LACTOFERRIN IN HUMAN OCULAR TISSUES THOMAS
E. GILLETIE, M.D., AND MATHEA R. ALLANSMITH, M.D. Boston, Massachusetts
The natural protective factors identified in normal human tears may be categorized as specific (antibody.V lymphocytesv') or nonspecific (phagocytic cells,4,5 lactoferrin," lysozyme," nonlysozyme antibacterial factor," interferon"). Nonantibiotic approaches to the treatment of human external ocular infections could result from augmentation or manipulation of these factors. One of the natural protective factors present in tears, lactoferrin, which is an iron complexing protein, was identified in bovine milk in 1939,10 in human milk in 1951,11 and in human tears in 1966.6 Lactoferrin has both bacteriostatic" and bactericidal'" properties, and may interact with specific antibody to produce an antimicrobial system more powerful than either lactoferrin or specific antibody alone. 14 Additionally, lactoferrin has been shown to affect complement activity in vitro. 15 Thus, it may be important in the natural defense of the ocular surface. Broekuyse" confirmed the presence of lactoferrin in human tears in 1974. No postulates as to its origin were offered, but Franklin, Kenyon, and Tomasi'? had noted immunofluorescent staining for lactoferrin along the apical border of lacrimal acinar epithelial cells. Because the normal plasma lactoferrin level is 0.0015 mg/ml," and the normal tear lactoferrin level is 1.45 mg/ml," local From the Department of Ophthalmology, Harvard Medical School; and the Department of Cornea Research, Eye Research Institute of Retina Foundation, Boston, Massachusetts. This study was supported by Grant EY-02882 from the National Eye Institute, National Institutes of Health (Dr. Allansmith). Reprint requests to Thomas E. Gillette, M.D., Eye Research Institute of Retina Foundation, 20 Staniford St., Boston, MA 02114. 30
lactoferrin production at or near the ocular surface is likely. Evidence from immunohistologic studies of other secretory tissues (the bronchial submucosal glands." the submaxillary gland," and the pancreas'P) suggests that the acinar epithelium of the lacrimal gland may be a local source of tear lactoferrin. Accessory lacrimal tissue, which is histologically similar to the main lacrimal gland, is another likely source of tear lactoferrin, and the finding of secretory granules in normal conjunctival epithelium'" makes the conjunctiva another possibility. The study reported herein was undertaken to identify, by an immunofluorescence technique, the sources of human tear lactoferrin. MATERIAL AND METHODS
Autopsy specimens-Lacrimal tissues were obtained at autopsy from seven cadavers, age 54 to 73 years, with no known ocular disease (Table). The postmortem interval before enucleation varied between nine hours and five days. The globes were enucleated by peritomy close to the corneosclerallimbus. The bulbar and palpebral conjunctiva and the main and accessory lacrimal tissues were then excised whole from the orbit (Fig. 1). Conjunctivas from three of these subjects were examined for lactoferrin. Biopsy specimens-Specimens from normal-appearing lacrimal glands were obtained from two patients during orbital decompression for thyroid ophthalmopathy and from one patient during orbital exploration for venous aneurysm (Table). Conjunctival biopsy specimens were obtained from three patients free of ocular disease and ten patients with a variety of inflammatory ocular diseases,
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LACTOFERRIN IN OCULAR TISSUE TABLE DATA ON LACRIMAL TISSUE DONORS
No. of Hours Postmortem
Case No. Tissue from autopsy
Tissue from surgery 8 9
including episcleritis, marginal blepharitis, benign lymphoid hyperplasia, herpes simplex keratitis, atopic keratoconjunctivitis, and benign mucosal pemphigoid.
Diagnosis Alcoholic cirrhosis of liver with jaundice Severe rheumatoid arthritis; pneumonia; subacute bacterial endocarditis Carcinoma of pancreas with metastasis Progressive supranuclear palsy Cirrhosis; portal hypertension; no jaundice Acute renal tubular necrosis with renal failure; Staphylococcus bacteremia Heart failure Thyroid exophthalmos Epibulbar venous aneurysm Thyroid exophthalmos
fixed in 19 parts absolute alcohol to one part glacial acetic acid at room temperature for 48 hours, in a modified version of a technique described by Wolman and Preparation of tissues for immuno- Behar.P The tissue was dehydrated, emfluorescence-The excised tissue was bedded in paraffin, cut into 4-J-L sections, and then deparaffinized as described by Sainte-Marie. 23 Immunofluorescence-A two-layer (indirect) immunofluorescence technique was used. Deparaffinized tissue sections were incubated in a moist chamber with a commercially obtained rabbit antilaetoferrin antiserum diluted 1:10 in O.IM phosphate-buffered saline for 15 minutes, and then subjected to two fiveminute washings in phosphate-buffered saline. The tissue sections were then incubated with a commercially obtained fluorescein isothiocyanate-conjugated Fig. 1 (Gillette and AIIansmith). Autopsy specigoat antirabbit IgG diluted 1:40 in men. Orbital lobe of main lacrimal gland (short phosphate-buffered saline for 15 minwhite arrow); accessory lacrimal gland (black arutes, and given two more five-minute rowhead); upper fomiceal conjunctiva (long white arrow). washings in phosphate-buffered saline.
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Control sections were incubated with normal rabbit sera at a 1:10 dilution, washed, and then incubated with the fluorescein-conjugated goat antirabbit IgG diluted 1:40 in phosphate-buffered saline.
Characterization of antisera-Mono-
5 (excitation) filters, and a 41O-nm barrier filter. Photomicrographs were taken on Ektachrome ASA 200 or ASA 400 color film, through the same combination of filters used for observation. RESULTS
specific antiserum to human lactoferrin Brilliant, apple-green fluorescence, inwas obtained commercially from two dicating the presence of lactoferrin, was sources. Both antisera were found to be seen in approximately 90% of acinar one-line-specific by Ouchterlony analysis epithelial cells. Often, all cells of a single against both colostrum and tears. Both acinus were stained, but in some acini antisera showed no identity with antisera one or several unstained cells contrasted against human secretory component and strikingly with the brightly stained cells human 19A (Fig. 2). Goat antirabbit IgG (Fig. 3a). The unstained cells were diswas one-line-specific against normal rab- tributed randomly, and rarely accounted for most cells in a single acinus (Fig. 3a). bit serum. Observation and photography- The distribution of stained and unstained Stained sections were examined with a cells was uniform throughout the lacriZeiss fluorescence microscope fitted with mal gland. The distribution of specific an HBO 200-W high-pressure mercury fluorescence within the acinar cells was vapor lamp, BG 38 (suppression) and UC uniform, with strongly fluorescent material homogeneously filling stained cells (Fig. 3b). Lacrimal tubules (identified by large luminal diameter relative to epithelial circumference) were mostly unstained (Fig. 3c), but an occasional tubular epithelial cell was stained. We did not see any tubule with most of its epithelial cells stained. Overall, fewer than 1% of tubular cells were stained. Most acinar and tubular lumina were unstained, but stained material was seen adjacent to the apical surfaces of some epithelial cells. No staining of interstitial tissues, vascular components, or lymphoid elements was seen (Fig. 3). Lacrimal-gland biopsy specimens showed a staining pattern identical to that of the Fig. 2 (Gillette and Allansmith). Ouchterlony autopsy specimens. analysis of rabbit antihuman lactoferrin antiserum. Central well, colostrum (co). Well 1, rabbit antihuAccessory lacrimal tissue showed a man IgA. Well 2, goat antihuman secretory compostaining pattern identical to that of tissue nent (absorbed with human lactoferrin). Well 3, from the main lacrimal gland, with aprabbit antihuman lactoferrin. Well 4, rabbit antihuman secretory component (unabsorbed). Well 5, proximately 90% of acinar epithelial cells empty. Well 6, rabbit antihuman lactoferrin. Rabbit and fewer than 1% of the tubular epitheantihuman lactoferrin shows intersections of nonlial cells staining for lactoferrin (Fig. 3d). identity with rabbit antihuman secretory component (arrow) and with rabbit antihuman IgA (arrowhead). All sections of conjunctiva examined,
Fig. 3 (Gillette and Allansmith). Fluorescent staining for lactoferrin in human lacrimal gland, accessory lacrimal gland, and conjunctiva. (a) Main lacrimal gland. Majority of acinar cells (A) stain for lactoferrin (arrowhead), but some are completely unstained (large arrow). Small arrow indicates autoOuorescent llpofuschin granule. Bar gauge = 50 um (x200). (b) Acinus (A) of main lacrimal gland. Staining for lactoferrin within acinar cell (arrowhead) is diffuse throughout the cell, with the exception of the nucleus. Bar gauge = 10 um (x800). (c) Main lacrimal gland. Acinus (A) is stained; tubules (TI and blood vessels (VI are not. Bar gauge = 50 /lm (x200). (dl Conjunctiva and accessory lacrimal gland. Arrow indicates staining of acinar cells of accessory lacrimal gland. Arrowhead indicates unstained tubular cells. Epithelium (EI of conjunctiva is unstained. Bar gauge = 50 um (x200). [e] Conjunctiva. Epithelium [E] and substantia propria (sp) are unstained. Neutrophils in substantia propria are stained for lactoferrin (arrowheads). Bar gauge = 50 /lM (x200). Inset (xSOO) shows staining for lactoferrin in a neutrophil of the substantia propria. Epithelium (E) and blood vessel (VI are autofluorescent.
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including biopsy and autopsy specimens, revealed staining of occasional neutrophils (Fig. 3e). These neutrophils were usually located in the substantia propria. No staining above background of the epithelium or the substantia propria was seen (Fig. 3d). Other ocular tissues were examined for the presence of lactoferrin to ensure a complete study of the eye and its appendages. All tissues were fixed and processed in a fashion identical to that done for lacrimal tissues and conjunctiva. No staining for lactoferrin was observed in the cornea, the sclera, the choroid, the iris, the retina, or the extraocular muscles. DISCUSSION
We have confirmed the presence of lactoferrin in the main lacrimal gland, which was reported by Franklin, Kenyon, and Tomasi" in 1973. Our data extend their findings by showing that lactoferrin is present in approximately 90% of lacrimal acinar epithelial cells. Fewer than 1% of tubular epithelial cells stained for lactoferrin. Fluorescence was distributed within the acinar cells as a dense homogeneous filling like that seen in serous acinar cells of human pulmonary tissue by Masson and associates.'! However, serous acinar cells (as defined by the criteria of Allen, Wright, and Reid 24) constitute less than half of the lacrimal gland's volume, so lactoferrin synthesis in the lacrimal gland cannot be attributed to only serous cells. No group of cells exhibited a fluorescent peripheral rim like that seen by Masson and associates'[ in groups of bronchial mucous cells. The positive identification of lactoferrin in most lacrimal acinar epithelial cells is strong evidence for the synthesis of tear lactoferrin in the main lacrimal gland. The tubular epithelium of the lacrimal gland, however, does not appear to contribute significantly to tear lacto-
ferrin synthesis. The identical fluorescent staining pattern of accessory lacrimal tissue implicates the accessory lacrimal glands as an additional source of tear lactoferrin, particularly if normal basal tear secretion is solely from accessory lacrimal tissue as suggested by Jones. 26 The absence of fluorescent staining for lactoferrin in the human conjunctiva eliminates this tissue as a source of tear lactoferrin and leaves open for investigation the identity of the conjunctival secretory granules noted by Takakusaki." Neutrophilic granulocytes have been shown to synthesize and secrete lactoferrin in vitro. ll6 Therefore, the neutrophilic granulocytes present in the normal human tear film may be an additional source of tear lactoferrin. Because the average number of neutrophils in the normal tear film is less than 250, 4 total release of their contained lactoferrin could produce an expected maximum of only 1.1 X 10- 4 mg/ml lactoferrin, according to the following calculation (the lactoferrin/neutrophil value is taken from Masson, Heremans, and Schonne'[): (3
lactoferrinlneutrophil) x (250 neutrophils) 7 JLI preocular tear film X
10-4 JLg/jJ.I = 1.1 x 1()'4 mg/ml
The normal concentration of lactoferrin in tear film is 1.45 mg/ml." Tear-film neutrophils could therefore not be expected to contribute significantly to normal tear-film lactoferrin content. Another possible source of tear lactoFerrin is the mantle of neutrophils known to be present in the normal human conjunctiva.F Adding the average numbers of neutrophils present in epithelium and substantia propria of tarsal and forniceal conjunctiva gives 8,000 cells/rum" for tarsal conjunctiva and 6,000 cells/mm" for forniceal conjunctiva. Multiplying the average of7,000 cells/rum" by the conjuncti-
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val surface area, which is 350 mm 2 (forniceal) + 350 mm 2 (tarsal), gives 49.0 x 1()6 cells/mm. Multiplying this by the average conjunctival depth of 0.1 mm noted in routine histologic examinations yields a total ocular neutrophil count of 4.9 x 1()6 cells. This gives a total maximum lactoferrin production by periocular neutrophils (achieved by total intracellularcontent release) of 1.47 ~g (obtained by the calculation [4.9 x 1()6 cells] x [3 X 10- 6 ug lactoferrin/cell)). Dividing by the preocular tear-film volume of 7 ~128 gives 0.21 mg/ml of lactoferrin. This value clearly indicates that the periocular mantle of neutrophilic leukocytes could only contribute significantly to normal tear-film lactoferrin levels if they were to release their total intracellular content. The staining of conjunctival granulocytes for lactoferrin shows the sensitivity of our immunofluorescence technique (average neutrophillactoferrin content = 3 X 10- 6 ug). The absence of conjunctival staining in the presence of positively staining neutrophils is evidence against the possibility that the human conjunctiva or its contained neutrophils is a source of lactoferrin in human tears. The lack of staining for lactoferrin in the lacrimal interstitium and the substantia propria of the conjunctiva seems to preclude a vascular source of lactoferrin in human tears. This conclusion is further supported by the much greater concentration of lactoferrin in tears than in serum. However, selective permeability of the acinar basement membrane or the conjunctival basement membrane to small amounts of lactoferrin beyond the reach of our immunohistochemical technique cannot be eliminated. SUMMARY
The main lacrimal gland and accessory lacrimal tissue from seven autopsy cases, lacrimal biopsy specimens from three patients, and conjunctival biopsy speci-
mens from ten patients were examined for lactoferrin by an immunohistologic technique. Lactoferrin was identified and localized to acinar epithelial cells of both main and accessory lacrimal tissue. Lactoferrin was not found in conjunctival tissue except within conjunctival neutrophils. Other possible sources of human tear lactoferrin were considered, but we concluded from our data that the primary source of lactoferrin in normal human tears is the acinar epithelium of the main and accessory lacrimal glands.
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