The course of lung disease in patients with CF varies considerably, even among patients with the same CFTR genotype who are receiving standardized care. This variation is largely unexplained. We found that patients carrying 1 defective copy of the MBL gene may have an overall 11% decrease in lung function, and those carrying 2 copies have an overall 25% decrease in lung function, compared with patients homozygous for the A/A
genotype. As shown in Figure , the MBL serum levels can, in addition to the structural alleles, be further subdivided because of the effect of a base substitution in codon –221 (X/Y
In agreement with the MBL serum level findings, the lung function in the low-expressing heterozygotes is virtually identical to those with the homozygous defect. During the 9-year observation period, the lung function was more or less unchanged in the whole group of patients. However, when paired data were analyzed from data collected at 8 and 16 years of age, it became evident that patients with genotypes encoding low MBL serum levels (the MBL-insufficient group) had a slightly lower lung function at age 8, but then experienced a marked drop in lung function over the ensuing 8-year period, whereas it remained more or less constant in the MBL-sufficient group. Thus, the effect of MBL on lung function is likely initiated quite early in life and becomes progressively more prominent with increasing age.
When the data were further analyzed, we found that the difference in lung function between patients homozygous for the normal allele (A/A) and patients carrying 1 or 2 copies of defective alleles (A/0 and 0/0) was restricted to patients with chronic P. aeruginosa infection and high titers of anti–P. aeruginosa serum precipitins. MBL seemed to offer no protection against chronic colonization of P. aeruginosa, because there were no significant differences between patients with normal and defective alleles with respect to the prevalence of chronic infection or the age at onset.
The link between the molecular and cellular abnormalities in CF and colonization of respiratory epithelium with P. aeruginosa
and other pathogens is still unclear. Recent evidence has suggested that increased concentrations of sodium chloride in the lungs due to CFTR mutations may impede the killing of P. aeruginosa
by innate antibacterial defense systems (defensins) (33
). There is also evidence suggesting that the CFTR molecule itself is directly involved in clearance of P. aeruginosa
), but several other mechanisms have also been proposed. In the early phase of intermittent colonization, there is no detectable anti–P. aeruginosa
serum precipitins and presumably little local inflammation. Transition to chronic P. aeruginosa
infection coincides with an increase in serum precipitins and activation of genes coding for alginate production (2
). This results in growth of P. aeruginosa
microcolonies embedded in agar, which protects against host defense systems.
That MBL is synthesized exclusively in the liver may explain why it lacks a protective role against P. aeruginosa
colonization and subsequent infection (35
). It may reach a localized inflammatory focus by exudation relatively late in the pathophysiological process (36
). Unlike lung surfactant protein D, MBL is absent from the normal mouse lung, but appears in lung lavage fluid 3 days after experimental infection with influenza virus (37
). We found MBL (median levels: 200 μg/L) in lung expectoration from 4 CF patients (all A/A
individuals) out of 100 investigated patients (data not shown). However, it is likely that lysosomal enzymes from polymorphonuclear leukocytes degrade MBL in the lungs of CF patients, because the mixing of normal serum with expectoration or sputum was followed by a rapid decrease in detectable MBL antigen (data not shown), as has been shown for several other serum proteins (38
As there is probably little or no local inflammation in the early phase of colonization with P. aeruginosa, there may very little, if any, MBL leaving systemic circulation. Once the inflammatory process is initiated, as a consequence of the local reaction of circulating and secretory anti–P. aeruginosa antibodies with microbial antigens at foci of P. aeruginosa microcolonies, there may be exudation of MBL. However, the mechanism by which MBL may have a protective role against the inflammatory tissue destruction that is secondary to chronic P. aeruginosa infection remains elusive at this point, because MBL binds only weakly to whole P. aeruginosa bacteria in vitro (P. Garred, unpublished observations). Thus, a direct, MBL-mediated P. aeruginosa killing or opsonization is not likely in the vivo situation, but MBL might play a role in the clearance or neutralization of P. aeruginosa–derived LPS or other toxic substances released from the bacteria.
Alternatively, MBL may have a protective role against the viral infections suggested to precede P. aeruginosa
colonization and exacerbation, which in turn may slow the progression of the disease (39
). Therefore, MBL deficiency could be associated with common viral respiratory infections that render the lungs more susceptible to the damage caused by chronic P. aeruginosa
colonization. There is also the possibility that MBL deficiency may cause intrinsic immune disturbances: it has recently been shown that lack of C1q, a molecule very similar to MBL, in knockout mice is involved in the clearance of apoptotic material (40
). Likewise, MBL could be involved in the clearance of immune complexes of pathophysiological importance in CF by its interaction with a galactosylated IgG (41
). None of these hypotheses would appear to be mutually exclusive.
It is very interesting that out of 10 patients with B. cepacia
infection (2 already infected in 1989 and 8 who contracted this infection during the follow-up period), 4 were heterozygous and 3 were homozygous for MBL variant alleles, giving a highly significant increased risk of acquiring this complication in carriers of variant alleles. This is in contrast to our finding that the acquisition of P. aeruginosa
does not seem to be related to the MBL levels. However, B. cepacia
is known to lead to a much higher degree of inflammation than P. aeruginosa
, as evidenced by the quite frequent septic form of infection that is rarely, if ever, seen with P. aeruginosa
). Therefore, MBL may appear on the bronchial surface much earlier in the course of B. cepacia
infection than in P. aeruginosa
infection. MBL’s protective role against the progression of B. cepacia
infection may therefore be disclosed in carriers of variant alleles.
It is of interest that the genes for lung surfactant proteins A and D, which are proteins with similar functions as MBL, are located near the MBL2
gene on chromosome 10 (43
). Therefore, it could be argued that the effect of the MBL variant alleles is due to linkage disequilibrium with polymorphisms in one of these or in another gene. Indeed, decreased levels of lung surfactant proteins A and D have been detected recently in lung lavage fluid from CF patients (44
). Although we cannot discount the possibility that the MBL effect on lung function and survival is due to linkage disequilibrium, the fact that each of the B
alleles are significantly associated with decreased lung function argues against this notion.
Because MBL variant alleles are so frequent in the healthy population, it is conceivable that multiple genetic factors may influence susceptibilities and outcomes in which MBL deficiency plays a role. It has been shown that concomitant occurrence of MBL variant alleles and IgG subclass deficiency may increase risk of infections (12
), and that concomitant occurrence of polymorphisms in the FcγRIIa receptor and MBL deficiency is associated with autoimmunity in chronic granulomatous disease (46
). Likewise, the possession of both complement C4-null alleles and MBL variant alleles has been shown to increase susceptibility to systemic lupus erythematosus (18
The high frequency of MBL variant alleles in different populations indicates that MBL polymorphisms represent a balanced genetic system favoring variant alleles arising from genetic selection (47
). In sub-Saharan Africa, more than 50% of the population carry the C
allele, and in certain South American Indian tribes, more than 70% of the population carry the B
). Thus, the normal A
allele may, under some circumstances, confer disadvantages to the host.
During the 10-year follow-up period, 14 patients died and 12 underwent lung transplantation. Of the 26 patients, 24 had chronic P. aeruginosa infection and 2 had chronic B. cepacia infection. Of these same 26, 12 were heterozygous and 3 were homozygous for MBL variant alleles. When death and lung transplantation are viewed as end-stage disease, this group of patients had a marked increase in risk of end-stage disease and a significantly reduced survival time. Taking their age at inclusion into consideration, and using a modified life table analysis, revealed that the expected median survival was about 8 years shorter in those carrying MBL variant alleles.
Our data indicate that a shortened life-span in carriers of variant alleles results primarily from the more aggressive course of lung disease caused by chronic P. aeruginosa infection, but they also indicate that carriers of variant alleles are at high risk of acquiring B. cepacia infection and this infection is often associated with an even greater mortality than chronic P. aeruginosa colonization. However, our data also raise the possibility that future patients may benefit from substitution therapy with purified or recombinant MBL.