Aging-associated shifts in functional status of mast cells located by adult and aged mesenteric lymphatic vessels

Abstract

We had previously proposed the presence of permanent stimulatory influences in the tissue microenvironment surrounding the aged mesenteric lymphatic vessels (MLV), which influence aged lymphatic function. In this study, we performed immunohistochemical labeling of proteins known to be present in mast cells (mast cell tryptase, c-kit, prostaglandin D(2) synthase, histidine decarboxylase, histamine, transmembrane protein 16A, and TNF-α) with double verification of mast cells in the same segment of rat mesentery containing MLV by labeling with Alexa Fluor 488-conjugated avidin followed by toluidine blue staining. Additionally, we evaluated the aging-associated changes in the number of mast cells located by MLV and in their functional status by inducing mast cell activation by various activators (substance P; anti-rat DNP Immunoglobulin E; peptidoglycan from Staphyloccus aureus and compound 48/80) in the presence of ruthenium red followed by subsequent staining by toluidine blue. We found that there was a 27% aging-associated increase in the total number of mast cells, with an ∼400% increase in the number of activated mast cells in aged mesenteric tissue in resting conditions with diminished ability of mast cells to be newly activated in the presence of inflammatory or chemical stimuli. We conclude that higher degree of preactivation of mast cells in aged mesenteric tissue is important for development of aging-associated impairment of function of mesenteric lymphatic vessels. The limited number of intact aged mast cells located close to the mesenteric lymphatic compartments to react to the presence of acute stimuli may be considered contributory to the aging-associated deteriorations in immune response.

Fig. 1.

Immunohistochemical labeling (representative images of segments of rat mesentery in close proximity to mesenteric lymphatic vessels) of protein targets shown to be present in mast cells (A, D, G, J, M, P, S) together with the labeling of the same segment by Alexa Fluor 488-conjugated avidin (B, E, H, K, N, Q, T) followed by staining of the same segment with toluidine blue (C, F, I, L, O, R, U). Specimens are labeled for mast cell tryptase (A), c-kit (CD 117) (D), prostaglandin D₂ synthase (G), histidine decarboxylase (J), histamine (M), transmembrane protein 16 A (P), and TNF-α (S). V, primary antibodies control. W, same segment labeled by Alexa Fluor 488-conjugated avidin to confirm presence of mast cells. X, secondary antibodies control. Scale bar on S corresponds to 100 μm and applies to all images. G, H, and I: images from 24-mo-old rat; the rest are taken from 9-mo-old animals. Mast cells were always found to lie on or outside the lymphatic wall. MLV, mesenteric lymphatic vessel.

Fig. 2.

Representative images of the activation of mast cells located in close proximity to rat mesenteric lymphatic vessels in adult and aged segments of mesentery. A: segment (9 mo old) of mesentery before activation. B: same segment with mast cells activated by compound 48/80 stained with the ruthenium red. C: same segment stained with toluidine blue. D: segment (9 mo old) of mesentery before activation. E: same segment activated by substance P in 10⁻⁵ M. F: segment (24 mo old) of mesentery before activation. G: same segment activated by substance P in 10⁻⁵ M. Scale bar on A corresponds to 100 μm and applies to all images.

Fig. 3.

A-J: Quantitative analysis of mast cells activation induced by various activators in adult (9 mo) and aged (24 mo) segments of rat mesentery. 48/80, compound 48/80; subs P, substance P. Values are means ± SE. *Significant differences (P < 0.05) before and after treatment with same age group; **significant differences between 9- and 24-mo age groups.

Fig. 4.

Aging-associated alterations in the fraction of mast cells activated by various treatments (FCAT) in adult (9-mo-old) and aged (24-mo-old) segments of rat mesentery. Values are means ± SE. **Significant differences (P < 0.05) between 9- and 24-mo-old age groups. Subst P, substance P.