotype and allele frequencies for these variations.

As mentioned above, the CCR2 SNP at position 190 G/A (Exon, coding sequence) was also genotyped. The relative SNP positions in the CCR2 and CCR5 genes are shown in Figure 1. Primer sequences detecting these polymorphisms are shown in Table 1. The genotype frequencies of the investigated CCR2 and CCR5 polymorphisms for subjects with sarcoidosis and for control subjects, summarized in Table 2, were all in Hardy-Weinberg equilibrium. Fifteen haplotypes were deduced, including five highly prevalent haplotypes (29), as shown in Table 3. CCR5 haplotype frequencies are given in Table E2. After correcting for multiple comparisons, there were no differences in the genotype, phenotype, and allele frequencies of the CCR2 and CCR5 polymorphisms, or in the haplotype frequencies between patients and control subjects in either the British or Dutch population.

Association between CCR5 Haplotype HHC and the Presence of Parenchymal Disease

After correcting for multiple comparisons, in the British population the presence of parenchymal disease (radiographie stage ≥ II versus stages 0 and I) was strongly associated with CCR5 haplotype HHC at presentation (odds ratio [OR], 5.2; 95% confidence interval [CI], 1.96-13.7; p = 0.02), at 2 (OR, 6.6; 95% CI, 2.5-17.6; p = 0.006), and at 4 years (OR, 6.8; 95% CI, 2.5-18.0; p = 0.0045). In the Dutch population the same association was seen at the 2- (OR, 6.7; 95% CI, 2.8-16.4; p = 0.002) and 4-year follow-up (OR, 9.0; 95% CI, 3.5-23.1; p = 0.0009) (Table 4). All these findings remained significant after adjustment for sex, age, treatment at presentation, and smoking status on multivariate logistic regression in each population. When genotype data for haplotype HHC was investigated, there was a trend toward a significant effect of haplotype copy number, more evident in the Dutch population (Table E3). The association also remained significant after adjustment for carriage of CCR2 haplotype 2, a haplotype in moderate linkage disequilibrium with HHC (D' = 0.66) (33); CCR2 haplotype 2 was not itself independently linked to stage greater than or equal to II.

Among patients with parenchymal involvement, changes in stage were more frequent in those with stage II at presentation. Therefore, to exclude bias, we repeated logistic regression after removing patients with stage II at presentation and a change in stage at 2 or 4 years (n = 29, 19 Dutch and 10 British) or no X-ray at either 2 or 4 years (n = 2, both Dutch). The association between HHC and parenchymal disease remained significant both at 2 and 4 years in both populations (p

To evaluate whether HHC was associated with separate measures of parenchymal involvement, both pulmonary function tests and BAL neutrophilia were also analyzed. Carriers of CCR5 HHC showed a lower FEV^sub 1^ (median: 82% vs. 92%, p = 0.008) and FVC (90% vs. 100%, p = 0.02) at presentation compared with non-HHC carriers. Furthermore, the prevalence of BAL neutrophilia (neutrophils > 4%) was significantly higher in HHC compared with non-HHC carriers (22.2% vs. 6.1%, p = 0.017). HHC carriage was not associated with the presence of other organ involvement (heart, liver, kidney, CNS, skin, or uveitis).

The T allele at position -3458 showed an association as strong as haplotype HHC in both Dutch and British patients at all time points with the alleles -2086 G, -2135 T, -2459 G, -3458 T, -3900 C, and -5663 A also associated with advanced radiographic stages of disease (see Table E4).

CCR5 haplotype HHA was higher in patients with stages 0-I compared with those with stages greater than or equal to II at presentation, when both populations were considered together (21.9 vs. 9.9%, p = 0.02); however, this association did not reach statistical significance after correction for multiple comparisons (p = 0.3). Furthermore, this weak association was present only in the Dutch population (33% of HHA carriers had stage 0-I compared with 12.7% of stage ≥ II; OR, 3.4; 95% CI, 1.3-8.9; p = 0.01, corrected p = 0.15), whereas in the British population, where the frequency of HHA was lower, an opposite trend was seen, with only six patients, all with radiologic stage greater than or equal to II, carrying haplotype HHA.

CCR5 Haplotype HHC and the Nature of Parenchymal Disease

When the analysis was limited to patients with parenchymal lung involvement on X-ray (stage ≥ II), HHC carriage did not increase significantly from stages II to IV (Table 5). Similarly, in this subgroup of patients, BAL neutrophil percentages and FEV^sub 1^ levels did not differ between carriers and noncarriers of haplotype HHC (data not shown). BAL neutrophil levels (p = 0.002) and FEV^sub 1^ levels (p

CCR5 HHC Carriage and Persistence of Lung Involvement

To investigate whether HHC carriage was associated with radiologic persistence of lung disease, we performed a subgroup analysis limited to the 124 patients who had lung involvement (stage ≥ II) at presentation and who also had radiologic staging after 4 years. At 4 years after presentation, CCR5 HHC carriage was observed in 31.8% of patients whose lung disease had regressed (either stage 0 or I), compared with 76.5% of patients with persistent lung involvement (stage ≥ II) (OR, 6.9; p

DISCUSSION

In the present study, we report a strong association between CCR5 haplotype HHC and persistent parenchymal lung involvement in sarcoidosis, as assessed by chest X-ray, suggesting an important pathophysiologic role for this chemokine receptor. However, despite clear statistical evidence, it remains uncertain whether this haplotype per se, or a single SNP within it, is critical to the development of parenchymal disease. Alternatively, the SNPs included in HHC could be in linkage disequilibrium with other unidentified functional variant(s) within the CCR5 gene or in neighboring genes.

Our conclusions are based on CCR5 haplotypes; therefore, the reliability of the method used for haplotype reconstruction is critical. Although errors in haplotype assignment can originate from genotyping or calculation error as well as inherent marker ambiguity in the presence of SNP heterozygosity, computational methods of haplotype inference such as PHASE (30), the statistical method used in this study, have been shown to have high accuracy (34, 35). Moreover, the frequencies of the deduced CCR5 haplotypes in our cohort are consistent with those previously reported by Gonzalez and coworkers (29) strongly supporting the reliability of PHASE.

Despite the interest in CCR5 genetic polymorphisms, relatively little is known about the possible functional consequences of the SNPs in the promoter region. We speculate that the variants included in haplotype HHC have effects on CCR5 transcriptional activity. This haplotype comprises several promoter polymorphisms, which can modify gene expression by altering transcription factor binding (36-44). The -2459 G/A polymorphism has been associated with variations in gene expression in vitro, albeit with controversial results according to the cell type studied (39, 40). Therefore, the effect of the variants may depend on the cell type, and this could partially explain the apparently contradictory results of functional studies. The SNPs at positions -3458 T/G, -3900 C/A, and -5663 A/G are also located within a region predicted to differentially bind transcription factors according to the presence of the wild-type or mutant alleles, as assessed by using the TESS transcription factor database (http://www.cbil.upenn.edu/tess). However, whether and how these polymorphisms affect transcriptional activity of the CCR5 promoter needs to be elucidated.

A large part of the information available on the functional consequences of CCR5 gene polymorphisms in vivo stems from studies of HIV-1-infected individuals as CCR5 is the major coreceptor for the HIV type-1 virus. Individuals homozygous for a 32-base pair deletion (Δ32/Δ32), resulting in a truncated protein that fails to reach the cell surface, are highly resistant to HIV infection (45, 46), whereas Δ32/wt heterozygous individuals express lower levels of CCR5 and show slower progression to AIDS (36, 47). However, substantial variations in gene expression among Δ32/wt and wt/wt subjects have been described (48), suggesting that polymorphisms other than Δ32 are likely to influence CCR5 expression. Interestingly, CCR5 haplotype HHC does not include the Δ32 deletion variant, suggesting that decreased gene expression is not the key mechanism of persistent lung disease in sarcoidosis and supporting the hypothesis that promoter variants might result in increased CCR5 expression, at least in some cell types. Indeed, whereas the reduction in gene expression caused by the Δ32 allele, for instance, is likely to have similar effects in all cell types, the effects on mRNA transcription of CCR5 promoter polymorphisms may be cell type-specific or otherwise affected by the local milieu within a particular tissue.

An increased frequency of the Δ32 allele in patients with sarcoidosis from Czech Republic has been reported (49). We could not replicate the same finding in either British or Dutch populations, and this is probably due to the small number of patients in the Czech study (n = 66) rather than to ethnic differences, as the allelic frequencies of the mutant allele are identical in the three control groups (British 11.3%, Dutch 12.1%, Czech 10.8%) and therefore in keeping with a previously reported genotype frequency of the CCR5 Δ32/wt of about 20% in whites (36).

The CCR5 gene is in close proximity to and in linkage disequilibrium with CCR2 (36). We have recently shown that a particular combination of SNPs in the CCR2 gene (haplotype 2) is associated with Löfgren's syndrome, a sarcoidosis subset characterized by an excellent prognosis (33). Intriguingly, CCR2 haplotype 2 is in moderate linkage disequilibrium with CCR5 HHC (D' = 0.66). The most plausible explanation for these apparently contradictory results is in the impact of HLA haplotypes. The HLA DRB1*0301-DQB1*0201 (DR3) haplotype is strongly associated with Löfgren's syndrome but not with "classic" sarcoidosis (84% vs. 19%, p

Although linkage disequilibrium across CCR5 is very similar between whites, African Americans, and Hispanics, the actual frequencies of haplotypes do differ (29). For instance, HHC/ HHC and HHC/HHE are very common in whites, but represent only a small proportion of African-American haplotype pairs. Our results cannot therefore be generalized to other ethnicities.

CCR5 is expressed at high levels in CD4^sup +^ Th1 lymphocytes; it plays a crucial role in recruiting mononuclear cells to inflammatory sites acting as modulator in leukocyte migration and activation. Recently, it has been also shown that CCR5 enhances T cell activation by improving T cell-antigen-presenting cell attraction at the immunologie synapse, suggesting a previously unknown function for this chemokine receptor (51). Plasma levels of CCR5 and its ligands CCL3 and CCL5, both chemoattractants for monocytes/macrophages and T cells (52), are increased in the BAL fluid of patients with sarcoidosis (18, 22, 23, 53, 54). Human monocytes and T cells show aberrant migration toward CCL5 and CCL3 as result of CCR5 overexpression, and this effect is inhibited by anti-CCR5 antibodies (55, 56). Therefore, we hypothesize that CCR5 dysregulation could lead to aberrant trafficking of T cells to the lung resulting in persistent parenchymal involvement in sarcoidosis. In agreement with this, several reports have shown an important role for CCR5 in attracting Th1 cells to the sites of inflammation in rheumatoid arthritis, multiple sclerosis, and glomerulonephritis, all diseases associated with a Th1-type immune response (56-59).

The role played by CCR5 in apoptosis might also be pathogenetically important. A dysregulation of the Fas/Fas-L system, activated through CCR5 (60), could lead to an abnormal persistence of the sarcoidosis inflammatory process and delayed resolution of the granulomatous response, as a consequence of an altered T cells clearance (61). Alveolar macrophages and lymphocytes expressing apoptotic receptors are also increased in patients with sarcoidosis (62, 63). Alternatively, variants of CCR5 may influence the binding and processing of microbes other than HIV, which, in turn, may be specific triggers of persistent lung impairment.

In the Dutch patients, CCR5 HHC was not associated with parenchymal disease at presentation. However, this is not surprising. Indeed, the Dutch patients, recruited from a secondary center, had earlier and milder disease as shown by functional indices and radiographic stage (Table E1) and therefore a period of follow-up was required to disclose an association with persistent lung disease. By contrast, the British patients were recruited from a tertiary center, with preselection of a larger subset with persistent lung involvement, and therefore the cardinal associations were already evident at presentation.

The importance of CCR5 HHC in sarcoidosis pathogenesis clearly remains to be clarified and confirmed in an independent study. It is important to state that our results indicate that the CCR5 HHC is specifically associated with the presence or absence of parenchymal disease only after classical sarcoid is contracted. In particular, HHC carriage was not linked to radiologic stage in patients with overt lung involvement (stages ≥ II). It is therefore likely that to develop pulmonary fibrosis, gene variants other than CCR5 HHC are required.

The molecular mechanisms through which CCR5 genetic polymorphisms influence disease severity require further study; nevertheless, our study strongly suggests a role for CCR5 haplotype HHC in the pathogenesis of persistent lung involvement in sarcoidosis.

Conflict of Interest Statement: P.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; E.A.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; A.U.W. received $35,000 in January 2004 as a consultancy fee from Centricorp and currently serves, in an unpaid capacity, on an Advisory Board for Actelion; S.J.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; S.R.D. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; H.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; J.C.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; A.A. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; A.T. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; R.M.d.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; K.I.W. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

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Paolo Spagnolo, Elisabetta A. Renzoni, Athol U. Wells, Susan J. Copley, Sujal R. Desai, Hiroe Sato, Jan C. Grutters, Atiyeh Abdallah, Anne Taegtmeyer, Roland M. du Bois, and Kenneth I. Welsh

Clinical Genomic Group, National Heart and Lung Institute, Department of Occupational and Environmental Medicine, Imperial College of Science, Technology and Medicine; Department of Radiology, Hammersmith Hospital; Department of Radiology, King's College Hospital; Heart Science Center, Harefield, National Heart and Lung Institute, Imperial College, London, United Kingdom; and Department of Pulmonology, Sint Antonius Hospital, Nieuwegein, The Netherlands

(Received in original form December 20, 2004; accepted in final form June 18, 2005)

Correspondence and requests for reprints should be addressed to Paolo Spagnolo, Clinical Cenomics Group, Imperial College, 1B Manresa Road, London SW3 6LR, UK. E-mail: p.spagnolo@imperial.ac.uk

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Am J Respir Crit Care Med Vol 172. pp 721-728, 2005

Originally Published in Press as DOI: 10.1164/rccm.200412-1707OC on June 23, 2005

Internet address: www.atsjournals.org

Copyright American Thoracic Society Sep 15, 2005
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