Using samples simultaneously obtained from the two major reservoirs of SP-D, the current study presents new data demonstrating that pulmonary and serum SP-D levels appear to be stably expressed in both patients with COPD and controls, can be influenced by smoking, and reflect the degree of airway obstruction and disease state. The highest pulmonary and the lowest serum SP-D concentrations were detected in healthy subjects. Smoking reduced the level of SP-D in BAL and increased the concentration in serum, apparently independent of age and smoking history. In active smokers with COPD, changes in BAL/serum SP-D ratio were most pronounced and for the first time, within minutes after the start of moderate exercise, an increase in serum SP-D levels with a reproducible kinetic profile was observed in smokers and in patients with COPD. Finally, we found changes in the quaternary structure of SP-D in these two groups suggesting a previously unappreciated smoking related effect. Together the data support the hypothesis for the translocation of SP-D from airways to serum and underscore the importance of concentration gradients, barrier integrity, and potentially quaternary structure in influencing the quantitative expression levels in these compartments.
The majority of SP-D production occurs in the lung with spatial localization to type II pneumocytes and Clara cells lining the distal airways . The fact that SP-D can also be detected in serum moved it into focus as a potential biomarker [12, 26]. SP-D has been analysed in BAL and serum of different patient groups before, however so far, only indirect evidence suggests lower than normal values in BAL and higher than normal values in serum of COPD patients [12, 16]. Our data show that the BAL/serum ratio is markedly changed in COPD patients. In our present study we demonstrate that drastic changes with respect to the BAL/serum ratio are especially evident in COPD patients. Under the conditions used in our study, the BAL/serum ratio was about 10-fold higher in healthy subjects as compared to COPD patients.
BAL fluid recovery from COPD patients is often potentially more variable than healthy volunteers, as indicated in Table 2. However, protein levels were not statistically different between groups making it rather unlikely that differences in BAL dilution are responsible for the observed differences in BAL SP-D concentrations. In addition, the measurement of SP-D in BAL showed a decent reproducibility and was in a similar range as described by Sims et al. . The SP-D concentrations in serum showed an even better reproducibility and were comparable to those reported by Lomas et al. . Furthermore, the stability of the BAL/serum ratios over a time period of 4 weeks indicates that this ratio reflects individual physiological conditions and disease states.
In line with previous studies we found a weak but significant correlation between SP-D levels in BAL and in serum with the degree of airway obstruction . Interestingly our correlation confirm data from a recently published study, where the ratio of SP-D in BAL and serum was shown to correlate significantly with the degree of airway obstruction in a trial with smoking subjects . These data are encouraging to use SP-D as a biomarker/surrogate marker for clinical read outs and would also justify larger validation trials.
The function of SP-D in serum if any as well as its source is still unclear. Serum SP-D levels have been shown to be steroid sensitive and to reflect an increased risk for exacerbations in patients with COPD . The origin of serum SP-D is currently considered to be the lung and raised serum levels have been related to increased concentrations in the lung e.g. during allergic inflammation , or have been suggested to be due to an increased permeability of the lung and leakage from the pulmonary site . The changes in the BAL/serum ratio observed in our study support the permeability hypothesis. However, the reproducible rapid increase during exercise in smokers and COPD patients in serum SP-D concentrations would then require a similar rapid change in alveolar/vascular permeability, which at least in healthy subjects  has not been detected before.
The similar protein concentration in BAL fluid between groups further suggest that the translocation of molecules between serum and the lung is rather complex. Our data does not solve this issue and it is important to keep in mind that SP-D is not only expressed in the lung and could therefore also be derived from other extrapulmonary sources .
Beside alterations of SP-D levels, we found remarkable changes in protein patterns after native separation and immunoblotting indicating a loss of its multimeric structure in smokers and smokers with COPD. The disruption of the multimeric structure of SP-D can have several deleterious consequences regarding its function in host defence and innate immunity , lowering the binding affinity to pathogen ligands  and might also reduce its anti-oxidant functions . The loss of multimeric integrity towards smaller subunits might also play a role in the hypothesized increased translocation of pulmonary SP-D into the circulation because SP-D molecules with lower molecular weight might more easily translocate into the systemic circulation.
Although native gel electrophoresis does not allow a prediction of the precise molecular weight of a protein, we found SP-D fragments in BAL in a range of about 200-800 kDa, indicating that it was not completely disrupted to monomers (43 kDa) but rather de-multimerized. Such degradation products have not been observed or reported so far, and it is tempting to speculate that oxidative and nitrosative stress might be the causes to initiate disruption by amino acid modification. Whether the appearance of lower multimeric forms or the susceptibility to post-tranlational modification by SNO, ONOO or crosslinking of SP-D are affected by the DNA polymorphism Thr/Thr11 described by Leth-Larsen remains speculative, since we did not analyse for polymorphisms . Further, we found smaller multimeric forms in smokers and significantly increased SP-D serum levels, whereas Leth-Larsen reported an reduction of SP-D serum levels associated with Thr/Thr11 genotypes.
Indeed, we found more nitrite in smoker compared to non-smoker BAL and first evidence for more S-nitrosylated SP-D (SNO-SP-D) in smokers (data not shown). The formation of S-nitrosothiol occurs through reactive nitrogen species and causes loss of multimerization and additional pro-inflammatory signalling activity on macrophages .
Radical oxygen species have also the potential to modify proteins, introducing carbonyl groups at certain amino acids . This modification can alter the quaternary structure, and increase the susceptibility to further degradation by proteinases. ROS are elevated in smokers , derived either directly from inhaled cigarette smoke or are released in response to smoking from various cells like neutrophils and macrophages. We could show in vitro that the native structure of SP-D can be modified by an oxidant but we were not able to detect carbonyl groups in BAL from smokers due to methodical limitations. However, in cystic fibrosis these oxidative modifications of SP-D have been observed and they were associated with a loss of functional properties i.e. a reduced agglutination of Pseudomonas aeruginosa . Increased bacterial and viral colonisation are common in patients with COPD , which might be linked to reduced pulmonary SP-D levels as well as to a potentially impaired functionality due to the observed disrupted structure . To further elucidate the role of SP-D structural modifications in COPD, a quantitative evaluation between all groups with respect to the proportions of disrupted relative to the total SP-D level will be required, and there appears to be a need to clarify if ELISA measurements are affected by these SP-D modifications.
In conclusion, we showed that pulmonary and serum SP-D levels are stable markers that are related to smoking, airway obstruction, and disease state. In addition, we demonstrated that cigarette smoke is capable to disrupt SP-D's quaternary structure, which might play a role in an impaired immunological function and an increased translocation of SP-D from the lung into the circulation.