Treatment with selectin blocking antibodies after lengthening contractions of mouse muscle blunts neutrophil accumulation but does not reduce damage

Abstract

P- and E-selectins are expressed on the surface of endothelial cells and may contribute to neutrophil recruitment following injurious lengthening contractions of skeletal muscle. Blunting neutrophil, but not macrophage, accumulation after lengthening contractions may provide a therapeutic benefit as neutrophils exacerbate damage to muscle fibers, while macrophages promote repair. In this study, we tested the hypothesis that P- and E-selectins contribute to neutrophil, but not macrophage, accumulation in muscles after contraction-induced injury, and that reducing neutrophil accumulation by blocking the selectins would be sufficient to reduce damage to muscle fibers. To test our hypothesis, we treated mice with antibodies to block P- and E-selectin function and assessed leukocyte accumulation and damage in muscles 2 days after lengthening contractions. Treatment with P/E-selectin blocking antibodies reduced neutrophil content by about half in muscles subjected to lengthening contractions. In spite of the reduction in neutrophil accumulation, we did not detect a decrease in damage 2 days after lengthening contractions. We conclude that P- and/or E-selectin contribute to the neutrophil accumulation associated with contraction-induced muscle damage and that only a portion of the neutrophils that typically accumulate following injurious lengthening contractions is sufficient to induce muscle fiber damage and force deficits. Thus, therapeutic interventions based on blocking the selectins or other adhesion proteins will have to reduce neutrophil numbers by more than 50% in order to provide a benefit.

Keywords: Injury; lengthening contraction; muscle; neutrophil; selectin.

Figure 1

Injection of irrelevant control antibody A110‐1 has no significant effect on damage or inflammatory cells in muscles 2 days after lengthening contractions. Force deficit (A). Damaged fibers (B). Neutrophils (Gr‐1+ cells) (C). Macrophages (CD68+ cells) (D).

Figure 2

Treatment with blocking antibodies against P/E‐selectin decreases neutrophil content in muscles 2 days after lengthening contractions. Neutrophils (Gr‐1+ cells) *P = 0.005 by Mann–Whitney Rank Sum Test (A). Representative partial section of muscle from uninjected mouse (B) and mouse injected with blocking antibodies (C) with scale bars = 200 μm. Examples of neutrophil (Gr‐1+ cells, brown) and blood vessel (GSL I, gray) co‐labeling with scale bars = 50 μm (D, E). Neutrophils outside of vessels (D). Neutrophils on the inner surface of large vessels (E).

Figure 3

Treatment with blocking antibodies against P/E‐selectin does not significantly decrease muscle damage 2 days after lengthening contractions, as assessed by force deficit.

Figure 4

Treatment with blocking antibodies does not reduce the total number of damaged fibers, but reduces the percentage of damaged fibers that are invaded by inflammatory cells, 2 days after lengthening contractions. Total damaged fibers expressed as a percentage of the total number of fibers in a muscle section (A). Representative partial section of muscle from uninjured mouse (B), uninjected mouse (C), and mouse injected with P/E‐selectin blocking antibodies (D). Damaged fibers expressed as a percentage of the total number of damaged fibers in a muscle section (E). Damaged fibers fell into one of three categories. Category 1 consisted of round and often swollen fibers stained dark with eosin Y. Category 2 consisted of fibers with variable or pale staining with eosin Y. Category 3 consisted of fibers similar to those in category 2, but with several nuclei within the fiber, assumed to be inflammatory cells. *Significantly different from category 1 within experimental group. #Significantly different from category 2 within experimental group. ψSignificantly different from control within category. Significance was determined by a two‐way analysis of variance (P < 0.05) (E). Magnified views of fibers from C and D (F–I). Examples of category 1 (F), category 2 (G, H) and category 3 (I) fibers. Scale bars = 200 μm.

Figure 5

Treatment with blocking antibodies against P/E‐selectin decreases macrophage content in muscles 2 days after lengthening contractions, although the decrease is not statistically significant (P = 0.076 by Mann–Whitney Rank Sum Test). Macrophages (CD68+ cells) (A). Representative section of muscle from uninjected mouse (B) and mouse injected with blocking antibodies (C). Scale bars = 200 μm.

Figure 6

Treatment with blocking antibodies against P/E‐selectin increases neutrophil, but not macrophage content after 2 days, in contralateral muscles that have not been subjected to lengthening contractions. Neutrophils (Gr‐1+ cells) *P < 0.001 (A). Representative partial section of muscle from uninjured mouse (B) and mouse injected with P/E‐selectin blocking antibodies (C), Gr‐1 labeled. Macrophages (CD68+ cells) P = 0.854 by Mann–Whitney Rank Sum Test (D). Representative partial section of muscle from uninjured mouse (E) and mouse injected with P/E‐selectin blocking antibodies (F), CD68 labeled. Scale bars = 200 μm.

Figure 7

Circulating levels of major populations of white blood cells before and after lengthening contractions (LC). Note that injection of blocking antibodies occurred within an hour of lengthening contractions. Neutrophils increased significantly with blocking antibody treatment *significantly different from control, #significantly different from 1 day pre‐LC value ψsignificantly different from 1 day post‐LC value (A). Monocytes increased with blocking antibody treatment but the increase did not reach significance (P = 0.066) (B). Lymphocytes increased 2 days after lengthening contractions in both experimental groups # significantly different from 1 day pre‐LC value (C). Data was analyzed by two‐way repeated measures analysis of variance tests (significance set at P < 0.05).