ARTICLE |
Correspondence to: Lennart E. Lögdberg, Laboratory of Stem Cell Biology, Lindsay F. Kimball Research Inst., New York Blood Center, 310 East 67th Street, New York, NY 10021. E-mail: Logdberg@nybc.org or Lindalennart@msn.com
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Summary |
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Alpha-1 microglobulin (1m), a lipocalin, is an evolutionarily conserved immunomodulatory plasma protein. In all species studied,
1m is synthesized by hepatocytes and catabolized in the renal proximal tubular cells.
1m deficiency has not been reported in any species, suggesting that its absence is lethal and indicating an important physiological role for this protein To clarify its functional role, tissue distribution studies are crucial. Such studies in humans have been restricted largely to adult fresh/frozen tissue. Formalin-fixed, paraffin-embedded multi-organ block tissue from aborted fetuses (gestational age range 722 weeks) was immunohistochemically examined for
1m reactivity. Moderate to strong reactivity was seen at all ages in hepatocytes, renal proximal tubule cells, and a subset of pancreatic islet cells. Muscle (cardiac, skeletal, or smooth), adrenal cortex, a scattered subset of intestinal mucosal cells, tips of small intestinal villi, and Leydig cells showed weaker and/or variable levels of reactivity. Connective tissue stained with variable location and intensity. The following cells/sites were consistently negative: thymus, spleen, hematopoietic cells, lung parenchyma, glomeruli, exocrine pancreas, epidermis, cartilage/bone, ovary, seminiferous tubules, epididymis, thyroid, and parathyroid. The results underscore the dominant role of liver and kidney in fetal
1m metabolism and provide a framework for understanding the functional role of this immunoregulatory protein.
(J Histochem Cytochem 48:15451552, 2000)
Key Words: alpha-1-microglobulin, fetal, tissue distribution, lipocalin
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Introduction |
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Alpha-1 MICROGLOBULIN (1m), a member of the lipocalin protein superfamily, is a low molecular weight (26-kD) plasma glycoprotein with intriguing biochemical and biological properties (
1m is known to form high molecular weight complexes, notably with immunoglobulin A (IgA), prothrombin, and albumin (
In addition to its presence in mammals and birds (1m gene has been identified in fish and amphibians (
1m (
1m deficiency has not been reported in any species, suggesting that its absence is lethal. Like other lipocalins, such as the retinol-binding protein,
1m may have a plasma transport function and might carry a hydrophobic ligand (
1m has immunomodulatory properties, including inhibition of antigen-stimulated lymphocyte and of granulocyte functions (
1m gene, present in the lipocalin cluster on chromosome 9 (q3233) (
1m gene expression during amphibian metamorphosis (
1m during growth and development.
Many studies of 1m in humans have measured its concentration in serum under a variety of normal and pathological conditions, or in urine as a marker of kidney pathology (
1m levels in serum (increasing with reduced glomerular filtration rate) and urine (increasing with proximal tubule damage). However, levels of
1m in biological fluids are not markedly influenced by other pathological conditions (
1m concentrations throughout the body and about changes in the relative distribution of
1m among its diverse complexes in health and disease.
To begin to clarify these issues, tissue distribution studies of the protein are crucial. Several biochemical and immunohistochemical studies have been published but are limited mainly to adult fresh or frozen tissue (1m in hepatocytes (the primary site of synthesis) and in proximal renal tubules (the major site of catabolism).
Because of the limited supply of fresh and frozen tissue, little is known about the developmental expression of 1m in humans. The recent development (
1m in formalin-fixed tissue and the enhanced detection with an antigen retrieval technique (unpublished observations) paved the way for investigating archival material. Using these methods, we have systematically explored the localization of
1m in the human fetus at various stages of development. These data will bear on the evolving understanding of the physiological and pathological role of
1m.
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Materials and Methods |
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Tissues
Archival, formalin-fixed fetal tissues were obtained from 22 aborted fetuses (age range 722 weeks estimated gestational age) after approval by the Montefiore Medical Center Institutional Review Board. These fetuses were the subjects of surgical pathology review, and special care had been taken with the diagnostic tissue blocks to include representative tissues from several organs from each fetus, sampled by a 3-mm diameter skin punch. Organs sampled included liver, kidney, pancreas, heart, skeletal muscle, lung, esophagus, stomach, small and large intestine, bone/cartilage, skin, testis, ovary, thymus, spleen, adrenal, and thyroid. Multiple tissue samples (1015) from an individual were placed in one cassette and processed according to standard protocol to generate paraffin blocks. Tissue sections for immunohistochemistry were cut at 4-µm thickness.
Antibodies
Chromatographically purified human urinary 1m (
1m antibodies (
1m were obtained. The binding specificities were analyzed by Western blotting (
1m (31 kD), IgA-
1m (90 kD, 180 kD), and albumin
1m (120 kD). IgG from the rabbit sera was purified by anion exchange chromatography as described (
1m IgG, exhaustively adsorbed on immobilized, purified urinary human
1m (3 mg
1m:1 ml CNBr-activated Sepharose; Pharmacia Fine Biochemicals, Uppsala, Sweden), served as a negative control in these experiments. In additional control experiments, we also employed anti-
1m IgG similarly adsorbed on immobilized, purified recombinant human
1m (
1m activity in radioimmunoassays or on Western blots. In initial experiments, a monoclonal mouse anti-
1m, BN11.10 (
1m antibodies/antisera (K107, K108, K109, K201, K202, and K319+323).
Immunohistochemistry for 1m
1m was readily and specifically detected by immunohistochemistry on formalin-fixed, paraffin-embedded tissues using polyclonal antibodies, with improvement in staining after antigen retrieval. In contrast, the monoclonal anti-
1m antibodies (BN11.10) failed to detect the protein under the same conditions. Therefore, polyclonal antibodies and antigen retrieval on tissue sections were employed throughout this investigation. The immunohistochemical assay used the avidinbiotinperoxidase-method with diaminobenzidine (DAB) as the substrate (LSAB kit; DAKO, Carpinteria, CA). Sections were counterstained with hematoxylin. Briefly, 4-µm-thick sections were stained with anti-
1m IgG, in most experiments at a final concentration of 10 µg/ml (K322), at 100 µl/slide (30-min incubation at room temperature in a humid chamber). Before the staining, after blocking of endogenous peroxidase with H2O2 (15-min incubation in a 37C water bath), the tissue sections had been heat-reduced (by microwave) for 5 min in citrate buffer, pH 6.0, for antigen retrieval. Antigen retrieval conditions and antibody dilutions were standardized using tissue sections from normal adult kidney (tubules positive; glomeruli negative) and from a liver (hepatocytes positive) with metastatic colon adenocarcinoma (tumor negative), exploiting the high concentrations of
1m in adult human kidney and liver. Kinetic studies showed that near-complete antigen retrieval was achieved as early as after 5 min of microwave heating. Staining was scored as strong (3+), moderate (2+), weak (1+), or absent (0) by two independent observers.
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Results |
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1m Distribution in Human Fetal Tissues: Overview
1m staining was found in organs at all ages studied (722 gestational weeks). As in adult tissues of many species, particularly strong staining was seen in the proximal tubules of the evolving kidney (Fig 1a), and cytoplasmic staining of fetal hepatocytes was also consistently present (Fig 1d). As detailed below,
1m staining was found at a number of other sites.
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In all cases, staining for 1m was specific and was not seen with anti-
1m antibodies that had been exhaustively absorbed against purified
1m, as demonstrated for kidney in Fig 1b. Table 1 summarizes the localization of
1m in fetal tissues using anti-
1m K322 on tissue from 22 cases. In addition to hepatocytes and renal proximal tubules cells, moderate anti-
1m staining of a subset of pancreatic islet cells (Fig 1c) was observed in most cases. Other sites with weaker and/or variable staining included the tips of small intestinal villi (Fig 1e), a scattered subset of intestinal mucosal cells (Fig 1f), cardiac muscle (Fig 1g), skeletal muscle (Fig 1h), smooth muscle, adrenal cortex, and testicular Leydig cells. In addition, the perivascular connective tissue was variably positive in most organs throughout the fetal ages studied.
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The following cells/sites were consistently negative: thymus, spleen, hematopoietic cells (Fig 1d), lung, glomeruli (Fig 1a), exocrine pancreas (Fig 1c), epidermis, cartilage/bone, ovary, seminiferous tubules, epididymis, thyroid, and parathyroid. It is noteworthy that 1m reactivity is not restricted to tissues originating from one germ layer, because cells from both endoderm (liver, intestines) and mesoderm (testis, muscle) appear to express
1m during one or more stages of fetal development.
1m Distribution in Human Fetal Tissues: Analysis by Organ System
Gastrointestinal System.
There was no staining for 1m in the superficial squamous epithelium of the oral cavity, pharynx, or esophagus at any age. In the stomach, the parietal cells showed positive staining but the columnar cells of the surface epithelium and the neck glands were negative. The small intestine showed granular reactivity within the cytoplasm of the surface absorptive cells at the tips of the intestinal villi (Fig 1e), beginning at about the 14th week of gestational age. The intensity of this staining and the number of reactive villous cells increased in number with further development, but goblet cells and crypts were consistently negative. Moderate-intensity positive staining was seen in scattered individual cells located at the base of the colon crypts as early as 7 weeks of gestational age (Fig 1f). These cells had the morphological characteristics of neuroendocrine cells. All the other cells of the colon surface and glandular epithelium were negative at all ages.
At the age of 7 weeks, there was moderately intense 1m staining of a few hepatocytes scattered throughout the liver, with low-intensity staining of more than 50% of the hepatocytes. No staining of the extrahepatic bile duct was seen at this age. With development of the portal triads, reactivity for
1m was seen in all the hepatocytes (Fig 1d), with slightly stronger staining of the periportal hepatocytes. This pattern of staining persisted even after development of the ductal plates. In the region of the portal triads, staining was seen in the periportal hepatocytes, ductulocanalicular cells, and "oval cells." The bile duct epithelium and the hematopoietic cells did not react with the anti-
1m antibodies at any age.
In the pancreas (see also below), moderate and occasionally strong 1m staining was seen in a subset of the cells in the islets of Langerhans (Fig 1c). These moderately staining cells were located mainly at the periphery of the islets but were also scattered within the center of the islets. The distribution of these cells did not follow that expected of any of the known subpopulations of islet cells. The rest of the cells in the islets showed very weak intensity staining or, more commonly, were unstained. Pancreatic acini and the ductule epithelium did not exhibit significant staining.
Genitourinary System.
1m staining in the kidney was restricted to the convoluted tubules and was strongly intense in this site (Fig 1a) and weakly intense in the collecting ducts. The glomerular tuft and Bowman's capsule were consistently negative (Fig 1a). There was moderately increased staining of the proximal tubule cells with increasing age. Weak staining of the interstitial Leydig cells of the testis was seen. No significant staining was seen in the seminiferous tubules or the rete testis.
Cardiovascular System.
1m staining of the myocardium was evident as early as 7 to 8 weeks of gestation (Fig 1g). This persisted with development. The endocardium and pericardium were negative. A variable- intensity staining of the intima and media of the medium-sized blood vessels was seen.
Respiratory System.
There was no significant 1m staining of the bronchial epithelium or the lung in fetuses below the age of 1516 weeks. In some cases, with development of the acini, the alveolar epithelium showed weak surface staining of the luminal cytoplasmic membrane. Staining of the pleura was not evident.
Reticuloendothelial System.
No 1m staining was seen in the thymus, lymph nodes, spleen, or bone marrow.
Endocrine System.
Diffuse weak-intensity cytoplasmic 1m staining was seen in the deeper layers of the adrenal cortex. No significant staining was seen in the thyroid or parathyroid tissues examined.
Musculoskeletal System.
There was no 1m staining of the skin or the cutaneous appendages. Positive staining of weak to moderate intensity was seen in skeletal (Fig 1h) and smooth muscle at all ages of development. Although strong expression of
1m was seen in the primitive mesenchyme, the bone and cartilage did not react with the antibody. Perivascular connective tissue and connective tissue at the hilus of the organs, such as the porta hepatis, showed moderate expression. There was no staining of the basement membranes of the epithelia or the collagen in the skin or bone.
Staining of Pancreatic Islet Cells and Scattered Neuroendocrine-like Cells of the Large Intestine Was Restricted to Some 1m Antibodies and Was Not Seen with Others
When some of the staining experiments were repeated with a different source of anti-1m antibodies (anti-
1m K4069), we noted that the majority of tissues exhibited staining patterns identical to those previously observed with anti-
1m K322. However, the anti-
1m K4069 antibodies stained neither the pancreatic islet cells nor the gastrointestinal "neuroendocrine" cells. Testing of additional antibodies/antisera raised against different preparations of purified human
1m revealed that some (5/8) lots were pancreas-reactive, whereas other lots (3/8) were nonreactive (Table 2). In these experiments, reactivity to kidney or liver was uniformly moderate to strong for all the antibodies/antisera. It was noted that adsorption of anti-
1m K322, not only with purified urinary
1m but, alternatively, with purified recombinant human
1m, completely eliminated its ability to stain the various fetal tissues, including pancreatic islet cells and gastrointestinal "neuroendocrine" cells.
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Discussion |
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The primary site of synthesis of 1m in the adult is the liver, where it is translated as a diprotein precursor with bikunin, a Kunitz-type proteinase inhibitor and subunit of inter-
-inhibitor (
1mbikunin gene is divided into two separate clusters of exons, indicating an origin from independent lipocalin and Kunitz-type ancestral genes, respectively. This tandem arrangement is conserved among mammals and fishes (
1m before they are released from the hepatocytes as separate proteins (
1m is the proximal convoluted tubules of the kidney.
The synthesis and/or expression pattern of 1m in other organs has not been well studied. This is largely due to the fact that tissue distribution studies have mostly been limited to fresh and frozen tissue, and conflicting data were reported (
1m to be readily and specifically detected on formalin-fixed, paraffin-embedded tissues by the use of polyclonal anti-
1m antibodies (
1m in the human fetus in a comprehensive manner and to elucidate its developmental expression, using a multi-organ block arrangement that allows efficient screening of large numbers of tissues.
1m staining was found in fetal liver and proximal convoluted tubules of the kidney in all cases and at all developmental stages (our earliest stage was Week 7 of gestation). This suggests a dominant role for these organs in the metabolism of
1m in fetal as in adult life. The dominant role of the liver in the synthesis of
1m suggests that
1m expression may be useful as an indicator of hepatocyte differentiation. Our preliminary data demonstrate that
1m can be employed as an immunohistochemical marker to aid in distinguishing primary from secondary liver tumors (unpublished observations).
In certain other organs (cardiac, skeletal, or smooth muscle; adrenal cortex; testicular Leydig cells), specific cytoplasmic staining for 1m was noted in most cases. In the adult, using the same staining protocol,
1m is essentially absent from the parenchyma at these sites (own unpublished observations; see Table 1) and its synthesis cannot be demonstrated in heart, skeletal muscle, or adrenal gland by RT-PCR (
1m may exert functions specific to the fetus in these tissues. Staining of these organs was generally weaker than that of hepatocytes and proximal tubule cells, and a clear developmental trend in staining intensity, could not be ascertained for the time period studied (722 weeks).
The presence of 1m in fetal intestinal villi may correspond to the weak staining by anti-
1m in adult rat intestine (
1m does not appear to be synthesized at this site in the adult, as determined by RT-PCR (
1m present as a result of local synthesis and where is it present due to uptake/deposition of circulating
1m? For example, the intestinal
1m could originate from fetal swallowing of amniotic fluid, which contains the protein. Additional questions concern the extent to which putative
1m receptors (
1m may reflect a specialized functional involvement of the molecule, possibly by providing immunoregulation as a component of the gut-related immune system.
Significant synthesis of 1m has not been demonstrated in adult human tissues other than liver and possibly pancreas (
1m at some of the other tissue sites, notably the perivascular extracellular matrix in adults (
1m. The transport pathways for such deposition remain to be clarified but may involve certain high molecular weight
1m complexes (notably those with IgA, albumin, and some proteinase inhibitors;
1m may exert different functions via different high molecular weight complexes, a form of tissue-specific functional specialization.
Related to this possibility are the immunoregulatory properties of 1m. The protein can inhibit antigen-induced lymphocyte activation, leukocyte migration and chemotaxis (
1m may provide tissues with protection from damage caused by inflammatory and/or immune reactions. Such local immunoprotection could be differentially mediated by different
1m complexes. Although overall levels of
1m in biological fluids are not markedly influenced by pathological conditions other than kidney disease (
1m concentrations in health and disease remains to be elucidated.
In this context, it is intriguing to find high local concentrations of 1m in extracellular matrix at boundaries of strategic immunological importance, such as the skin (epidermodermal basement membranes) (
Staining of "neuroendocrine" cells of the gastrointestinal tract or pancreatic islets was noted with some but not other 1m antibodies. Possible explanations include that the moiety detected could represent (a) a processed form of
1m, exhibiting only a unique epitope(-s) or (b) a different molecule crossreactive with a unique
1m epitope. The observation that anti-
1m K322 antibodies, adsorbed on immobilized urinary or recombinant
1m both lost their reactivity argues against a reactivity of the antibodies to a contaminant in the
1m preparation used for immunization because urinary and recombinant human
1m would not be expected to contain the same minor impurities. An RT-PCR study performed on adult human tissues (liver, kidney, lung, heart, spleen, muscle, thyroid, pancreas, and adrenal gland) (
1m mRNA only in the liver and, to a lesser extent, the pancreas, lending support to the first explanation. Other studies in humans (
1m synthesis by pancreas. These authors (
1m and bikunin are synthesized as diproteins, the later study will hopefully clarify the synthesis/ uptake issues.
In summary, we have used a reproducible immunohistochemical method for detection of 1m in archival fetal human tissue to create the first map of the distribution of
1m in developing tissues. Although its function remains to be clarified,
1m, on the basis of structural, histological, immunological, and evolutionary evidence, is likely to play a role in development, molecular transport, and immunomodulation. With respect to these and related issues, this report sets the stage for further histological studies in the functional characterization of
1m and its role in pathological processes.
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Acknowledgments |
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Supported by the Department of Pathology, Albert Einstein College of Medicine, and by a grant from the Swedish Medical Research Council (project no. 7144).
The study was approved by the Institutional Review Board at the Albert Einstein College of Medicine. Drs M. Prystowsky, C.M. Salafia, and S. Gupta gave critical comments to various versions of the manuscript, and Dr L. PaulLögdberg reviewed the language.
We thank Rachel Tucker (Immunohistochemistry), Ernestine Middleton, Dinah Carroll (both Histology), Maria Allhorn, and Ann-Sophie Johnson (both Lund) for technical support.
Received for publication April 21, 2000; accepted May 17, 2000.
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