Recent studies on SP-D have highlighted the protein's immunomodulatory function. In addition to its ability to enhance the clearance of pathogens, a dampening effect on inflammatory processes has become more apparent [7
]. First applications of rfhSP-D to mice suffering from allergic asthma also showed a dampening of the allergic response due to allergen inhalation with a modification of cytokine levels and a decrease in airway hyper-responsiveness on allergen challenge [19
]. Similar results were achieved in preterm newborn lambs which were exposed to LPS and treated with recombinant full length dodecamer SP-D or received no SP-D: in contrast to control animals the lambs which were treated with SP-D had stable lung function, a decreased systemic inflammatory response and survived [36
]. Several patient groups show decreased levels of SP-D in their BAL, such as smokers [2
], and patients with cystic fibrosis [4
]. It is possible that the administration of recombinant SP-D might become a therapeutic option for patients suffering from diseases in which native SP-D levels are low [37
]. Whilst the precise pathogenic impact of decreased SP-D levels in BAL in such diseases has not so far been clearly elucidated, it is clear that low SP-D levels are a feature of COPD, becoming more significant with years of smoking [3
Native SP-D consists of four trimeric subunits. Within each subunit four domains can be distinguished: an N-terminal cysteine rich domain, a collagen-like domain, an α-helical coiled-coil neck domain and a CRD. Possible mechanisms for immunomodulation based on the CRD and collagen-like domain have been suggested recently [39
]. Compared to native SP-D, the rfhSP-D used in the present study consists only of a small collagen-chain, the α-helical coiled-coil region and the CRD. Nevertheless, rfhSP-D expressed in E. coli possesses structural features that might be effective in binding ligands and recognizing immune cells, as revealed by high-resolution crystal structure analysis [20
]. Furthermore, previous studies demonstrated its biological activity in vivo
, although beneficial effects on lung structure have not been depicted so far [11
The present study was performed to test the hypothesis whether the findings in SP-D knock-out mice after rfhSP-D treatment which demonstrate anti-inflammatory features of this truncated protein, were accompanied by an attenuation of the structural alterations present in SP-D deficient mice. The anti-inflammatory features which have been reported previously were characterized by decreased levels of chemokines such as MCP-1 and a diminished number of apoptotic and necrotic cells. The design of the present study included two variables: total dose and onset of treatment. Thus, the specific influence of each of these variables on the overall results can not be dissected, and the possibility that distinct developmental windows may differentially influence certain parameters can not be excluded. Our data provide evidence for the biological activity of rfhSP-D on the structural remodeling of the lung. In particular, we show that 1) the pulmonary emphysema is less severe, 2) both hyperplasia and hypertrophy of type II cells are less severe and 3) the excess intracellular surfactant pool is decreased due to rfhSP-D treatment.
With respect to the emphysema, we found a significantly higher alveolar number after only 3 weeks of treatment compared to the PBS treated D0 group. Compared to wild type mice, the number of alveoli was normalized. On the other hand the total surface area of alveolar epithelium was not influenced, independent of the duration of treatment. Former studies demonstrated that the alveolar surface area of human lungs compared to healthy lungs was only decreased in severe but not in moderate or mild emphysema so that this parameter is not sensitive enough to find slight emphysematous alterations [40
]. Therefore, the total surface area of alveolar epithelium in the present study might not be appropriate as a parameter to distinguish between mild to moderate differences of the degree of emphysema. Moreover, in view of the fact that the treated mice have smaller lung volumes but the same alveolar surface area than the untreated mice it seems to be reasonable that the number of alveoli is higher in the treated mice: in smaller lungs the alveolar surface area is maintained by a higher number of alveolar septa. Consistent with this, we found a smaller mean alveolar volume, indicating smaller dimensions of the distal airspace as a result of the therapy with rfhSP-D.
As the higher number of alveoli after treatment with rfhSP-D is accompanied by a lower number of apoptotic and necrotic alveolar macrophages (which is also apparent after 3 weeks of treatment [14
]) delayed corpse clearance with attendant consequences like increased ROS-production of bystander alveolar macrophages in SP-D knock-out mice might at least partly be responsible for the remodeling processes resulting in decreased alveolar numbers. Whether the increase in alveolar number after treatment results from a decreased destruction due to suppressed inflammatory activity or a generation of new alveoli or both is not clear. Wert et al. demonstrated the progressive character of the pulmonary emphysema in SP-D knock-out mice from the third week of live on [16
]. As there was no difference in the degree of pulmonary emphysema between the three rfhSP-D treated groups, a generation of new alveoli as a result of the treatment with rfhSP-D can not be excluded. A previous study by Zhang et al. showed that a conditional expression of native rat SP-D was not able to correct an existing pulmonary emphysema according to qualitative findings, suggesting that the higher number of alveoli in our study is in part a consequence of an inhibition of alveolar destruction [41
]. Kingma et al. provided evidence that the collagenous domain of SP-D is important to prevent mice lungs from emphysema development [42
]. This study seems to contradict findings in the present study. Although rfhSP-D is missing a proper collagenous domain it was able to maintain lung structure, presumably by contributing to an anti-inflammatory environment. The anti-inflammatory effect of rfhSP-D can be explained by the model of Gardai et al. [39
]. By binding of the CRD to the signal inhibitory regulatory protein α (SIRP α) rfhSP-D inhibits NFκB and consecutively immune cell activation and MMP-expression. Kingma et al. showed however that SP-D lacking the collagenous domain was not able to both generate an anti-inflammatory environment and prevent SP-D knock-out mice from developing pulmonary emphysema [42
]. This contradiction to Gardai et al. [39
] and other recently published data [11
] was discussed as a consequence of an unanticipated change in the structure of the CRD [42
]. Moreover rfhSP-D expressed in yeast which, opposed to E. coli expressed rfhSP-D, does not have any portion of the collagen region, was not able to prevent SP-D knock-out mice lungs from emphysema development after intranasal application (our unpublished observations), indicating a role of the expression-system for the biological activity of the fragment. Alternatively, the short collagen domain of rfhSP-D used in this study might be effective in maintaining its function. As previously shown, nearly 40% of the intranasally administered rfhSP-D could be detected one hour after injection in cell free lavage (4 μg rfhSP-D) [11
]. The level of native SP-D in control mice amounts to 3 μg [11
]. According to measurements in the study by Kingma et al. the levels of the collagenous domain deprived SP-D in BAL were 11 μg/ml [42
]. Therefore a higher concentration of rfhSP-D in the present study is no appropriate explanation for the observed beneficial effect on lung structure.
Like the conditional replacement of native SP-D [41
], the treatment with rfhSP-D led to lower surfactant phospholipid levels which, in case of the treatment with rfhSP-D, were significant after 6 weeks of therapy [14
] demonstrating a reversibility of the disturbed surfactant homeostasis. In the present study, a decrease in the number of type II cells and the total volume of intracellular lamellar bodies per lung was not observed until after the same period of treatment, namely 6 weeks. These parameters were normalized after 9 weeks of treatment. This different behavior of the number of type II cells which declines after 6 weeks of treatment and the number of alveoli which is already influenced after 3 weeks of treatment might be a consequence of recently developed concepts, stating that the pulmonary emphysema and the alterations relating to type II cell and intracellular surfactant homeostasis are mediated by different mechanisms [9
]. The treatment with rfhSP-D seems to influence both mechanisms. Botas et al. first observed alterations of type II cells in SP-D knock-out mice in the third week of life, indicating a progress of these alterations with age [10
]. The fact that the type II cell number in knock-out mice is normalized even if treatment with rfhSP-D begins after the third week of life, suggests that the proliferation of these cells may be inhibited by the truncated SP-D-fragment. Alternatively, the anti-inflammatory effects of rfhSP-D may limit epithelial damage and subsequent epithelial repair.
The stereological findings in mice treated with rfhSP-D with respect to the type II cell alterations, as well as the intracellular surfactant pool, are similar to results seen in SP-D knock-out mice with additional ablation of the GM-CSF-gene [9
]. The absence of both GM-CSF and SP-D in transgenic mice brought about a reduction of type II cell volume and number per lung, as well as a correction of the total volume of lamellar bodies per type II cell and lung, indicating that the alterations with respect to type II cells and the intracellular surfactant pool due to SP-D-deficiency are at least partly mediated by GM-CSF [9
]. Consistently, previous studies in mice over-expressing GM-CSF showed a type II cell hypertrophy and also a hyperplasia [44
]. Fisher et al. found that local recombinant rat SP-D expression in the lungs of SP-D knock-out mice was able to inhibit the synthesis of saturated phosphatidylcholine by type II cells, meaning that in SP-D-deficient mice the synthesis of surfactant material is increased [45
]. The exact mechanism of this inhibition is not known but it is possible that it is a secondary effect due to a decrease of moderately elevated GM-CSF levels in SP-D knock-out mice [9
]. At any rate, the earlier the therapy with rfhSP-D starts, and with it presumably the inhibition of the synthesis of saturated phosphatidylcholine, the lower are the amounts of intracellular surfactant content. Moreover, the absence of giant lamellar bodies and the decrease in volume of the intracellular surfactant per lung and cell as a consequence of rfhSP-D therapy could be considered a result of a reduced inflammatory state.