Elsevier

Clinica Chimica Acta

Volume 272, Issue 2, 27 April 1998, Pages 101-110
Clinica Chimica Acta

Determinants of Clara cell protein (CC16) concentration in serum: a reassessment with two different immunoassays

https://doi.org/10.1016/S0009-8981(98)00006-0Get rights and content

Abstract

Clara cell protein (CC16) is a 16 kiloDalton protein secreted by Clara cells in the lining fluid of bronchiolar and bronchial epithelium. Recently, Nomori et al., using a nephelometric latex immunoassay, reported a strong correlation between serum CC16 (sCC16) and serum lipids as well as the body mass index (BMI) [Nomori H, Horio H, Takagi M Kobayashi Y, Hirabayashi Y. Clara cell protein correlation with hyperlipidemia. Chest 1996;110:680–4]. The same authors found higher values of sCC16 in males compared to females and did not detect any significant influence of tobacco smoking. Since these results are in disagreement with previous observations showing consistently a decrease of sCC16 in smokers and no influence of sex, we have reassessed in healthy subjects the determinants of sCC16 using two different assays: a particle counting-based latex immunoassay (LIA) using polyclonal antibodies and a fluorescence enzyme immunoassay (FEIA) using monoclonal antibodies. sCC16 was determined in a group of 52 female and 44 male healthy subjects (age 18 to 66 years), including 35 smokers and 61 nonsmokers. sCC16 measured by LIA and FEIA were well correlated (r=0.92, n=96, P<0.0001) with values (geometric mean and range) of 13.3 (5.2–34.5) and 14.7 (4.1–53.1) μg/l, respectively. The determinants of sCC16 measured by both techniques were traced by stepwise regression analysis using as independent variables age, sex, smoking status, BMI or serum lipids (total cholesterol and triglycerides) and the glomerular filtration rate (GFR) estimated on the basis of serum creatinine or β2-microglobulin. Only two significant determinants emerged: tobacco smoking which correlated negatively and the GFR which correlated positively with sCC16. No influence of serum lipids, BMI, age and sex on sCC16 was detected. We think that an analytical interference with serum lipids explains the results by Nomori et al. which are not confirmed here by two independent techniques and are inconsistent with the current understanding of the physiopathology of the Clara cell and its main secretory product, CC16.

Introduction

Clara cell protein is the main secretory product of Clara cells which are nonciliated cells localized predominantly in terminal and respiratory bronchioles [1]. Because of anomalous migration properties, Clara cell protein shows by sodium dodecyl sulfate polyacrylamide gel electrophoresis an apparent molecular size of 10 kiloDalton (kDa) and has been referred to as CC10 [1]. However, the exact molecular mass determined by electrospray/mass spectrometry is 15 840 and the protein is thus more correctly abbreviated as CC16 [2]. CC16 is one of the most abundant respiratory tract-derived proteins with values averaging 2% of the total protein content of lung lavage fluids [3]. From the airways, CC16 passively diffuses into plasma across the bronchoalveolar–blood barrier [4]. Like other low-molecular-weight proteins, plasma CC16 is rapidly eliminated by glomerular filtration before being taken up and catabolized by renal tubules [4]. CC16 is identical to urinary protein 1, a microprotein isolated from urine of patients with tubular proteinuria 5, 6. The exact functions of CC16 are still unknown but there is increasing evidence that CC16 has immunosuppressive and antiinflammatory properties and could play a role in lung inflammatory processes [4].

The main interest of CC16 stems from the fact that it occurs also in serum (sCC16) where it increasingly appears as a specific lung marker, reflecting the number of Clara cells and the integrity of the bronchoalveolar–blood barrier [4]. For instance, the concentrations of sCC16 are significantly reduced by tobacco smoking, a situation in which the number of Clara cells and the CC16 secretion in airways are known to be diminished 7, 8, 9, 10, 11. This effect of tobacco, consistently found in several independent studies has been recently questioned by Nomori and coworkers who, on the basis of a slight elevation of sCC16 measured by a nephelometric latex immunoassay, raised the opposite hypothesis of a stimulation of CC16 secretion induced by tobacco smoking [12]. These authors also observed higher sCC16 in males compared to females which again contradicts previous studies showing no influence of sex [12]. But the most striking finding of Nomori and coworkers is the very strong association between sCC16 and both serum lipids and the body mass index (BMI) [13]. The purpose of the present study was to resolve these discrepancies by carefully reassessing the influence of each of these factors on sCC16 among healthy subjects by using two independent immunoassays, a particle counting-based latex immunoassay (LIA) using polyclonal CC16 antibodies and a fluorescence enzyme immunoassay (FEIA) using monoclonal CC16 antibodies.

Section snippets

Population

We studied 96 blood donors (52 females and 44 males), aged from 18 to 66 years (mean, 42), including 35 current smokers and 61 nonsmokers. BMI was under 20 in 12 subjects (19.6±0.4), between 21 and 25 in 44 (22.6±1.3) and exceeded 25 among 34 (28.6±2.5). Smokers had an average (geometric mean) cigarette consumption of 18.5 cigarettes/day (range, 10–40) and an average smoking history of 13.7 pack-years (range, 2–42).

CC16 LIA

The concentration of sCC16 was determined by a sensitive immunoassay relying on

Results

The concentration of sCC16 measured by LIA of healthy subjects showed a log-normal distribution around a mean of 13.35 μg/l with values ranging from 5.2 to 34.5 μg/l. When measured by FEIA, the sCC16 geometric mean was 14.8 μg/l with a range comprised between 4.1 and 53.1 μg/l. There was a good correlation between the two immunoassays (r=0.92, n=96, P=0.0001) (Fig. 1). All subjects had a normal renal function whether estimated on the basis of serum creatinine (86.6±13.2 μmol/l) or serum β2

Discussion

The aim of the present study was to reevaluate the influence of different factors on the concentration of sCC16 in healthy subjects using two different immunoassays. Values of sCC16 in healthy subjects obtained with both assays were well correlated and in keeping with those determined with various enzyme-linked immunosorbent assay methods in Asian populations 10, 15. They were however on average four- to fivetimes lower than those reported by Nomori and coworkers 12, 13, 16. Our range of normal

Acknowledgements

This study was supported by the European Union (EV4-CT96-0171) and the National Fund for Scientific Research (Belgium). C. Hermans is Research Fellow and A. Bernard Research Director of the National Fund for Scientific Research. Dr J.C. Daniel from Rhône-Poulenc Industrie, France, is gratefully acknowledged for providing us with batches of polystrene Latex particles (ESTAPOR K109).

References (29)

  • A. Bernard et al.

    Early decrease of serum Clara cell in silica-exposed workers

    Eur Respir J

    (1994)
  • N. Shijubo et al.

    Serum and BAL Clara cell 10 kDa protein (CC10) levels and CC10 positive bronchiolar cells are decreased in smokers

    Eur Respir J

    (1997)
  • A.B. Lumsden et al.

    Goblet and Clara cell of human distal airways, evidence for smoking induced changes in their number

    Thorax

    (1984)
  • H. Nomori et al.

    Protein 1 (Clara cell protein) serum levels in healthy subjects and patients with bacterial pneumonia

    Am J Respir Crit Care Med

    (1995)
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