Meibomian gland dysfunction (MGD) is believed to be the leading cause of dry eye disease (DED), which affects tens of millions of Americans.1 Of particular interest, the most common pharmaceutical treatment for the management of MGD in the United States is the off-label use of topical azithromycin.2 This macrolide antibiotic is presumed to be effective because of its anti-inflammatory and antibacterial actions, which may suppress the MGD-associated posterior blepharitis and growth of bacteria on the eyelid.3 However, to our knowledge, no published, peer-reviewed data demonstrate that azithromycin has the ability to act directly on the human meibomian gland to enhance this tissue’s function and to ameliorate the pathophysiology of MGD.

We hypothesize that azithromycin can act directly on human meibomian gland epithelial cells to stimulate their differentiation, enhance the quality and quantity of their lipid production, and promote their holocrine secretion. Our purpose was to begin to test our hypothesis.

Immortalized human meibomian gland epithelial cells (IHMGECs; passages 20-22) were cultured in the presence or absence of 10% fetal bovine serum as previously reported.4 Cells were treated with the ethanol vehicle or azithromycin (10 µg/mL; Santa Cruz Biotechnology) for varying periods. Cellular morphological appearance was recorded, cells were counted with a hemocytometer, and lipid accumulation was assessed by staining cells with LipidTOX green neutral lipid stain (Invitrogen Corp) according to reported methods.4 Staining fluorescent intensities were quantified using ImageJ software (http://rsbweb.nih.gov/ij/index.html). Statistical analyses were performed with ttest (2-tailed, unpaired).

Our results show that azithromycin induces a striking, time-dependent accumulation of lipid in IHMGECs (Figure 1A). Within 3 days of azithromycin exposure, the number, size, and staining intensity of intracellular lipid-containing vesicles had markedly increased as compared with those of vehicle-treated control cells. This azithromycin effect on lipids appeared to become maximal at days 3 to 7 of the study (Figure 1B).

Evaluation of cellular morphology indicated that azithromycin may promote terminal maturation of IHMGECs given that vesicle accumulation was often followed by a cell break-up and vesicle release (Figure 1C).

In contrast to these effects, azithromycin reduced the proliferation of IHMGECs. As shown in Figure 2, this result was found irrespective of whether IHMGECs were cultured under proliferation or differentiation conditions.

This study supports our hypothesis that azithromycin can act on human meibomian gland epithelial cells and stimulate their lipid accumulation. This azithromycin effect appears to be paralleled by a cellular maturation, a decreased proliferation, and a holocrine-like secretion.

This azithromycin action is quite notable because MGD is thought to be the most common cause of DED.1 Typically, the meibomian glands produce and release a lipid mixture that promotes the stability and prevents the evaporation of the tear film, thereby playing an essential role in ocular surface health. Conversely, MGD destabilizes the tear film and increases its evaporation. Meibomian gland dysfunction is caused primarily by hyperkeratinization of the terminal duct epithelium and reduced secretion quality, and it leads to cystic dilatation of glandular ducts, acinar cell death, and lipid deficiency.1 The end result is DED, characterized by a cycle of tear film hyperosmolarity and ocular surface stress and leading to increased friction, inflammation, and damage to the eye.5 The effect of moderate to severe DED is analogous to conditions such as dialysis and severe angina and is associated with significant pain, role limitations, low vitality, and poor general health.5

Given our finding that azithromycin stimulates the function and differentiation of IHMGECs in vitro, it is possible that this antibiotic may prove beneficial as a treatment for MGD and its associated DED in vivo.