Print version ISSN 0037-8682
Rev. Soc. Bras. Med. Trop. vol.45 no.1 Uberaba Jan./Feb. 2012
CASE REPORT RELATO DE CASO
Expressão espontânea anômala do receptor de interleucina 8: um breve relato de dois casos
Paulo AntasI, II, III; Steven HollandII; Timothy SterlingIII, IV
ILaboratório de Imunologia Clínica, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ
IILaboratory of Clinical Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
IIIDivision of Infectious Diseases, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN, USA
IVCenter for Health Services Research, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN, USA
Interleukin 8 (CXCL8) is an autocrine chemokine specific for the chemoattraction and activation of granulocytes, NKT cells and T lymphocytes. Patients with tuberculosis and latent Mycobacterium tuberculosis infection were assessed for the spontaneous expression of CXCR1 (CD128) and CXCR2 on lymphocytes and monocytes. Compared with ex vivo profiles, increased spontaneous CXCR2 expression and normal CXCR1 expression were found on lymphocytes in two out of 59 individuals. Monocytes showed normal ex vivo profiles for both receptors. After stimulation with purified protein derivative, the in vitro levels of CXCL8 were below the median levels of all patients with prior tuberculosis. Spontaneous CXCR2 modulation did not cause notable variation in the in vitro levels of CXCL8.
Keywords: Interleukin 8 receptor. Lymphocytes. Cytokines.
Interleucina 8 (CXCL8) é quimiocina autócrina específica para atração e ativação de granulócitos, assim como NKT e linfócitos T. Pacientes com infecção por Mycobacterium tuberculosis e latentes foram recrutados para comparar expressão espontânea dos receptores CXCR1 (CD128) e CXCR2 nos mononucleares. Comparado com perfis ex vivo dos linfócitos, observou-se aumento em CXCR2; porém, expressão normal de CXCR1 em dois dos 59 indivíduos. Monócitos mostraram perfis ex vivo normais; após estimulação específica in vitro das citocinas estudadas com extrato bruto total, não se encontrou correspondência na anomalia observada ex vivo. Modulação espontânea de CXCR2 não causou grande variação in vitro nos níveis de CXCL8.
Palavras-chaves: Receptor de interleucina 8. Linfócitos. Citocinas.
Interleukin 8 (CXCL8), an important autocrine chemokine, belongs to the glutamic acid-leucine-arginine-CXC chemokine family that specifically chemoattracts and activates human neutrophilic granulocytes (as previously reviewed1). To date, mice do not appear to produce or respond to this putative chemokine. Two CXCL8 receptor subtypes have been identified on human leukocytes: receptor A, or CXCR1 (later designated CD128), and receptor B, or CXCR22. Both receptors share 77% amino acid homology but differ in their binding properties. CXCR1 is specific for CXCL6 and CXCL8 with high binding affinity, whereas CXCR2 binds to CXCL8 and other CXC chemokine family members with equally high affinity. Binding to CXCR2 usually promotes angiogenesis and endothelial cell chemotaxis. Normal granulocytes not only produce a large amount of CXCL8 but also promptly respond to CXCL8 stimulation via these two receptors (as previously reviewed1). In a pioneering study, staining of T cells for induced CXCR1 and CXCR2 expression showed strongly elevated levels of expression exclusively in NKT cells and CD8+ T cells3. In addition, CD8+ T lymphocytes, but not B cells or CD4+ T cells, expressed CXCL8 receptors. Lymphocytes also expressed higher levels of CXCR2 than CXCR13.
In this study, the specific and spontaneous ex vivo CXCR1 and CXCR2 expression levels were compared by flow cytometry. In parallel, cytokine levels were also measured in stimulated primary cell cultures. We compared 2 cohorts of individuals and determined the presence of any abnormality in either CXCL8 receptors or spontaneous expression.
Twenty-nine prior tuberculosis (TB) patients and 30 control patients with latent Mycobacterium tuberculosis infection were identified through the Baltimore City Health Department Eastern Chest Clinic and the Nashville Metropolitan Health Department Tuberculosis Clinic (USA). The eligibility and exclusion criteria have been described elsewhere4. This study was approved by the institutional review boards (IRB) of both sites. All study participants provided written informed consent. Peripheral blood mononuclear cells (PBMC) were purified (to > 92% purity) within 24h of obtaining the specimens from study participants using a previously described protocol4, and the flow cytometric staining was assayed and analyzed as described before with slight modifications5. Cytokine levels were measured by a multiplex bead array, and CXCL8 was detected at 96h via enzyme-linked immunosorbent assay (ELISA) after stimulation with purified protein derivative (PPD). Anti-HLA monoclonal antibodies were purchased from Pharmingen Inc. (USA). IgG2a-isotype control antibodies and anti-CXCR1 and anti-CXCR2 monoclonal antibodies were obtained from Becton & Dickinson Biosciences Co. (USA). PPD was regularly purchased from Statens Serum Institut (Denmark). Escherichia coli-derived lipopolysaccharide (LPS) was obtained from Sigma-Aldrich (USA). Recombinant human IFN-Γ was obtained from Genentech (USA), and a commercial CXCL8 ELISA kit was obtained from Pierce (USA).
The levels of ex vivo CXCR1 and CXCR2 expression at the screening and the single-cell levels were compared among persons who had had been previously treated for TB and among control patients with latent M. tuberculosis infections. Table 1 depicts the spontaneous levels found for each patient group. Due to the homogeneous pattern, all data were further combined for receptor expression (below). As an internal control, negative responses to isotype control antibodies were detected in all individuals tested (Figure 1). All individuals assayed demonstrated standard staining for CXCR1, showing means (+ SEM) values of 3.7% (+ 0.3) and 63.6% (+ 2.1) in the lymphocyte and monocyte (R1) gated populations, respectively (Figures 1A and B). Likewise, 57 examined individuals showed a similar, regular pattern for CXCR2 expression on both lymphocytes (Figure 1C) and monocytes (2.2% + 0.1 and 65.2% + 2.3, respectively). In contrast, two clinically characterized prior extrapulmonary TB patients showed spontaneously high CXCR2 expression ex vivo. The first patient was a 47-year-old male (#ID: 125) who was culture-positive for M. tuberculosis with a PPD reaction of 18 mm, who responded well to the standard treatment and who had had pleural effusion plus extensive infection (Table 1). The second subject was a 52-year-old male (#ID: 162) who was culture-positive for M. tuberculosis with no reaction to PPD, who responded well to standard treatment and who exhibited bone/joint lesions (Table 1; Figure 1C). During sample handling, the latter patient showed an increased number of erythrocytes when the PBMCs were purified. However, a normal blood pattern was exhibited when the monocytes of those cases were also compared, indicating that the abnormality did not extend to the other mononuclear populations, making it unlikely that an artifact was observed during the cell purification and staining procedures (Figure 1D). In addition, the remote possibility of cross-staining of the granulocyte population was ruled out because all studied patients showed similar light scattering profiles (Figure 2). Remarkably, the cytokine profile did not change significantly (data not shown), even though the CXCL8 level was slightly decreased at 96h when PBMCs (106 cells/ml) were cultured after PPD (10mg/ml) stimulation (Table 1). To better explore this abnormality, staining for HLA was also performed in parallel for one patient (#ID: 162). As expected, monocytes exhibited a positive modulation for HLA when LPS (200ng/ml) and IFNg (1,000U/ml) were used alone or in combination: 19.0-fold, 92.6-fold and 67.4-fold for LPS, IFNg and LPS+IFNg, respectively. This result ruled out possible non-responsiveness during the cell culture procedures. Unfortunately, because of IRB conflicts at the time that this study was performed, no additional analysis could be carried out.
It is hypothesized that increased serum CXCL8 regulates its own receptors on granulocytes by inducing receptor internalization and proteolytic cleavage (as previously reviewed1). The same hypothesis could be extended to other cell populations, such as T lymphocytes. In addition, blocking experiments with neutralizing anti-CXCL8 antibodies and pretreatment with bacterial LPS, which rapidly downmodulates CXCL8 receptor levels, showed that the inducing activity was due to the presence of CXCL8 in the conditioned medium6. Nevertheless, the almost consistent in vitro CXCL8 levels that were found in the present study cannot explain the disparities in ex vivo CXCR2 expression. A recent study showed that CXCR2 expression is affected by medications (as previously reviewed1). In another setting, increased gene expression of CXCL8 and the corresponding receptors was detected in PBMCs, mainly during chronic heart failure, resulting in the selective recruitment of leukocyte subpopulations into inflamed tissues7. However, marked abnormalities in genes from those patients were significantly modulated in a normal direction during specific therapy. Three single-nucleotide polymorphisms (SNP) have been reported in the CXCR2 gene. The +1208 T/C SNP, located in the non-coding region of the CXCR2 gene, might provide valuable information for the pathogenesis of and susceptibility to chronic inflammatory disease8. In addition, a highly significant association has been found between the homozygous +1208 TT genotype and an aggressive phenotype of breast carcinoma8. Furthermore, the +1440 G/A SNP and some haplotypes are associated with periodontitis in Brazilian individuals. In contrast, the +785 C/T SNP, located in exon 11 and resulting in a silent codon change (no amino-acid substitution), may be important in protecting against pulmonary inflammation, despite exhibiting no association with Kawasaki disease. Thus, CXCR2 is an important and interesting new candidate gene that is potentially involved in regulating the inflammatory process in the airways9. It has been postulated previously that the CXCR2 +785 T allele may be associated with protection against decreased expiratory flow rates and anomalous gas exchange in chronic obstructive pulmonary disease (COPD) and with a number of quantitative spirometric abnormalities, such as reduced lung function and respiratory symptoms10. Conversely, this SNP is associated with angiogenesis in systemic sclerosis in individuals homozygous for the CXCR2 +785 C allele8. Although differential expression in both PBMC populations ruled out a primary deficiency, any association of the CXCR2 gene SNPs and the 2 individuals described here deserve further investigation, as does the analysis of the intrinsic regulation of monocytes and lymphocytes by CXCR2 because chemokine receptors are selectively and differentially expressed on different leukocyte subsets in individuals with arthritis.
In the current study, 2 patients showed stimulated-CXCL8 production that fell within the range of all patients tested and the normal range of CXCR1 expression, but those 2 patients showed abnormal CXCR2 expression when compared with the other analyzed individuals. A probable pharmacological explanation seems unlikely because those 2 individuals were medication-free (except for TB treatment) for more than two years. Unfortunately, for logistical reasons, neither patient could be followed after the completion of this study. Detailed clinical histories were not available. However, previous complete blood count analyses did not show any discrepancies between the 2 cases and the rest of the cohort. Unlike CXCR1, CXCR2 can bind other CXC chemokines with high affinity. Accordingly, CXCR2 plays more a pleiotropic role in inflammation than does CXCR1 (as previously reviewed1). This difference may be detected in the 2 patients found here. It is worth noting that the regular cytokine levels found in the 2 patients do not seem to explain the disparities found in this study. In addition, the CXCR2 observed on granulocytes in patients suffering from active systemic lupus erythematosus is more important than CXCR1 in CXCL8-mediated inflammatory responses. The real mechanism of the increased spontaneous CXCR2 expression on lymphocytes from the 2 patients remains to be elucidated.
In conclusion, two original results were found: I) increased ex vivo spontaneous expression of the CXCL8 receptor CXCR2, but not CXCR1, was observed in two individuals, whereas II) the secretion of CXCL8 and other cytokines was not substantially impaired. This abnormal modulation of CXCR2 in these atypical cases warrants further investigation.
The authors are grateful to Mrs. Liza Krassner and Dr. Stuart Krassner (UCI, Irvine, USA) for editing the text of this manuscript, the staff for assistance with laboratory facilities, and the nursing staff of the TB ward for helping with patient recruitment.
This study was partly financed by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (Brazil). P.A. is currently under a Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) research fellowship program (PQ-2, Brazil).
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Dr. Paulo Antas
Lab. Imunologia Clínica/IOC/FIOCRUZ
Av. Brasil 4365
21045-900 Rio de Janeiro, RJ, Brasil
Fax: 55 21 2290-0479
Received in 26/11/2009
Accepted in 17/01/2011