Human eyelid meibomian glands and tarsal muscle are recognized by autoantibodies from patients affected by a new variant of endemic pemphigus foliaceus in El-Bagre, Colombia, South America

Abstract

Background: Previously, we described a new variant of endemic pemphigus foliaceus (EPF) in Colombia, South America (El Bagre-EPF).

Objective: Continuing our characterization of this variant of EPF, we now focus on one of our previously reported clinical findings: the presence of ocular lesions. These ocular lesions are seen in patients having extensive skin involvement, as measured by the Lund and Browder scale, which is generally used for patients with skin burns.

Methods: We specifically searched for evidence of autoreactivity to various eyelid structures in these patients and correlated our immunologic data with the clinical findings. We performed indirect immunofluorescence studies using normal-appearing human eyelid skin from routine blepharoplasties as substrate tissue. We tested sera from 12 patients with El Bagre-EPF and ocular lesions, 5 patients with sporadic (nonendemic) pemphigus foliaceus, and 20 healthy control subjects (10 from the El Bagre-EPF endemic area and 10 from nonendemic areas). We used fluorescein isothiocyanate conjugated goat antiserum to human total IgG/IgA/IgM as a secondary antibody. In addition, we used fluorescein isothiocyanate conjugated antibodies to human fibrinogen, albumin, IgG, IgE, C1q, and C3, Texas Red (Rockland Immunochemicals, Inc, Gilbertsville, PA), Alexa Fluor 555, or Alexa Fluor 594 (Invitrogen, Carlsbad, CA). Ki-67 (a cell proliferation marker) was used to determine the cell proliferation rate, and nuclear counterstaining was performed with either 4', 6-diamidino-2-phenylindole or Topro III (Invitrogen, Carlsbad, CA).

Results: We observed autoreactivity to multiple eyelid structures, including meibomian glands and tarsal muscle bundles at different levels, and some areas of the epidermis and the dermis close to the isthmus of the eyelids. Tarsal plate autoreactivity was seen in 10 of 12 of the El Bagre-EPF sera and in one control with pemphigus erythematosus. Furthermore, immunoprecipitation using an eyelid sample as a substrate with 1 mmol/L of sodium orthovanodate showed autoreactivity to several antigens, including some of possible lipid origin.

Limitations: The main limitation of this study is the fact that the antigen or antigens remain unknown.

Conclusion: We identified for the first time to our knowledge autoantibodies to meibomian glands and tarsal muscle in El Bagre-EPF. Our findings suggest that the autoantibodies to the ocular structures cause the clinical and histopathological findings in the ocular lesions in El Bagre-EPF.

Fig 1

Clinical images of El Bagre-endemic pemphigus foliaceus (EPF). A, Edema, erythema, and scaling of eyelids with severe ectropion. B, Hyperemia, edema, erythema, and conjunctival injection without mucoid discharge. C, Dry eye and atrophy of superior and inferior tarsal muscles. D, Catarrhal conjunctivitis-like alterations characterized by significant edema of superior and inferior eyelids. Small internal synechiae (as rare finding) resembling nasal ptyrigia in internal cantus of left eye. E, Most common clinical findings include meibomianitis of lower eyelid and partial occlusion of meibomian ducts. F (as in B), Hyperemia, edema, erythema, and conjunctival injection, in this case with mucoid discharge. A, D, C, and F, Thinning of eyebrows.

Fig 2

A, B, D, and E, Results of indirect IF (IF) performed after partial fixation with paraformaldehyde. C and F, Results of indirect immunofluorescence performed using paraffin-fixed samples followed by antigen retrieval techniques. A, Using conjuctiva as the antigen source, antihuman conjugated IgG-Alexa 488 (Invitrogen) showed positive ICS and basement membrane zone (BMZ) staining (white arrows). B, Positive intercellular (IC), BMZ, and hair follicle staining using antihuman total IgG antiserum as secondary antibody (green) (fluorescein isothiocyanate [FITC]) (white arrows). Nuclei of cells were stained with Topro III (Invitrogen) (red ). In addition, note strong reactivity of hair follicle bulb to Ki-67 proliferating antigen (red) (Texas Red) (blue arrow). C, Structure (top) that resembles secretion near tear duct, likely of mixed material including mucins, lipids, and other tear components. These components contribute to high, non-Newtonian viscosity of tear film and its low surface tension, features essential for tear film stability (white arrows). In same figure, Ki-67 antigen demonstrated clumped elongated pattern around eyelid base, within isthmus, and in some parts of epidermal layer (red) (green arrows). Positive autoreactivity as small and large dots (red) using Alexa Fluor 555 (Invitrogen) against human IgG (yellow arrows). Nuclei were counterstained with DAPI (Pierce) (blue). D and F, IC and BMZ staining were seen in El Bagre-endemic pemphigus foliaceus using antihuman conjugated IgG FITC (white arrows)(×20). D, BMZ staining of meibomian glands (×100). E, Secretory portion of meibomian gland, in yellow dots, as part of intrinsic fluorescence of these structures (purple arrows). Ki-67 antigen showed positive clumped pattern surrounding involving base of gland ducts on eyelid (white arrows).

Fig 3

A, A diagram of the eye superior tarsal muscle (musculus tarsalis superior) showing a panoramic view of a tarsal plaque. (Reprinted from Gray H. Gray's Anatomy of the Human Body. 20th ed. Philadelphia: Lea & Febiger;1918.) B, Direct IF shows a panoramic view of a tarsal plaque with small continuously yellow-greenish dots around the plaque (red arrow). In addition to this, reactivity to larger dots (blue arrows) and smaller ones (white arrow) was observed. These findings were seen using a secondary goat antihuman IgG/IgA/IgM antiserum conjugated with FITC. C, Positive red dots, seen in bundles of tarsal muscle, using antihuman IgG antiserum conjugated with Alexa Fluor 594 (white arrows). D, Using conjugated antihuman IgG Alexa 488 (yellow stains dots), larger bundles of tarsal muscle show positive reactivity of different shapes inside the myocytes (purple arrows). In this case, the reactivity was seen with antihuman fibrinogen FITC conjugate. In addition, thin zig-zag yellowish positivity was seen inside the muscle bundles resembling the staining of sarcoplasmic structures. E, When repeating the experiments using antihuman fibrinogen FITC conjugate alone, the staining shows a positive reactivity outside the myocytes, as seen at higher magnification (green staining) (blue arrow). In addition, there is a structure that resembles a “large-cell junction” (red staining)(yellow arrow) using antihuman IgM antiserum conjugated with rhodamine. The white arrow indicates the extracellular space. F, The white arrow shows autoreactivity to a large structure inside the myocytes using antihuman IgG FITC conjugated. The yellow arrow shows autoreactivity to the plasma membrane. G, We performed IIF testing on heart muscle tissue from sheep, beef, rat, and human, to determine if the reactivity could be observed in multiple species. Positive staining was noted among the muscle bundles (green staining) utilizing FITC conjugated antihuman fibrinogen (red arrows). This staining was noted when rabbit antihuman fibrinogen and albumin antisera were used. H, We used tissue microarray slides as the antigen source with Alexa 488 conjugated and antihuman IgG heavy and light chain (H&L) antiserum. We again showed positivity within muscle bundles (green staining)(yellow arrow). To identify colocalization with the autoreactivity detected by antihuman IgG for gap junctions, we used an antibody to connexin-43 as a control (brown staining)(red arrow). However, the autoreactivity did not superimpose with connexin-43, although confocal microscopy was not performed. (Note that in D, E, F, and H, the cell nuclei were counterstained with DAPI [blue]). I, The sera were tested by IP, and an approximately 45 kDa protein band was strongly recognized by sera with numbers 2, 3, 4, 5, and 7 (and very weakly by number 8). In addition to this band, other antigenic bands were immunoprecipitated, mostly by the El Bagre-EPF patient sera (numbers 2, 3, 4, 5, 7). As a control, we also used normal human serum (NHS) (Lane 1). Lanes 3 to 7 correspond to sera from El Bagre-EPF patients, and lane 8 corresponds to serum from a sporadic PF patient. The molecular weight marker standards are shown in the first lane on the left (200, 117, 96, 66, 45, and 31 kDa, respectively). The arrows on the right point to protein bands of approximately 200, 117, 89, 67 and 34 kDa that were consistently recognized by the El Bagre-EPF sera, and to lesser extent, by the sera from patients with pemphigus erythematosus. Although we show the results of 7 sera, all 12 sera showed similar results.