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Departments of
* Internal Medicine,
Laboratory Medicine, and
Immunobiology, Yale University School of Medicine, New Haven, Connecticut, USA
1Correspondence: Yale University School of Medicine, Internal Medicine-Pulmonary and Critical Care Division, 333 Cedar St., TAC 441-S, New Haven CT 06511, USA. E-mail: erica.herzog{at}yale.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: plasticity • fusion
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. While the precise mechanisms for this phenotypic change remain unclear, one known process is that of heterokaryon formation between circulating donor hematopoietic cells and preexisting recipient somatic cells, which has been found in such diverse populations as Purkinje cells (2
, 3)
, hepatocytes (3
4
5)
, myocytes (6)
, and specialized epithelial populations of the colon (7)
and kidney (8)
. In epithelial populations, heterokaryon formation appears to require secondary inflammation or aging to develop in post-transplant models that use radiation for preconditioning. However, fusion is not always detectable (9)
. While low-level marrow contribution to the lung epithelia has been found both by our group (10
, 11)
and others (12
13
14
15)
, prior work has failed to detect heterokaryon formation in the lungs of transplanted mice (3
, 9)
. Whether or not fusion occurs in the lung is important, for this concept affects the therapeutic potential and diseases for which such an approach could be clinically meaningful. We tested the hypothesis that secondary inflammation after total body irradiation and sex mismatched wild-type (WT) BMT into male and female mice that lack Sp-C results in the appearance of heterokaryons comprised of transplanted BMDCs and recipient lung epithelia. We selected Sp-C null mice as recipients for two reasons: first, because the ability of BMDC to activate a previously silent gene (Sp-C) could be used to assess for lung-specific nuclear reprogramming of marrow-derived lung epithelial cells (MDLE); and second, to assess for amelioration of the inflammation and alveolar destruction caused by the lack of Sp-C. Furthermore, we tested the nuclear stability of male BM-derived epithelial cells by fluorescence in situ hybridization (FISH) for maintenance of the Y chromosome. Last, we sought a therapeutic benefit for any restoration of Sp-C expression in the recipient lung.
Our findings indicate that lung-specific reprogramming of transplanted BMDCs occurs after bone marrow transplant (BMT) and secondary injury; low-level Sp-C restoration was found in 50% of transplant recipients regardless of sex. In male recipients of female BM, the majority of Sp-C-positive cells contained the Y chromosome, indicating they had been derived by fusion. In female recipients of male BM, fusion was confirmed by the presence of Y chromosome-positive, cytokeratin-expressing (CK+) cells with unequal numbers of the sex chromosomes. We also found that Y chromosome loss was a common event in BM-derived Sp-C+ cells in female recipients. Low-level Sp-C restoration had no effect on the lung inflammation caused by the Sp-C null state. These results show that lung-specific reprogramming of BMDCs is accompanied by heterokaryon formation and Y chromosome loss after BMT and secondary lung inflammation and, although not therapeutic in this model, may be beneficial in other types of lung disease.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. In these mice, targeted disruption of exon 2 causes complete abrogation of normal Sp-C expression at the mRNA and protein levels. The resulting strain-dependent lung inflammation manifests as the mice age, with a mild increase in age-related emphysema and interstitial pneumonitis. Wild-type littermates were used as BM donors and controls.
Lethal irradiation and bone marrow transplantation
After myeloablation with 850 cGy total body irradiation using a 137Cs source, 4- to 6-wk-old male and female Sp-C null mice received a tail vein injection of 3 million unfractionated nucleated BM cells harvested from age-matched opposite sex WT or Sp-C null donors as described previously (10)
. Controls included Sp-C null recipients of sex mismatched Sp-C null marrow, untransplanted Sp-C null mice, and untransplanted WT mice. Following BMT, the mice received antibiotics and autoclaved food and water for 1 month, after which they received routine daily care. Each group contained 4–7 mice and each experiment was performed three times. As pilot work had demonstrated that 
50% of Sp-C null recipients of WT marrow showed restored Sp-C expression, the group containing WT 
Sp-C null recipients contained approximately twice as many mice as the controls in order to have equivalent numbers of WT marrow recipients that did and did not show Sp-C restoration.
Sacrifice and lung harvest
Six months after BMT, mice anesthetized with ketamine/xylazine underwent bronchoalveolar lavage (BAL) as described (17)
via insertion of a 23-gauge angiocatheter into the trachea, followed by instillation of 1.5 ml ice-cold PBS. The BAL cell count was performed using a Neubauer hemacytometer. Right ventricular perfusion was performed using the standard method of median sternotomy, right heart puncture, and infusion of 10 ml ice-cold PBS into the pulmonary circulation. The left lung was then singly cannulated and removed for enzymatic digestion (see below). The right lung was inflated to 20 cm water pressure and fixed in 10% buffered formalin for 4 h. It was then transferred to 70% ethanol prior to paraffin embedding, sectioning, and H&E and Sirius red staining.
Single cell preparations and cytospins
Single cell suspensions and cytospin preparation of the left lung were undertaken as described previously (10)
. Briefly, the left mainstem bronchus was cannulated with a 23-gauge angiocatheter (Becton-Dickinson, Franklin Lakes, NJ, USA) inflated with 2.4U/ml dispase (Roche, Mannheim, Germany), followed by warm 1% low melt agarose, placed on ice for 2 min, then immersed in 1 ml of dispase solution for 45 min at room temperature. It was then minced in DMEM supplemented with100U/ml DNAseI (Roche) and sequentially sieved through a 40 µ filter (Falcon, Oxnard, CA, USA) and 22 µ nytex mesh (Sefar America, Kansas City, MO, USA). The resulting homogenate was centrifuged at 800 g for 10 min, washed with PBS, resuspended in PBS with 3% BSA and 5 mM EDTA, spun onto Surgipath (Richmond, IL, USA) precleaned slides in a Thermo-Shandon cytospin (Pittsburgh, PA, USA) at 800 rpm for 5 min, fixed in 2% paraformaldehyde (PFA) for 5 min, air dried overnight, and stored at –80°C.
RNA isolation, reverse transcriptase PCR for Sp-C and ß-actin
Total RNA was extracted from 500,000 unfractionated lung or BM cells using the RNeasy Protect Mini kit (Qiagen, Valencia, CA, USA). RNA was treated with DNase I (Roche) and reverse transcribed using Superscript II reverse transcriptase with random hexamer primers (Invitrogen, Carlsbad, CA, USA). PCR for Sp-C was performed on 2 µg of the cDNA with a forward primer spanning the ApaIII site used to insert the neomycin resistance cassette into exon 2 of the native Sp-C gene and reverse primer in exon 5. The sequences are Fwd 5' ATG GAG AGT CCA CCG GAT TAC Rev 5' ACA GAC TTC CAC CGG TTT CTG. Control PCR for ß-actin was also performed using Fwd 5'-GTG GGC CGC TCT AGG CAC CA-3'; Rev 5'-CGG TTG GCC TTA GGG TTC AGG-3'. As these ß-actin primers do not cross an exon boundary, they were used as a control for both RNA stability and detection of genomic DNA contamination when used in the absence of the reverse transcriptase enzyme. All PCR products were analyzed on 2% agarose gels.
Pro-Sp-C and Sp-B immunofluorescence on sections
Deparaffinized and rehydrated 12 µ sections were placed in PBS/0.25% Triton-X (Tx) for 30 min at room temperature, blocked for nonspecific staining for 2 h with PBS/0.25%Tx/2% normal goat serum, and incubated with 1:500 primary antibody (either Sp-B or pro-Sp-C, both from Chemicon, Temecula, CA, USA) overnight at 4°C. Slides were washed in PBS for 30 min and detected using 1:500 anti-rabbit Texas red or anti-rabbit FITC (both from Invitrogen), washed, and mounted with vectashield containing DAPI (Vector Labs, Burlingame, CA, USA). Slides were alternately detected with Texas red or FITC antibodies in order to avoid artifact related to autofluorescence in one channel. To quantify total type II cells per region of lung, serial sections were stained for Sp-B, which is expressed at normal levels in these mice.
Fluorescence in situ hybridization after pro-Sp-C staining
To evaluate for fusion and Y chromosome loss, the 12 µ paraffin sections on which pro-Sp-C staining had been detected were subjected to fluorescence in situ hybridization (FISH) for the Y chromosomes using a modification of published protocols (10)
. After coverslips were washed off using PBS, the slides were immersed in 1M sodium thiocyanate at 80°C for 20 min, digested with 0.2N HCl at room temperature for 12 min, washed in PBS, serially dehydrated in 70%/95%/100% ethanol, and air dried. Digoxigenin-labeled Y chromosome probe (template kind gift of Michael Speicher, Germany) (18)
was applied in standard hybridization buffer. Slides were coverslipped, sealed with rubber cement, and denatured at 73°C for 5 min, followed by overnight hybridization at 37°C. Slides were then washed in 2 x SSC/50% formamide at 42°C for 15 min followed by 0.2 x SSC at 42°C for 15 min, blocked and detected with 1:10 rhodamine or FITC-conjugated antidigoxigenin antibody (Roche) for 30 min at 37°C, washed in PBS, air dried, and coverslipped using vectashield with DAPI.
Cytokeratin immunofluorescence followed by X-Y FISH
PFA-fixed cytospins were thawed, air dried, permeabilized with 0.25% Triton-X in PBS for 5 min, washed with PBS, incubated with anticytokeratin (DAKO, Carpinteria, CA, USA) overnight at 4°C, washed in PBS, incubated with 1:500 anti-rabbit FITC, serially dehydrated in 70/95/100% ethanol, and air dried. Biotinylated X probe (Cambio, Cambridge, UK) and digoxigenin-labeled Y probe were applied and the slides were sealed, denatured, and hybridized as above. The next morning, slides were washed and the chromosomal probes were detected using 1:400 SA-647 (Invitrogen) and 1:10 rhodamine-conjugated antidigoxigenin antibody for 30 min at 37°C, PBS washed, and coverslipped using vectashield with DAPI.
Fluorescence in situ hybridization on bone marrow cytospins
BM cytospins were brought to room temperature, permeabilized, and analyzed by Y chromosome FISH as described above.
Quantification of alveolar chord length on sections
Nonoverlapping pictures of H&E-stained lung sections were taken using the 10 x objective. Where necessary, vessels and large airways were excluded. Pictures were then analyzed on a Macintosh computer using the public domain NIH Image program available from the web site http://rsb.info.nih.gov/nih-image. Analysis was performed with sequential logical image match and operations with a horizontal, then vertical, grid. Five measurements per field were obtained for all animals including the positive and negative controls. One slide was analyzed per mouse. The length of the lines overlying air spaces was averaged as the mean chord length.
Data acquisition and analysis
Light microscopy of sections and fluorescence microscopy of sections and cytospins were performed on an Olympus BX51 microscope equipped with a SensicamQEcamera. IPLAB software was used to capture images. When Sp-C+ cells were identified, they were marked and rephotographed using a Zeiss LSM-10 confocal microscope equipped with argon, 543, 633, and chameleon two photon lasers. Confocal microscopy with 3-dimensional z-stack analysis was used to reimage these cells following Y chromosome FISH. To avoid error related to partial sampling of nuclei, only those cells whose complete nucleus was included within the 12 µm section were counted. All microscopic analyses were performed with the investigator blinded regarding the identity of the slides analyzed.
Statistical analysis
Differences between means were calculated by the Student’s t test using Microsoft Excel Software. Statistical significance was achieved when P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Results for males and females are discussed separately.
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The Sp-C null male recipients of WT female marrow contained 93.85 ± 1.21% marrow cells that lacked a Y. This did not differ significantly from the Sp-C null recipients of Sp-C null female marrow (94.18±2.48, P<0.67). These Y chromosome counts differed from untransplanted male WT and Sp-C null controls, which contained occasional cells that lacked the Y chromosome (0.8±0.8%, P<0.001, 0.92±1.03%, P<0.001).
In female Sp-C null recipients of WT male marrow, 94.18 ± 2.48% of BM cells contained the Y chromosome. This did not differ significantly from Sp-C null recipients of Sp-C null male marrow (93.28±1.78%, P<0.46). There was no signal for Y chromosome in the untransplanted WT or Sp-C null females.
Sp-C mRNA is detected in Sp-C null recipients of WT marrow
Sp-C mRNA was detected by RT-PCR in the lungs of 19/38 (50%) of Sp-C null recipients of WT marrow. All of the WT mice were positive for Sp-C gene expression. No Sp-C mRNA was detected in the lungs of Sp-C null recipients of Sp-C null marrow or in untransplanted Sp-C null mice (Table 1
, Fig. 1
). Sp-C mRNA was not detected in the bone marrow of any mice (data not shown).
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Pro-Sp-C-positive cells are detected in Sp-C null recipients of wild-type marrow
Pro-Sp-C immunofluorescence and 3-dimensional confocal microscopy on sections of lungs from Sp-C null male recipients of female WT marrow identified 1.08 ± 1.38 pro-Sp-C-expressing cells per region of lung containing 1000 Sp-B + type II cells (as assessed by serial sections). These results differed significantly from the Sp-C null recipients of Sp-C null marrow and untransplanted Sp-C null mice, which contained no pro-Sp-C-expressing cells (P<0.001 for both). Wild-type mice contained pro-Sp-C-expressing cells at a frequency of 1000 cells per 1000 type II cells. All data are presented in Table 1
(males and females pooled) and Fig. 2
.
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Similar to the male Sp-C null recipients, female Sp-C null recipients of WT male marrow expressed pro-Sp-C at a rate of 0.8 ± 1.41 per 1000 type II cells, which was significantly different from controls. Sp-C null recipients of Sp-C null male marrow and untransplanted Sp-C null mice contained no pro-Sp-C-expressing cells, and WT mice contained 1000 Sp-C + cells per 1000 type II cells. Data are presented in Table 1
. For all of the Sp-C null recipients, Sp-C proprotein was detected only in mice that had a positive signal for Sp-C mRNA by RT-PCR. One mouse that exhibited PCR product for Sp-C did not exhibit immunostaining for Sp-C proprotein.
Fusion detected in the majority of Sp-C-expressing cells in male recipients
Fusion was detected in the Sp-C null male recipients of WT female marrow by identification of pro-Sp-C-expressing cells that contained the Y chromosome using confocal analysis of 12 µ sections. In the male recipients of female marrow, 65.4 ± 8.9% of Sp-C-expressing cells contained the Y chromosome (Fig. 3
, Table 2
). This analysis could not be performed in the male recipients of Sp-C null female marrow or untransplanted Sp-C null males as there were no pro-Sp-C-expressing cells. In untransplanted WT males, all Sp-C+ cells contained the Y chromosome (Table 2)
.
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Fusion detected in the majority of marrow-derived epithelial cells in female recipients
In the female Sp-C null recipients of WT male marrow, CK-expressing cells containing the Y chromosome were found at a frequency of nearly 0.1% (0.95±0.63 cells/1000 CK+ cells), which is similar to the frequency of marrow-derived Sp-C+ cells. Fusion (assessed by an unequal number of the sex chromosomes) was found at a frequency of 0.63 ± 0.43 per thousand, or 63% of MDLE (Table 3
). CK+ Y chromosome+ cells were found as binucleate cells with two distinct nuclei, one containing and one not containing the Y chromosome (XX/XY or any variation thereof), and as mononuclear cells containing unequal numbers of the sex chromosomes (Fig. 4A
and Fig. 4B
, respectively). Detection of Sp-C expression by RT-PCR or immunostaining did not affect the level of fusion (data not shown). The frequency of fusion was similar in female Sp-C null recipients of Sp-C null male marrow (1±0.48 Y-containing cells per 1000 CK cells [P>0.14]), of which 71 ± 48% had an unequal number of X and Y chromosomes, P>0.36). No Y chromosome+ cells were found in untransplanted Sp-C-null or WT female mice. (Table 3)
.
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Y chromosome loss detected in most Sp-C-expressing cells in female recipients of male marrow
Y chromosome loss was quantified by assessing the Y chromosome content of pro-Sp-C + cells in the lungs of female Sp-C null recipients of WT male marrow. In these mice, Sp-C expression can only be derived from a transplanted cell, which in this case was male and contained a Y chromosome. Any cell that expresses Sp-C without a Y chromosome is a marrow-derived cell that has lost its Y. (Note that only those cells for which the entire nucleus was present in the 12 µm section were counted.) The frequency of Sp-C+ Y chromosome negative cells was 0.53 ± 1.09 per 1000 type II cells or 72% of the pro-Sp-C + cells (Table 3
, Fig. 4
C, D). No Sp-C-positive cells in the untransplanted WT females contained the Y chromosome. This analysis was not performed in the untransplanted Sp-C null and Sp-C null recipients of Sp-C null marrow due to the lack of pro-Sp-C detection.
Low-level Sp-C gene transfer does not decrease lung inflammation caused by lack of Sp-C
As shown in Fig. 5
A, among male mice the transplant recipients showing Sp-C restoration had a total BAL cell count of 4.16 ± 1.26 x 104 cells, which was significantly decreased compared with Sp-C null recipients of WT BM, in which no Sp-C was detected (6.28±2.0x104, P<0.04), and compared with untransplanted Sp-C null controls (8.11.22x104, P<0.01), but still significantly higher than the BAL cell count of WT controls (8.7±5x103, P<0.0005). However, there was no difference between the total BAL cell count of the recipients with restored Sp-C expression and the Sp-C null recipients of Sp-C null marrow (3.75±2.61x104).
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In female Sp-C null recipients of WT marrow in which Sp-C was detected at the mRNA and/or protein level, the mean total BAL cell count was 5.18 ± 3.11 x 104 (Fig. 5B
). Sp-C null recipients of WT marrow that did not show evidence of Sp-C expression had a mean cell count of 6.87 ± 3.6 x 104. These numbers were not statistically different (P<0.29), indicating that a low level of Sp-C restoration did not affect the inflammatory phenotype. Sp-C null recipients of Sp-C null marrow had a mean BAL cell count of 8.09 ± .44 x 104. The difference between this value and those of the recipients of WT BM that demonstrated Sp-C restoration approached statistical significance (P<0.06). There was no difference between these mice and the untransplanted Sp-C null mice (5.5±10.07x104, P<0.79). All of these Sp-C null mice had significantly higher BAL cell levels than WT female mice (1.22±1.08x104, P<0.05 for all values). Data are shown in Fig. 5B
.
Low-level Sp-C transfer does not affect histological indices of lung damage
Qualitative review of H&E-stained lung sections revealed that all Sp-C null mice exhibited regional emphysema and a monocytic interstitial inflammation with thickened alveolar septa and regions of atelectasis compared with the WT mice (Fig. 6
). Interstitial inflammation was greater in the Sp-C null than WT mice regardless of transplant status and/or restoration of Sp-C gene expression (Fig. 6B-E
). Age-matched WT mice exhibited moderate emphysema with little inflammation. Sirius red staining did not reveal subepithelial or interstitial fibrosis in any of these mice (data not shown).
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Low-level Sp-C transfer does not affect alveolar chord length
There was only a very modest, but statistically significant, increase in chord length due to Sp-C deficiency in female mice (Fig. 5D
) and no effect of Sp-C deficiency on chord length in male mice (Fig. 5C
). WT male and female mice showed age-related alveolar enlargement. There was no significant difference in alveolar chord length between transplanted and untransplanted Sp-C null mice.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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20
21)
.
Two earlier papers failed to find marrow-derived cells expressing Sp-C or a transgene expressed from the Sp-C promoter (22
, 23)
. The reasons for the differences between these papers and our current findings are likely due to the secondary inflammation related to the Sp-C null phenotype at the late post-transplant time point at which we performed our analysis and/or transgene failure in the prior studies. Thus, while not directly comparable to these negative studies, our results provide insight into the conditions necessary for the induction and detection of lung-specific reprogramming of marrow-derived cells.
The important role of Sp-C in pulmonary homeostasis is highlighted by the fact that Sp-C deficiency causes chronic lung disease in humans and mice (16
, 24)
. Sp-C is expressed uniquely in pulmonary epithelial progenitors during embryogenesis (25)
and, in the adult lung, in type II pneumocytes (20)
(which repopulate both alveolar epithelial lineages in response to injury) and bronchoalveolar stem cells (BASCs, which repopulate the damaged airway after inhalational injury; ref. 26
). The location and morphology of the Sp-C+ cells in our study reveal them to be type II cells. It is intriguing to consider these marrow-derived, Sp-C-expressing cells in the light of recent data showing that BMDCs may fuse with crypt cells to give rise to all four epithelial lineages in the colon (7)
. Whether Sp-C-expressing cells derived from BM can act as progenitor cells in the lung remains to be seen.
Mechanism
In the male recipients of female BM, fusion of transplanted marrow with resident cells in the recipient lung accounted for > 50% of BM-derived Sp-C+ cells. Detection of Y chromosome+ cells expressing Sp-C shows that reprogramming of the donor cell has occurred by fusion with the recipient cell. In female recipients of male BM, the presence of Y chromosome+, CK+ cells with an unequal number of X and Y chromosomes may provide evidence of fusion in the female lung as well. The fate of these heterokaryons is variable, as some were binucleate cells with distinct XX and XY nuclei and others were mononuclear with unequal X/Y chromosomal content. This aneuploidy could also result from endomitosis of a male-derived cell, followed by Y chromosome loss rather than fusion. However, as fusion has been found to be the main mechanism for the appearance of marrow-derived muscle, liver, and GI tract cells, it is more likely that the non-2N cells resulted from heterokaryon formation.
To date, this type of in vivo fusion has been shown under the conditions of stress (9
, 27)
. It is not clear whether fusion of BM cells with transformed colonic epithelia (7)
contributes to ongoing pathology or represents a repair mechanism. The fact that many of these cells have lost the Y chromosome may be an indication of accelerated senescence related to abnormalities associated with cell fusion. Whether genomic instability and mutagenesis accompanies this Y chromosome loss requires further study. Further study is also needed to determine whether cellular reprogramming can be used to rescue injured epithelia or whether it poses a novel therapeutic target for the prevention of lung disease.
A number of explanations exist for the BM-derived epithelial cells that apparently formed without fusion. The finding of fused and nonfused populations has been reported for epithelial populations in the colon (7)
, liver (4)
, and kidney (8)
as well as nonepithelial cells in the brain (2)
, muscle (6
, 28
, 29)
, and heart (30)
. First, it is possible that two different mechanisms exist for the development of mature somatic cells from marrow: fusion and differentiation. This would imply that two different BM subpopulations are involved that undergo different types of nuclear reprogramming in response to the local environment in the target organ. Another explanation for these seemingly divergent mechanisms would be loss or gain of the genomic markers (such as the Y chromosome) used to define the fused cells.
The results reported herein differ greatly from our previous publications, in which MDLE did not appear to form by fusion (9
, 10)
. It is likely that in the current study, unlike previous work, the chronic inflammation related to Sp-C deficiency and the advanced age of the mice created a fusogenic milieu in the recipient lungs that enhanced heterokaryon formation. However, it is also possible that the techniques used in our earlier studies, which relied on expression of transgenes and/or the Y chromosome, lacked the sensitivity needed to detect this relatively rare event. Such uncertainty highlights the need for development of more reliable and efficient assays to detect stable heterokaryons and products of reductive divisions.
Lack of a therapeutic benefit
We investigated multiple aspects of the Sp-C null phenotype and found no changes due to marrow-derived Sp-C restoration. Our primary comparison was between the Sp-C null recipients of WT marrow that did and did not exhibit Sp-C restoration. Using BAL cell counts, which quantify the total cellular content of the epithelial lining fluid of the lung, we found no decrease in the degree of inflammation in the female transplant recipients. There was a mild decrease in the degree of inflammation between the transplanted Sp-C null males that did and did not show Sp-C restoration. However, compared with the Sp-C null recipients of Sp-C null BM the values were similar. This group was included to control for effects of irradiation and transplantation. Had it not been included, the mild decrease in BAL cell counts seen in the Sp-C null males would have been erroneously ascribed to the restoration of Sp-C expression.
We were particularly interested in histological changes related to Sp-C expression since these are a surrogate for respiratory function. Gas exchange in the alveolus depends in part on an adequate number of alveoli. When alveoli are destroyed, such as occurs with emphysema, the alveolar spaces enlarge. The distance between alveolar walls is quantified with chord length measurements normally in the 20–40 µ range (17)
. We had hoped to find an effect of Sp-C restoration on the mild increase in age-related emphysema that is attributable to Sp-C deficiency. This was not the case, as within both sexes Sp-C restoration showed only a trend toward decreased chord length that was not statistically significant. These data correlated with the qualitative review of the H&E-stained slides, which were indistinguishable. Sirius red staining of these slides, which detects collagen deposition, was uniformly negative, showing that significant fibrosis had not occurred in these animals at this time point.
While disappointing, these results were not surprising for at least two reasons. First, the low number of Sp-C-expressing type II cells (1/1000) would be unlikely to make a biologically relevant contribution to lung homeostasis. Second, as we hypothesize that inflammation and injury are key factors driving the development of blood cell:lung cell heterokaryons, these cells may not develop until inflammation and cell death processes are well established. Because Sp-C is not known to have anti-inflammatory effects or induce lung regeneration, restoration of this protein would not be expected to be ameliorative once the process had already begun.
Another issue may be the ability of the cells to process pro-Sp-C properly. We were unable to test for the presence of the mature, fully cleaved Sp-C protein in BAL fluid since the antibody for mature Sp-C was not available to us for this project. It is also likely that the very low level of lung-specific reprogramming would be below the sensitivity cutoff for Western blot of BAL fluid. Thus, while pro-Sp-C was used as a marker of type II cell gene expression, it does not define the secretory capabilities of these cells. Further work is needed to define whether heterokaryons formed in this manner display the full secretory phenotype of alveolar type II cells.
It may also be that the Sp-C null phenotype is too mild or the wrong type of injury to induce therapeutic levels of fusion and reprogramming. High levels of stable fusion (>50%) have been found in the livers (4)
and kidneys (8)
of FAH null mice. In the lung, the paucity of structural cells caused by emphysema represents one end of the tissue response to Th2 inflammation whereas fibrosis is a hypercellular response to similar inflammatory cues (31)
. At the 6 month point we chose, there was mild to moderate emphysema in all mice, even the WT, with no fibrosis, indicating that Sp-C deficiency only resulted in a chronic inflammatory state. It has also been shown for kidney and liver that the severe tissue damage caused by FAH deficiency coupled with the profound selective advantage of FAH+ hepatocytes derived from transplanted BM leads to robust heterokaryon survival/expansion with efficient therapeutic results (4
, 8)
. Application of similar amplification strategies to the lung may result in identification of pulmonary diseases likely to benefit from therapeutic heterokaryon formation.
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ACKNOWLEDGMENTS |
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Received for publication January 25, 2007. Accepted for publication March 1, 2007.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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