Induction of the Histamine-Forming Enzyme Histidine Decarboxylase in Skeletal Muscles by Prolonged Muscular Work: Histological Demonstration and Mediation by Cytokines

Abstract

Recent studies suggest that histamine—a regulator of the microcirculation—may play important roles in exercise. We have shown that the histamine-forming enzyme histidine decarboxylase (HDC) is induced in skeletal muscles by prolonged muscular work (PMW). However, histological analysis of such HDC induction is lacking due to appropriate anti-HDC antibodies being unavailable. We also showed that the inflammatory cytokines interleukin (IL)-1 and tumor necrosis factor (TNF)-α can induce HDC, and that PMW increases both IL-1α and IL-1β in skeletal muscles. Here, we examined the effects (a) of PMW on the histological evidence of HDC induction and (b) of IL-1β and TNF-α on HDC activity in skeletal muscles. By immunostaining using a recently introduced commercial polyclonal anti-HDC antibody, we found that cells in the endomysium and around blood vessels, and also some muscle fibers themselves, became HDC-positive after PMW. After PMW, TNF-α, but not IL-1α or IL-1β, was detected in the blood serum. The minimum intravenous dose of IL-1β that would induce HDC activity was about 1/10 that of TNF-α, while in combination they synergistically augmented HDC activity. These results suggest that PMW induces HDC in skeletal muscles, including cells in the endomysium and around blood vessels, and also some muscle fibers themselves, and that IL-1β and TNF-α may cooperatively mediate this induction.

Prolonged muscular work (PMW) requires a large supply of O₂ and nutrients as well as the rapid removal of CO₂ and waste products. Thus, it is of interest to elucidate the mechanisms involved in supporting such functions. Histamine is known to regulate the microcirculation (i.e., histamine dilates precapillary arterioles and increases capillary permeability).^1–3) Histidine decarboxylase (HDC) is the histamine-forming enzyme. HDC activity is induced in response to a variety of stimuli,^4–7) and the induction of HDC activity is due to a de novo formation of HDC.⁸⁾ Recent studies suggest that histamine may play important physiological roles in skeletal muscles.^9–12)

Interestingly, PMW induces HDC in skeletal muscles in mice.^13–16) However, published histological analyses of such HDC induction are lacking because appropriate anti-HDC antibodies have not previously been available. It is of considerable interest to know in what types of cells within skeletal muscles HDC is induced by PMW because this might help explain the role of histamine as a regulator of the microcirculation.

Various cytokines [interleukin (IL)-1, tumor necrosis factor (TNF)-α, IL-6, IL-8, etc.] are reportedly released during and/or after exercise in humans.^17,18) Among these cytokines, IL-1 and TNF-α (but not IL-6 or IL-8) are capable of inducing HDC in various tissues,⁷⁾ and intraperitoneal injection into mice of IL-1α or IL-1β, at as little as 1 ng/g, produces a significant elevation of HDC activity in skeletal muscles.^14,19) Although in an earlier study on mice we could detect neither IL-1α nor IL-1β in the blood serum after PMW, prolonged mastication increased both IL-1α and IL-1β in the masseter muscles, and IL-1-KO mice exhibited reduced endurance to prolonged mastication.²⁰⁾ Here, based on the background mentioned above, we examined, in mice, the effects of PMW on the histological evidence of HDC induction and the effects of IL-1β and TNF-α on HDC activity in skeletal muscles.

MATERIALS AND METHODS

Animals

BALB/c mice were bred in our laboratory and used as a normal strain in the present study. The mice (male) used for the present experiments were 6 to 8 weeks old (23–27 g body weight). All experiments complied with the Guidelines for Care and Use of Laboratory Animals issued by Tohoku University.

PMW

At 09:00 on the morning of the day of the experiments, all mice were moved to cages with new wood-chip bedding and kept for 5 h without food but with free access to water. Prolonged walking in a cylindrical cage (37 cm diameter) turning at 5.2 rpm was started at 14:00 to 16:00, as described previously.¹⁴⁾ In each experiment, control mice were kept in the cages without food or water for the same period as the duration of the PMW.

Immunostaining of HDC in Murine Skeletal Muscle Tissues

After 2 h of PMW, mice were anesthetized and decapitated. Then, gastrocnemius muscles were removed, resected and immediately fixed by immersion in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at 4°C overnight. After being dehydrated using a graded series of ethanol solutions and infiltrated with xylene, specimens were embedded in paraffin. Serial sections of 4 µm thickness were cut and immunostaining was performed as described previously.²¹ After deparaffinization, antigen retrieval was performed using Target retrieval buffer (DAKO Corp., Carpinteria, CA, U.S.A.) for 1 h at 100°C. Sections were incubated at 4°C overnight with primary antibody for HDC (dilution 1 : 40; Abcam, Cambridge, MA, U.S.A., Cat. #: ab37291) diluted with 5% normal goat serum in phosphate-buffered saline containing 0.05% Triton-X and 5% bovine albumin. Next, the sections were incubated with Alexa⁴⁸⁸-conjugated goat anti-rabbit antibody (1 : 750; Molecular Probes, Eugene, OR, U.S.A.) for 1 h at room temperature, and the cell nuclei were stained with 4′-6-diamidino-2-phenylindole (DAPI) (Dojin Chem, Kumamoto, Japan).

Injection of IL-1 and TNF

IL-1β (Ohtsuka Pharmaceutical Co., Tokushima, Japan), TNF-α (Dainippon Pharmaceutical Co., Osaka, Japan), or a mixture of those two cytokines in sterile saline was injected intravenously into mice (0.01 mL/g body weight).

Determination of IL-1α, IL-1β, and TNFα in Blood Serum

Blood was collected directly into test tubes following decapitation. Serum was recovered by centrifugation at 2000 ×g at 4°C, then stored at −80°C until used. The IL-1α, IL-1β, and TNF-α in the serum were assayed using enzyme-linked immunosorbent assay (ELISA) kits (Endogen, Cambridge, MA, U.S.A.), the assay procedures being performed exactly as described by the manufacturer.

Assay for HDC Activity

For the assay of HDC activity, mice were decapitated, and the masseter, quadriceps femoris, and pectoralis superficial muscles were removed and stored at −80°C until assayed. HDC activity was assayed as described previously^14,22) and expressed as nmol of histamine formed during a 1 h period of incubation by the enzyme contained in 1 g (wet weight) of each tissue (nmol/h/g).

Data Analysis

Experimental values are given as mean±standard deviation (S.D.) The statistical significance of the difference between two means was evaluated using a Student’s unpaired t-test after ANOVA, p values less than 0.05 being considered to be significant.

RESULTS

Detection of HDC in Skeletal Muscles after PMW by Immunostaining

We have repeatedly shown in our previous reports that PMW (prolonged walking or prolonged gnawing) induces HDC activity in skeletal muscles within a few hours.^{9,10,14–16)} To extend those observations, in the present study we histologically confirmed the induction of HDC protein by PMW. Immunostaining of HDC in mouse gastrocnemius muscle clearly indicated that PMW increases HDC-positive cells (Figs. 1A, B). HDC-positivity was localized to cells in the endomysium and to several muscle fibers, and at high magnification it was also confirmed around blood vessels within the perimysium (Fig. 1C).

Fig. 1. Immunostaining of HDC in Gastrocnemius Muscle

(A) Control without PMW. (B and C) After 2 h PMW. HDC-positive cells are detected in the endomysium and in some muscle fibers only in the PMW group. In the enlarged view, HDC-positive cells were confirmed around blood vessels within the perimysium.

Serum Levels of IL-1 and TNF-α

Although we could detect neither IL-1α nor IL-1β in the blood serum after PMW (data not shown), TNF-α was significantly increased within 1 h of the start of PMW (Fig. 2).

Fig. 2. Exercise-Induced Elevation of TNF-α in the Blood Serum

Mice were subjected to PMW and sacrificed at the indicated times. Each value is the mean±S.D. from 4 mice. * p<0.05 vs. time 0.

Effects of Intravenous Injection of IL-1β, TNF-α, or Their Combination on HDC Activity in Skeletal Muscles

IL-1α and IL-1β recognize the same receptors.²³⁾ Thus, we tested the effect of IL-1β on the HDC activity in skeletal muscles. IL-1β (10 ng/g) increased HDC activity in all the muscles tested (masseter, pectoralis, and quadriceps femoris). HDC activity in these tissues peaked at around 5 h after the injection of IL-1β (Fig. 3 upper). Injection of TNF-α (100 ng/g) also increased HDC activity in all of these muscles (Fig. 3 lower). It should be noted that the time–course of the increase in HDC activity was similar between IL-1β and TNF-α. As shown in Fig. 4, the inductions of HDC activity by IL-1β and TNF-α were dose-dependent. The minimum doses of IL-1β and TNF-α that would induce HDC activity in this study were around 1 and 10 ng/g, respectively, indicating that IL-1β has a more potent ability to induce HDC than TNF-α. As shown in Fig. 5, although 1 ng/g of IL-1β produced no significant HDC elevation in this experiment, injection of that dose in combination with 10 ng/g of TNF-α produced a markedly augmented HDC elevation (vs. 10 ng/g TNF-α alone) in all the skeletal muscles examined.

Fig. 3. Time Course of the Elevation of HDC Activity in Skeletal Muscles Following an Injection of IL-1β or TNF-α

IL-1β (10 ng/g) or TNF-α (100 ng/g) was intravenously injected into mice, and the mice were sacrificed at the indicated times. Each value is the mean±S.D. from 4 mice. * p<0.05 vs. time 0.

Fig. 4. Dose-Dependent Elevation of HDC Activity Induced by IL-1β and TNF-α

IL-1β or TNF-α was intravenously injected at the doses indicated, and the mice were sacrificed 5 h later. Each value is the mean±S.D. from 4 mice. * p<0.05 vs. dose 0.

Fig. 5. Synergistic Elevation of HDC Activity by Co-injection of IL-1β and TNF-α

IL-1β, TNF-α, or a mixture of the two was intravenously injected into mice at the doses indicated, and the mice were sacrificed 5 h later. Each value is the mean±S.D. from 4 mice. * p<0.05 vs. saline and #p<0.05 vs. TNF-α alone.

DISCUSSION

Histamine is a known regulator of the microcirculation.^1–3) We found previously that mice given histamine H1-receptor antagonists or an HDC inhibitor, and also mice deficient in HDC or in histamine H1-receptors, display reduced endurance to PMW.^9,10) In humans, histamine H1- and H2-receptors have been shown to be involved in mediating post-exercise hyperemia and vasodilation in skeletal muscles.^11,12) These findings suggested that histamine might support PMW by maintaining or enhancing the functions of the microcirculation.

PMW increases HDC mRNA in skeletal muscles.^9,10) In the present study, by immunostaining skeletal muscles using a recently introduced commercial polyclonal anti-HDC antibody, we found that cells in the endomysium and around blood vessels, and indeed some muscle fibers themselves, became HDC-positive after PMW. In normal mice, the amount of histamine in the skeletal muscles, as in skin, is much higher (about 100 times) than that found in mast cell-deficient mice,¹⁴⁾ suggesting that skeletal muscles are rich in mast cells. In our previous study, we suggested that mast cells are present around blood vessels and nerves in the epimysium of skeletal muscles, and that histamine may be supplied by its release from, and/or its production via HDC induction in, mast cells.¹⁰⁾ However, we have also reported that HDC induction in skeletal muscles after electrical stimulation of the muscles was similar between normal mice and mast cell-deficient mice.¹⁴⁾ In addition, the HDC induction in various tissues following intravenous injection of IL-1α was similar between normal mice and mast cell-deficient mice.²⁴⁾ We suppose that histamine may be supplied from mast cells under conditions of mild PMW, but also supplied by cells other than mast cells under other conditions (such as more severe PMW).

Although, earlier, we could not detect IL-1α or IL-1β in the blood serum after PMW, we found that PMW did induce IL-1β mRNA in skeletal muscles,²⁰⁾ suggesting that an undetectable level of IL-1β may be released by PMW. Indeed, immunostaining detected both IL-1α and IL-1β around blood vessels within the perimysium of skeletal muscles after PMW.²⁰⁾ IL-1α and IL-1β recognize the same cell-surface receptors and stimulate them in an autocrine or paracrine manner.²³⁾ In the present study, TNF-α was detected in the blood serum after PMW. The minimum intravenous dose of IL-1β that would induce HDC activity in this study was about 1/10 that of TNF-α, while a combination of the two synergistically augmented HDC activity. Thus, it seems likely that both TNF-α and IL-1 (α and/or β) contribute to the elevation of HDC activity seen in skeletal muscles following PMW, even though the production of IL-1α and/or IL-1β is very small.

Incidentally, prolonged walking induces HDC activity in various skeletal muscles (masseter, pectoralis superficial, or quadriceps femoris)¹⁵⁾ and also in other tissues (lung, spleen, and bone marrow).¹⁴⁾ Those findings support the idea that PMW-induced TNF-α and/or undetectable levels of IL-1 (α and/or β) released into the serum may contribute to HDC induction in various tissues when their concentrations have reached to a sufficiently high level.

In humans, the reported effects of exercise on the plasma levels of IL-1 and TNF-α are controversial, and there may be only minor elevations, if any.¹⁷⁾ In contrast, IL-6 is elevated markedly and rapidly after exercise.¹⁷ In mice, PMW increases IL-6 in skeletal muscles and in the blood serum, too.²⁵⁾ However, IL-6 is inactive at inducing HDC in various tissues in mice.⁷⁾

Training increases the resistance to hard exercise. We have reported that training reduces HDC-induction by PMW.^14,16) Prolonged and/or unaccustomed hard muscle activity frequently cause pain in the muscle, and histamine is a typical pain-producing substance. Thus, it remains to be clarified whether histamine is involved in the above training-induced adaptation and/or in muscle pain. The discussion points above are summarized in Fig. 6.

Fig. 6. Summary of Discussion Exercise Stimulates the Production of IL-1 (α and/or β) and TNF-α

These cytokines induce histidine decarboxylase within cells in the endomysium and around blood vessels (including mast cells) in skeletal muscle tissues, and also within some muscle fibers themselves. The histamine released from such cells may support the microcirculation in skeletal muscles and contribute to anti-fatigue (thereby prolonging the ability of the muscles to perform work) and/or to the recovery from fatigue. It would be of interest to examine in future the possibility that histamine might also be involved in muscle pain, thereby serving as an alarm signal indicating the need for rest.

CONCLUSION

PMW induces HDC in skeletal muscles including cells in the endomysium and around blood vessels, and also some muscle fibers themselves, and IL-1β and TNF-α may cooperatively mediate this induction.

Acknowledgment

We are grateful to Dr. Robert Timms for editing the manuscript and for instructive discussion.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Beaven MA. Histamine: its role in physiological and pathological processes. Monographs in Allergy (Dukor P, Kallós P, Trnka Z, Waksman BH, de Weck AL eds.), Vol. 13, Karger, Basel, pp. 1–113 (1978).
• 2) Beer DJ, Matloff SM, Rocklin RE. The influence of histamine on immune and inflammatory responses. Adv. Immunol., 35, 209–268 (1984).
• 3) Garrison JC. Histamine, bradykinin, 5-hydroxytryptamine, and their antagonists. Goodman and Gilman’s The Pharmacological Basis of Therapeutics (Gilman AG, Rall TW, Nies AS, Taylor P eds.), 8th ed., Pergamon Press, New York, pp. 575–599 (1990).
• 4) Schayer RW. Histamine and microcirculation. Life Sci., 15, 391–401 (1974).
• 5) Kahlson G, Rosengren E. New approaches to the physiology of histamine. Physiol. Rev., 48, 155–196 (1968).
• 6) Endo Y. Induction of histidine decarboxylase in mouse tissues by mitogens in vivo. Biochem. Pharmacol., 32, 3835–3838 (1983).
• 7) Endo Y, Kikuchi T, Takeda Y, Nitta Y, Rikiishi H, Kumagai KGM-CSF. GM-CSF and G-CSF stimulate the synthesis of histamine and putrescine in the hematopoietic organs in vivo. Immunol. Lett., 33, 9–13 (1992).
• 8) Kikuchi H, Watanabe M, Endo Y. Induction by interleukin-1 (IL-1) of the mRNA of histidine decarboxylase, the histamine-forming enzyme, in the lung of mice in vivo and the effect of actinomycin D. Biochem. Pharmacol., 53, 1383–1388 (1997).
• 9) Niijima-Yaoita F, Tsuchiya M, Ohtsu H, Yanai K, Sugawara S, Endo Y, Tadano T. Roles of histamine in exercise-induced fatigue: Favouring endurance and protecting against exhaustion. Biol. Pharm. Bull., 35, 91–97 (2012).
• 10) Yoneda H, Niijima-Yaoita F, Tsuchiya M, Kumamoto H, Watanabe M, Ohtsu H, Yanai K, Tadano T, Sasaki K, Sugawara S, Endo Y. Roles played by histamine in strenuous or prolonged masseter muscle activity in mice. Clin. Exp. Pharmacol. Physiol., 40, 848–855 (2013).
• 11) Romero SA, Hocker AD, Mangum JE, Luttrell MJ, Turnbull DW, Struck AJ, Ely MR, Sieck DC, Dreyer HC, Halliwill JR. Evidence of a broad histamine footprint on the human exercise transcriptome. J. Physiol., 594, 5009–5023 (2016).
• 12) Romero SA, McCord JL, Ely MR, Sieck DC, Buck TM, Luttrell MJ, MacLean DA, Halliwill JR. Mast cell degranulation and de novo histamine formation contribute to sustained post-exercise vasodilation in humans. J. Appl. Physiol., 10.1152/japplphysiol.00633.2016
• 13) Graham P, Kahlson G, Rosengren E. Histamine formation in physical exercise, anoxia and under the influence of adrenaline and related substances. J. Physiol., 172, 174–188 (1964).
• 14) Endo Y, Tabata T, Kuroda H, Tadano T, Matsushima K, Watanabe M. Induction of histidine decarboxylase in skeletal muscle in mice by electrical stimulation, prolonged walking and interleukin-1. J. Physiol., 509, 587–598 (1998).
• 15) Ayada K, Watanabe M, Endo Y. Elevation of histidine decarboxylase activity in skeletal muscles and stomach in mice by stress and exercise. Am. J. Physiol. Regul. Integr. Comp. Physiol., 279, R2042–R2047 (2000).
• 16) Ayada K, Tadano T, Endo Y. Gnawing behavior of a mouse in a narrow cylinder: a simple system for the study of muscle activity, fatigue, and stress. Physiol. Behav., 77, 161–166 (2002).
• 17) Pedersen BK. Exercise and cytokines. Immunol. Cell Biol., 78, 532–535 (2000).
• 18) Pedersen BK, Hoffman-Goetz L. Exercise and immune system: regulation, integration, and adaptation. Physiol. Rev., 80, 1055–1081 (2000).
• 19) Watanabe M, Tabata T, Huh JI, Inai T, Tsuboi A, Sasaki K, Endo Y. Possible involvement of histamine in muscular fatigue in temporomandibular disorders: animal and human studies. J. Dent. Res., 78, 769–775 (1999).
• 20) Chiba K, Tsuchiya M, Koide M, Hagiwara Y, Sasaki K, Hattori Y, Watanabe M, Sugawara S, Kanzaki M, Endo Y. Involvement of IL-1 in the maintenance of masseter muscle activity and glucose homeostasis. PLoS One, 2015, Nov. 24; 10(11): e0143635. doi: 10.1371/journal.pone.0143635. eCollection 2015 (2015).
• 21) Sato S, Tsuchiya M, Komaki K, Kusunoki S, Tsuchiya S, Haruyama N, Takahashi I, Sasano Y, Watanabe M. Synthesis and intracellular transportation of type I procollagen during functional differentiation of odontoblasts. Histochem. Cell Biol., 131, 583–591 (2009).
• 22) Endo Y. A simple method for the determination of polyamines and histamine and its application to the assay of ornithine decarboxylase and histidine decarboxylase activities. Methods Enzymol., 94, 42–47 (1983).
• 23) Dinarello CA. The interleukin-1 family: 10 years of discovery. FASEB J., 8, 1314–1325 (1994).
• 24) Wu X, Yoshida A, Sasano T, Iwakura Y, Endo Y. Histamine production via mast cell-independent induction of histidine decarboxylase in response to lipopolysaccharide and interleukin-1. Int. Immunopharmacol., 4, 513–520 (2004).
• 25) Tsuchiya M, Kiyama T, Tsuchiya S, Takano H, Nemoto E, Sasaki K, Watanabe M, Sugawara S, Endo Y. Interleukin-6 maintains glucose homeostasis to support strenuous masseter muscle activity in mice. Tohoku J. Exp. Med., 227, 109–117 (2012).