BRIEF REPORT |
Correspondence to: Samuel Hawgood, Suite 150, U. of California San Francisco, Laurel Heights Campus, 3333 California Street, San Francisco, CA 94118-1245. E-mail: hawgood@itsa.ucsf.edu
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Summary |
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Surfactant proteins A and D, collagen-like lectins (collectins), were first isolated from the lung. In the lung, SP-A and SP-D have roles in surfactant homeostasis and innate immunity. In this study we show that SP-A and SP-D mRNA can be detected in a significant number of non-pulmonary tissues but the proteins have a more limited distribution. SP-D protein was detected in lung, uterus, ovary, and lacrimal gland, whereas SP-A protein was detected only in the lung. The results suggest that SP-D participates in mucosal immunity throughout the body. (J Histochem Cytochem 50:993996, 2002)
Key Words: surfactant proteins, collectins, innate immunity
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Introduction |
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SP-A and SP-D are members of the collectin protein family, defined by linked collagen-like and carbohydrate recognition domains (
Mice overexpressing or lacking SP-A and SP-D are useful models in which to explore the roles of these proteins in host defense. Mice lacking SP-D develop spontaneous pulmonary inflammation (
To define the tissue distribution of SP-A and SP-D in the mouse, we harvested tissues from both male and female C57/BL6 mice 812 weeks old. Tissues from gene-targeted C57/BL6 mice deficient in SP-A or SP-D gene expression, generated in our laboratory, were harvested as controls for antibody specificity. RNA was isolated by the guanidinium isothiocyanate method using RNA STAT-60 reagents (Tel-Test; Friendswood, TX). Two µg of total RNA from each tissue was reverse-transcribed using a RETROscript kit (Ambion, Austin, TX). The SP-A primers were located in exon V (5') and exon VI to yield a 276-bp product. The primers of SP-D were located in exon VII and exon VIII to yield a 469-bp product. RT-PCR was performed for 35 cycles using 500 ng of template cDNA and an annealing temperature of 60C. SP-A and SP-D PCR products were probed by Southern blotting with 32P-labeled nested oligonucleotides. Controls without reverse transcriptase or template were run with each reaction and were always negative. For Northern analysis, total RNA (20 µg for all samples) isolated from mouse tissues was electrophoresed, blotted, and probed sequentially for SP-A, SP-D, and 18S RNA. The SP-A cDNA probe was a 345-bp fragment from exon VI. The SP-D probe was a 270-bp Bgl II fragment of exon VIII. The specificity of the primers and probes was confirmed by negative amplification or hybridization to mRNA from the lungs of SP-A-/- and SP-D-/- mice using SP-A and SP-D primers and probes respectively (not shown).
For Western blotting analysis, freshly harvested mouse tissues (100 mg) from wild-type and gene-targeted mice were sonicated in 500 µl 50 mM NaHCO3 (pH 9.0) containing 20 µl protease inhibitor cocktail (Sigma P8340; St Louis, MO). The samples were centrifuged to clear and used immediately without any freezethaw cycles. Tears were also collected from anesthetized mice for Western analysis. The antibody to mouse SP-D was raised in rabbits against mouse SP-D expressed in Chinese hamster ovary cells. The antibody against mouse SP-A was raised in rabbits against the CRD fragment of mouse SP-A expressed in E. coli using the pET23 vector system (Novagen; Madison, WI). Antibody specificity was confirmed by Western analysis using recombinant SP-A and SP-D and by Western analysis and immunocytochemistry on tissues from SP-A-/- and SP-D-/- mice (not shown). Based on the strength of the mRNA and protein signals, the lung, uterus, and lacrimal gland were selected for immunocytochemistry using standard protocols that included antigen retrieval. Controls without the primary antibody and reactions run on -/- mice gave no reaction product.
SP-D mRNA was detected by RT-PCR in a wide range of tissues, including the salivary gland, lacrimal gland, ovary, uterus, esophagus, stomach, testes, thyroid, and heart (Fig 1). No SP-D mRNA was detected in the bladder, brain, colon, duodenum, jejunum, kidney, liver, mammary gland, muscle, pancreas, prostate, seminal vesicle, spleen or tongue. A more restricted pattern of SP-D expression (lung, uterus, and lacrimal gland with weaker signals in the ovary, esophagus, and trachea) was detected by Northern blotting analysis (Fig 2). SP-D protein was detected by Western analysis in an even more restricted set of tissues: lung, trachea, uterus, ovary, and lacrimal gland (Fig 3). Clearly, the sensitivity of the methods used for mRNA and protein detection is markedly different and low-level protein expression in some tissues could have been missed. In the lung, SP-D was localized to alveolar epithelial type II cells and non-ciliated bronchiolar cells (Fig 4) as previously described in the mouse (
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SP-D mRNA and protein were recently detected in a large number of human tissues (
SP-A mRNA was readily detected by RT-PCR only in lung, trachea, and esophagus of the mouse, a very different pattern of expression from that of SP-D (Fig 1). A weak signal for SP-A mRNA was also detected in liver, stomach, jejunum, pancreas, spleen, heart, testes, and ovary but Northern blotting analysis only detected SP-A mRNA in the lung (Fig 2). SP-A protein was also detected only in the lung by Western analysis (Fig 7), suggesting a much more restricted distribution of protein expression relative to SP-D. In the lung, SP-A and SP-D have similar cellular distributions (Fig 4). We are uncertain as to the significance of the patchy detection of SP-A in the lumen of uterine gland ducts (Fig 6B) because we were unable to confirm the presence of SP-A in the uterus by Western blotting analysis. We did not detect any reaction product in the uterus of SP-A-/- mice (not shown), suggesting that the finding was specific, and SP-A mRNA was detected in the uterus with 40 cycles of amplification (not shown). The more restricted distribution of SP-A compared to SP-D is consistent with most other reports, but there may be significant interspecies variation. In the rat, SP-A mRNA has been detected in mesentery and in selected sites throughout the gastrointestinal tract, and in the intestine and swim bladder of the carp (
In summary, we have shown that SP-D is expressed in several non-pulmonary sites, specifically the uterus and lacrimal gland, in the C57/BL6 mouse. It is possible that SP-D contributes to the immune defense, similar to the purported role of SP-D in the lung, or has other unrecognized functions in these tissues. In contrast, SP-A protein appears to be predominantly expressed in the lung in the mouse, at least within the group of 25 tissues examined in this study. Given that mRNA for both collectins was detected in many other tissues, we cannot exclude the possibility that the distribution and abundance of the proteins might be more widespread in the context of infection or inflammation. The mouse should prove a useful model in which to further study the function of SP-D and SP-A at non-pulmonary sites.
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Acknowledgments |
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Supported by NHLBI grants HL58047 and HL24075 and by the Howard Hughes Medical Institute.
We thank Erin Collins for excellent technical help and Dr Jo Rae Wright (Duke University) for the polyclonal antibody to mouse SP-D.
Received for publication December 12, 2001; accepted March 4, 2002.
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