Mast cell-directed recruitment of MHC class II positive cells and eosinophils towards mesenteric lymphatic vessels in adulthood and elderly

Abstract

Background: Aging impairs mesenteric lymph flow, which is crucial for fluid and macromolecule homeostasis, fat absorption, and immune function. Previously, we demonstrated that mast cells (MCs) line mesenteric lymphatic vessels (MLVs) with a greater degree of basal activation of MCs in aged mesentery. The number of intact MCs available to react acutely to inflammatory stimuli was decreased with age. However, the role of mast cells in recruiting other immune cells towards MLVs and its aging-associated alterations has not been explored before in great detail.

Methods and results: In this study we treated live mesenteric tissue isolated from Sprague Dawley (SD) rats, as well as adult 9-mo and aged 24-mo Fischer-344 (F-344) rats for 2 hours with MC activators (48/80 and Substance P) and performed whole mount IHC and vital dye staining of the mesenteric segments containing MLVs to identify immune cell recruitment towards MLVs after mast cell (MC) activation. Number of major histocompatibility complex (MHC) class II positive APCs and eosinophils near MLVs was counted and compared between treatments and ages.

Conclusions: With greater density of MCs near MLVs, we for the first time demonstrated that mesenteric MC activation by compound 48/80 and Substance P resulted in recruitment of MHC class II positive cells and eosinophils towards MLVs. This effect was reduced in cromolyn-injected rats, thus confirming that MCs are necessary for such recruitment. The immune cell presence near MLVs after MC activation was reduced in aged tissues. We link these findings to our previous report of lesser number of intact MCs available for initiating an acute immune response in aged mesentery. Cumulatively, these findings serve as the first step in study of the aging-associated mechanisms that link MCs, lymphatic vessels, and disordered immune function in the elderly.

FIG. 1.

Identification of mast cell-directed increase in presence of MHC class II positive cells by mesenteric lymphatic vessels. (A) Toluidine blue staining of mast cells with higher density of mast cells by MLV. (B) Higher density of MHC class II positive cells by MLV after MC activation by compound 48/80. (C) MHC class II positive cells by MLV (green) are not mast cells (red). (D–O) MHC class II positive APCs by MLV. D–F normal saline-injected SD rat; G–I cromolyn sodium-injected SD rat; J–L normal saline-injected 9-mo F-344 rat; M–O normal saline-injected 24-mo F-344 rat. D, G, J, M: control; E, H, K, N: compound 48/80 treatment; F, I, L, O: Substance P treatment. Scale bar on B corresponds to 200 μm and applies to A and B panels, scale bar on C corresponds to 100 μm and applies to panels C–O.

FIG. 2.

Quantitative analysis of number of MHC class II positive APCs near mesenteric lymphatic vessels in control conditions and after mast cell activation by compound 48/80 and Substance P. (A) Comparison of mesenteric segments from saline and cromolyn sodium-treated SD rats. (B) Comparison of mesenteric segments from 9-mo and 24-mo old F-344 rats. Values are means±SE; * indicates significant differences (p<0.05) before and after treatment in saline-injected SD rats (A) or within same age group; ** indicates significant differences between 9-mo and 24-mo age groups within same treatment.

FIG. 3.

Identification of mast cell-directed increase in the presence of eosinophils by mesenteric lymphatic vessels. Eosinophils marked by green arrows, (not all of these cells pointed by arrows). (A–C) normal saline-injected SD rats; (D–F) cromolyn sodium-injected SD rats; (G–I) 9-mo F-344 rats; (J–L) 24-mo F-344 rats. A, D, G, J: control; B, E, H, K: compound 48/80 treatment; C, F, I, L: Substance P treatment. C2: zoomed image of portion of panel C to demonstrate in closer view the color and shape of eosinophils. Scale bar on F corresponds to 50 μm and applies to all panels.

FIG. 4.

Quantitative analysis of number of eosinophils near mesenteric lymphatic vessels in control conditions and after mast cell activation by compound 48/80 and Substance P. (A) Comparison of mesenteric segments from saline and cromolyn sodium treated SD rats. (B) Comparison of mesenteric segments from 9-mo and 24-mo old F-344 rats. Values are means±SE; * indicates significant differences (p<0.05) before and after treatment in saline-injected SD rats (A) or within same age group; ** indicates significant differences between 9-mo and 24-mo age groups within same treatment.