Lactadherin orthologs inhibit migration of human, porcine and murine intestinal epithelial cells

Abstract

Lactadherin was originally described due to its appearance in milk, but is abundantly expressed especially by professional and nonprofessional phagocytes. The proteins has been shown to have a multitude of bioactive effects, including inhibition of inflammatory phospholipases, induction of effero- and phagocytosis, prevent rotavirus induced gastroenteritis, and modulate intestinal homeostasis by regulating epithelial cell migration. The level of expression seems to be important in a row of serious pathologies linked to the intestinal epithelial barrier function, vascular- and autoimmune disease. This study examines the ability of lactadherin to modulate migration of intestinal epithelium. A cell exclusion assay is used to quantify the ability of human, bovine and murine lactadherin orthologs to affect migration of primary small intestine epithelium cells. Previous reports show that recombinant murine lactadherin stimulate rat small intestine cell migration. The present study could not confirm this. Conversely, 10 μg/ml lactadherin inhibits migration. Therefore, as lactadherins enteroprotective properties is well established using in vivo models we conclude that the protective effects are linked to lactadherins ability operate as an opsonin, or other modulating effects, and not a direct lactadherin-cell induction of migration. Thus, the molecular mechanism behind the enteroprotective role of lactadherin remains to be established.

Keywords: IBD; MFG‐E8; intestinal cell migration; lactadherin; wound healing.

Figure 1

Rat small intestine cells (IEC‐18) were seeded in the cell exclusion assay, grown to confluence, the insert removed and the cells treated with a concentration gradient of lactadherin orthologs as per materials and methods. As seen, IEC‐18 migration is proportionally inhibited to a maximal 14.6% inhibition versus control monolayers using rmLact (a), 21% inhibition using hLact (b) and no statistically significant inhibition using bLact (c). * denotes p < .05, ***p < .001, and # not statistically significant

Figure 2

Human primary small intestine cells (FHS 74 int) were likewise grown in the cell exclusion assay and treated with a concentration gradient of lactadherin orthologs. Recombinant murine lactadherin exhibited an inhibitory trend, however was not statistically significant (a). Human lactadherin inhibited migration by 21.8% (b) and bLact did not affect migration (c). * denotes p < .05, **p < .01, ***p < .001, and # not statistically significant

Figure 3

Human colonic carcinoma cells (Caco‐2) were likewise treated with 10 μg/ml bLact and micrographs acquired of the cell exclusion areas. A striking inhibition was observed between control wounds (a) and bLact treated wound (c). As a positive control wounds were treated with 5 ng/ml epidermal growth factor which induced migration as expected (b). Quantification of remaining void area post treatment was done on sextuplicates and showed a 51% reduction of migration when Cac0‐2 monolayers were treated with 10 μg/ml bLact (d). **denotes p < .01, and ***p < .001

Figure 4

Caco‐2 monolayers were treated with 10 μg/ml bLact and examined using Differential Interference Contrast. Control monolayers (a) migrated collectively and displayed cobblestone morphology within the monolayer and a flattening of the pioneer cells with lamellapod extension in the axis of migration. Lactadherin‐inhibited pioneer cells (b) displayed no flattening or lamellapods and negligible wound closure was observed. This phenotype after treatment was similar to that of the pioneer cell in the IPEC‐J2 monolayers

Figure 5

Primary pig small intestine cells (IPEC‐J2) were treated with 10 μg/ml bLact per materials and methods, the nuclei stained and micrographs acquired. When comparing control monolayers (a) with lactadherin‐treated monolayers (b) a pronounced ring of nuclei as well as a significantly larger exclusion area was observed in the latter

Figure 6

IPEC‐J2 cells treated with bLact and the plasma membrane visualized using Cellmask orange. Left panels (a, c & e) are without and right panels (b, d & f) are with 10 μg/ml bLact treatment. Whole wound comparison of control versus treated monolayers (a vs. b) exhibited the same wound morphology as described previously with less collective migration and wound coverage in bLact treated wells. Further magnification revealed a pronounced decrease in leading edge (arrows in c), but not cryptic lamellapod formation (arrows in c), and an accumulation of cells at the wound edge compared to control (d vs. c respectively). Examination of the leading edge using Differential Interference Contrast imaging revealed that the pioneer cells lost their flattened morphology characteristic of adequate adherence when treated with bLact. compared to controls (f vs. e)

Figure 7

As lactadherin exhibits amphipathic properties and contains an integrin‐binding RGD domain it might induce migration by haptotaxia. To examine this, plates were coated with a 10 pmol/L–10 nmol/L bLact gradient and FHs‐74 int monolayers grown in the exclusion assay. All concentrations inhibited both cell adhesion when grown and cell migration into the voids. **denotes p < .01, and ***p < .001