513
Journal of Physiology (1990), 422, pp. 513-522 With 7figures Printed in Great Britain
NERVE-MEDIATED CONTRACTIONS OF SHEEP MESENTERIC LYMPH NODE CAPSULES
BY K. D. THORNBURY, N. G. McHALE, J. M. ALLEN* AND GWEN HUGHES* From the Department of Physiology, Queen's University ofBelfast, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland, and the *Biomedical Sciences Research Centre, University of Ulster, Newtownabbey BT37 OQB, Northern Ireland
(Received 11 September 1989) SUMMARY
1. Isometric tension was recorded in vitro from strips cut from the capsules of mesenteric lymph nodes of sheep. 2. One minute periods of field stimulation at frequencies of 1, 2, 4, 8 and 16 Hz (pulse duration, 0 3 ms) elicited tonic contractions of increasing force and duration. The stimulus frequency-response relationship began to flatten out at frequencies > 4 Hz, where the response was already 72 % of that at 16 Hz. 3. The response to field stimulation was abolished by tetrodotoxin (1 ,UM). 4. Phentolamine, rauwolscine and prazosin (all 1 IM) reduced the response to field stimulation, while desipramine (1 ,tM), potentiated it. 5. Atropine (1 /M) was without effect on the response. 6. These results suggest that sheep mesenteric lymph node capsules have a noradrenergic innervation which modulates their tone via an action on a-
adrenoceptors. INTRODUCTION
Morphological studies indicate that lymph nodes are innervated by a variety of fibre types including adrenergic (Giron, Crucher & Davis, 1980; Felten, Livnat, Felten, Carlsen, Bellinger & Yeh, 1984), substance P-containing and calcitonin generelated peptide-containing (Popper, Mantyh, Vigna, Maggio & Mantyh, 1988) types. It has been suggested that these nerves might modulate the immune system since sympathetic denervation of lymph nodes or the spleen alters the immune responsiveness of these organs (Besedovsky, Del Rey, Sorkin, Da Prada & Keller, 1979; Felten et al. 1984; Popper et al. 1988). Smooth muscle is also present in the capsules and trabeculae of lymph nodes in many species including cats, dogs, cattle, sheep and goats (Passantino, 1940). Adrenergic nerves appear to be associated with the trabeculae and there is evidence of a dense subcapsular adrenergic plexus which could conceivably innervate the capsular smooth muscle (Felten et al. 1984). At present the role of this muscle remains a matter for speculation, although one potentially important function is suggested by the recent demonstration that stimulation of the lumbar sympathetic chain in the sheep increases the output of MS 7933 17
PH Y 422
514
K. D. THORNBURY AND OTHERS
lymphocytes from the popliteal node (McHale & Thornbury, 1989). While the mechanism of this response has yet to be confirmed it may be mediated by contraction of the node capsule and/or trabeculae. A similar mechanism has been well described in the splenic capsules of dogs and sheep where contraction in response to stimulation by sympathetic nerves and circulating adrenaline results in expulsion of erythrocytes and a raised haematocrit (Barcroft, Harris, Orahovats & Weiss, 1925; Hodgetts, 1961). At present, however, there has been no functional study describing the effect of nerve stimulation on lymph node capsular smooth muscle; indeed it has not yet been confirmed that this tissue is even innervated. In the experiments described below we have used the technique of in vitro transmural field stimulation to study nervemediated contractions of capsular strips taken from sheep mesenteric lymph nodes. The evidence suggests that the smooth muscle of these node capsules possesses an adrenergic innervation which evokes contractions by an action on oc-adrenoceptors.
METHODS
Mesenteric lymph nodes were removed immediately after euthanasia with pentobarbitone (80 mg/kg, I.v.) from five sheep which had been previously anaesthetized for mesenteric lymph flow studies (anaesthesia induced with pentobarbitone 20-30 mg/kg, i.v. and maintained by breathing 1-3 % halothane in 02). Transversely orientated strips (approximately 15 mm long, 3 mm wide, 1-2 mm thick) were cut and stored until use at room temperature in Krebs solution composition (mM): NaCl, 120; NaHCO3, 25; KCl, 5 9; NaH2P04, 1-2; CaCl2, 2-5; MgCl2, 1-2, glucose, 11, gassed with 95% 02, 5% CO2. They were then mounted in water-jacketed organ baths (volume, 1 ml) maintained at 37 °C and perfused with Krebs solution. Initial resting tension was adjusted to 4 mN and a period of at least 30 min was allowed for equilibration. Tension changes were measured with Statham UC3 and Dynamometer UFI isometric transducers the outputs of which were written on Gould 2400S and Lectromed MX 216 chart recorders. Field stimulation was applied via platinum ring electrodes at each end of the organ bath. Pulses of 0 3 ms duration were delivered in trains at constant frequencies of 1, 2, 4, 8 or 16 Hz from Grass S88 and SI1 stimulators set at nominal output voltages of 50 V. Stimulation periods always lasted for 1 min and the recovery time between stimuli was usually 8-10 min. The drugs used were as follows: tetrodotoxin (Sigma); phentolamine mesylate (CIBA); rauwolscine hydrochloride (Carl Roth); prazosin hydrochloride (Pfizer); desipramine hydrochloride (Sigma); atropine sulphate (Sigma). All of these were made up to their final concentrations in Krebs solution. Summarized results are expressed throughout as the mean response+s.E. of the mean, and statistical comparisons of means were made using Student's paired t test.
RESULTS
Of the fifty-two preparations observed in the present study forty-eight showed spontaneous activity. This was mostly (forty-one preparations) of the type described by Tumer, Ozturk-Demir, Basar-Eroglu & Noyan (1983) and consisted of irregular contractions of 0-1-0-5 mN force (e.g. see small fluctuations in baseline tension of Figs 2 and 5). This activity was rarely present throughout the experiment, usually appearing in bursts lasting from several minutes to 05 h. In seven preparations the activity was more regular in force and frequency (0-5-1 beats/min) and lasted for over 30 min, while in four preparations there was no evidence of any spontaneous activity.
LYMPH NODE INNER VATION
515
Effect offield stimulation The typical effect of 1 min periods of field stimulation at frequencies of 1, 2, 4, 8 and 16 Hz is shown in the upper panel of Fig. 1. In contrast to the rather small spontaneous contractions described above, transmural field stimulation produced I 4mN
2min 2Hz
1 Hz
4Hz
8Hz
16Hz
6 5E 4 H
2
-
1
00
16 12 4 8 Stimulus frequency (Hz) Fig. 1. Upper panel: effect of stimulus frequency on isometric tension in a sheep mesenteric lymph node capsular strip. The preparation was stimulated for 1 min periods indicated by arrows. Lower panel: stimulus frequency-response relationship summarized for six preparations. Circles, mean responses; vertical bars, + standard error of mean.
relatively large increases in tone, ranging from 1P7 mN at 1 Hz to 5-9 mN at 16 Hz. These responses consisted of a rapid contraction phase which occurred during the first 10-15 s of stimulation, followed by a slower increase in tension to a plateau during the next 30-45 s. After the stimulus ceased, the muscle relaxed slowly, taking several minutes to reach its previous resting tension after stimulation at frequencies of 2 Hz or greater. A summary of the effect of stimulation on peak isometric tension (mN) in six preparations is presented in the lower panel of Fig. 1. The curve shows that the tension response increased steeply over the 1-4 Hz frequency range, but thereafter increases in stimulus frequency produced only slight improvement. Moreover, it was found that after stimulation at 16 Hz, subsequent responses to 4 Hz were reduced to approximately 60% of those obtained previously at this frequency. In contrast, consistent responses to repeated 4 Hz stimulations could be obtained for 1-2 h. For these reasons 4 Hz was chosen as the frequency with which to examine the effects of the various blocking drugs described below.
Effect of tetrodotoxin The effect of tetrodotoxin (1 /M) on the response to field stimulation at 4 Hz was tested to establish if the response was mediated by nerves. In the example shown in the upper panel of Fig. 2, two separate 1 min periods of 4 Hz stimulation elicited 17-2
K. D. THORNBUR Y AND OTHERS
516
TTX (1 uM)
t\
~
X
4Hz 4 Hz
#
I
~~
f' 4z 4 Hz
A
,
4z 4 Hz
mN ~~~~~4
2 min
4z 4 Hz
3-
E
2-
0 ._ C
H
U
Control
E
TTX (1 uM)
1-
0-
4 Hz
4 Hz
4 Hz
4 Hz
Fig. 2. Upper panel: effect of tetrodotoxin (TTX, 1 ,IM) on the response to stimulation at 4 Hz. Lower panel: summary of the effect of TTX in five preparations. f, mean response to a 1 min period of stimulation at 4 Hz in the absence of TTX; 0, responses after exposure to TTX; vertical bars, + standard error of mean.
Phentolamine (1
gM) 4 mN
5 min 4 Hz
4 Hz
4 Hz
4 Hz
41 3-
LII Control
2-
El
E
a)
Phentolamine (1
gM)
1-
0-
4 z 4 Hz
4 Hz
Fig. 3. Upper panel: effect of phentolamine (1 /iM) on the response to stimulation at 4 Hz. Lower panel: summary of the effect of phentolamine (n = 7). The format is similar to Fig. 2.
LYMPH NODE INNER VATION
517
similar responses. After infusion of tetrodotoxin for 10 min the response was almost completely abolished. This is confirmed in the summary of five such experiments represented by the column graph in the lower panel of Fig. 2. The open columns on the left refer to two successive stimulus periods before tetrodotoxin was infused, Rauwolscine (1 pM) 3 mN t7 4 Hz
4 Hz
4 Hz
+% 4 Hz
2 min
T
2-
zControl E
T
,0 1 X ;
Rauwolscine (1
pM)
CA
4Hz 4 Hz 4 Hz Fig. 4. Upper panel: effect of rauwolscine (1 /M) on the response to stimulation at 4 Hz. Lower panel: summary of the effect of rauwolscine (n = 5). The format is similar to Fig. 2. 4 Hz
while the two hatched columns represent two stimulus periods after infusion of the drug. Before tetrodotoxin the mean responses were not significantly different (P > 0-2), while in the presence of tetrodotoxin they were markedly depressed (P < 0 03). In contrast to stimulus-evoked contractions, spontaneous contractions persisted in the presence of tetrodotoxin (see small fluctuations in baseline, Fig. 2, upper panel).
Effect ofphentolamine, rauwolscine and prazosin In order to determine whether or not the above responses could have been mediated by adrenergic nerves acting on a-adrenoceptors the effects of the aadrenoceptor blocking agents phentolamine, rauwolscine and prazosin were tested. Of these phentolamine is thought to be non-selective with regard to oc-adrenoceptor subtype, while rauwolscine and prazosin are thought to be relatively selective for a2 and a, subtypes, respectively. Figure 3 (upper panel) shows a typical example of the action of phentolamine (1/UM) on the response to 4 Hz stimulation. Two 1 min periods of 4 Hz stimulation produced similar responses before phentolamine was perfused, but at 15 and 25 min after exposure to the blocker the contractions evoked by 4 Hz were markedly reduced. The effect of phentolamine on seven preparations is summarized in the column diagram in the lower panel of Fig. 3, the format of which is similar to that of Fig. 2. The open columns represent the mean responses of two stimulus periods, 10 min apart, in the absence of blocker, while the hatched columns refer to stimulus
K. D. THORNBUR Y AND OTHERS
518
Prazosin (1 PM)
i
(I
I--, j~
C-4
% 4 Hz
% 4 Hz
IL_ ¼
¼
¼
4 Hz
2 X 4mN
2min
4 Hz
2-
2
-I Control
E
c
m E Prazosin (1
1-
uM)
0
CD
4 Hz
4 Hz
4 Hz
4 Hz
Fig. 5. Upper panel: effect of prazosin (1 /M) on the response to stimulation at 4 Hz. Lower panel: summary of the effect of prazosin (n = 9). The format is similar to Fig. 2.
Desipramine (1 pM) 3 mN 1 Hz
2 Hz
¼1 Hz
¼ 2 Hz
2 min
T E 2-
Control
0 C c aD
H
E Desipramine
(1 pM)
-
0-
1 Hz
2 Hz
Fig. 6. Upper panel: effect of desipramine (1 #M) on the responses to stimulation at 1 and 2 Hz. Lower panel: summary of the effect of desipramine (n = 9). The format is similar to Fig. 2.
LYMPH NODE INNER VATION
519
periods 15 and 25 min after infusion of blocker. Phentolamine reduced the responses (P < 0-006 and P < 0-002, when compared with the second control period). Figure 4 shows that rauwolscine (1 /SM) had an effect similar to that of phentolamine when tested using a similar protocol. In the example shown in the upper panel two 1 min periods of 4 Hz stimulation elicited responses which were reduced after exposure to rauwolscine for 15 and 25 min. A summary of five experiments is shown in the lower panel of Fig. 4, confirming that rauwolscine reduced the responses (P < 0 03 at 15 min and P < 0003 at 25 min). Atropine (1
PiM) 4 mN 2 min
4 Hz
4 Hz
E c
4 Hz
4 Hz
3Z ~~~~~~~~~~~~]Control 2
Atropine (1
0
pM)
c
01
4 Hz
4 Hz
4 Hz
4 Hz
Fig. 7. Upper panel: effect of atropine (1 M) on the response to stimulation at 4 Hz. Lower panel: summary of the effect of atropine (n = 5). The format is similar to Fig. 2.
In comparison to phentolamine and rauwolscine, an equimolar concentration of the a,l-antagonist prazosin was less effective at blocking the effect of 4 Hz stimulation. Nevertheless, that the responses were partially blocked is evident from both the typical record and summary diagram (n = 9) shown in Fig. 5 (P < 003 after 15 min and P < 0-02 after 25 min of exposure to prazosin, respectively).
Effect of desipramine The above results with a-adrenoceptor antagonists suggest that the responses to field stimulation were mediated by adrenergic nerves. If this is the case, then one would also expect desipramine to potentiate the response, since this substance is known to block reuptake of noradrenaline into adrenergic nerve terminals. That the response was indeed enhanced in this way can be seen in Fig. 6. In these experiments responses to 1 and 2 Hz, rather than 4 Hz, were tested since these fell on the steep part of the stimulus frequency-response curve (see Fig. 1), and were thought more likely to show potentiation. In the upper panel of Fig. 6 an example is shown where, after exposure to desipramine (1 ,sM), the tension responses were both greater and more prolonged. Nine such experiments are summarized in the lower panel of Fig. 6
520
K. D. THORNBUR Y AND OTHERS
where it can be seen that the mean potentiation was more marked at 2 Hz (a 37 % increase, P < 0 02).
Effect of atropine Figure 7 (upper panel) shows the effect of atropine (1 /M) in a preparation where 4 Hz stimulation produced contractions of around 3 mN. Atropine made no obvious difference to the strength or duration of the response, suggesting that there was little or no cholinergic component. This conclusion is further supported by the five experiments summarized in the lower panel where responses in the presence of atropine were not significantly different (P > 0 56) from those obtained in the control period. DISCUSSION
Several investigators have previously examined the contractility of lymph nodes in vitro. There are early reports of isolated whole mesenteric nodes from cats (Florey, 1927) and dogs (Martin, 1932) contracting when adrenaline was added to their bathing medium. More recently Tumer et al. (1983) have described spontaneous activity in intact node preparations from the prescapular region in sheep and goats, of the guinea-pig mesentery, and of strips cut from calf prescapular nodes. Of these only the cattle nodes displayed regular, long-lasting activity characterized by discrete contractions and relaxations. The records of sheep nodes obtained by these authors were fairly similar to those obtained in the present study, despite a number of differences in the preparation (e.g. whole prescapular nodes versus strips of mesenteric node capsule). The fact that spontaneous activity was observed in the presence of tetrodotoxin in the present experiments suggests that it was of myogenic origin. So far as we are aware, this is the first study that has attempted to demonstrate the action of nerves on lymph node capsular smooth muscle. Transmural field stimulation with short pulses produced contractions which were effectively blocked by tetrodotoxin, suggesting that they were mediated by nerves. The stimulus frequency-response relationship was similar to those obtained for a variety of vascular smooth muscle preparations, with the greatest increases in response occurring in the 1-4 Hz range, and a levelling off at frequencies of 4-16 Hz (Johannsen & Ljung, 1967; Ljung, Bevan, Pegram, Purdy & Su, 1975). That the nerves concerned were adrenergic is suggested by two pieces of evidence. Firstly, the response to 2 Hz stimulation was potentiated by desipramine which blocks reuptake of noradrenaline into adrenergic nerve terminals and secondly, three different oadrenoceptor blocking agents reduced the effect of 4 Hz stimulation. Of these, phentolamine and rauwolscine, both of which block x2-receptors, were apparently more effective under the experimental conditions than was prazosin, a selective aclantagonist. This might suggest that the main postsynaptic receptor involved is of the °2 subtype. However, it is not possible to make this point confidently at this stage since all the blockers were used in a single dose, and the time dependence of the blockade exhibited in Figs 3, 4 and 5 suggests that equilibrium conditions may not been achieved by the end of the experiment (although the prazosin block appears to
LYMPH NODE INNER VATION
521
have come closer to this than the others, adding weight to the notion that it was less effective). Characterization of the a-receptor subtypes will therefore be the object of further study. It is interesting to note that the responses of lymph node capsules to adrenergic nerve stimulation more closely resemble those of mesenteric and portal veins (Holman & McLean, 1967; Johannsen & Ljung, 1967; Holman, Kasby, Suthers & Wilson, 1968) than those of mesenteric lymphatic vessels (McHale, Roddie & Thornbury, 1980). While the former characteristically take the form of relatively large increases in tone that dwarf the spontaneous activity inherent in these tissues, lymphatics show little increase in tone at stimulus frequencies of up to 4 Hz, but rather, shorten the period of their phasic contraction-relaxation cycle in a manner conducive to increased fluid propulsion. This suggests that the nerves of lymphatic vessels and lymph nodes serve different functions. While the former appear to modulate pumping activity of the vessels in the same way that sympathetic nerves do in the heart, the latter appear to produce increases in tone. The older workers thought that this might play a part in expelling lymphocytes from lymph nodes (Florey, 1927; Martin, 1932). Martin (1932) showed that intravenous adrenaline produced a lymphocytosis in man and, more recently, Shannon, Quin & Jones (1976) confirmed that emotional stimulation and intravenous adrenaline caused an increased efflux of lymphocytes from sheep prefemoral nodes. MeHale & Thornbury (1989) demonstrated a threefold increase in lymphocyte output from the sheep popliteal node when lumbar sympathetic chain was stimulated at 4 Hz. The effect of chain stimulation was almost abolished by phentolamine, suggesting that it was mediated by x-receptors. Taken together with the present results, it is conceivable that contraction of the lymph node capsule may play a part in this effect, either by simply squeezing out the node's contents, or perhaps by directing the incoming afferent lymph through the body of the node where it would pick up lymphocytes, rather than allowing it to skirt around the periphery via a low-resistance pathway provided by the subeapsular sinus (Heath & Spalding, 1987). In this way autonomic nerves may modulate the recirculation of lymphocytes and ultimately the immune response itself, thus providing one possible mechanism through which the nervous system and immune system interact (Besedovsky et al. 1979; Ader & Cohen, 1981; Felten et al. 1984). The authors wish to thank the Department of Health and Social Services (Northern Ireland) for providing financial support, and Pfizer for supplying prazosin free of charge. REFERENCES
ADER, R. & COHEN. N. (1982). Behaviourally conditioned immunosuppression and murine systemic lupus erythematosus. Science 215, 1534-1536. BARCROFT, J., HARRIS, H. A., ORAHOVATS, D. & WEISS, R. (1925). A contribution to the physiology of the spleen. Journal of Physiology 60, 443-456. BESEDOVSKY, H. O., DEL REY, A., SORKIN, E., DA PRADA, M. & KELLER, H. H. (1979). Immunoregulation mediated by the sympathetic nervous system. Cellular Immunology 48, 346-355.
FELTEN, D. L., LIVNAT, S., FELTEN, S. Y., CARLSEN, S. A., BELLINGER, D. L. & YEH, P. (1984). Sympathetic innervation of lymph nodes in mice. Brain Research Bulletin 13, 693-699.
K. D. THORNBUR Y AND OTHERS 522 FLOREY, H. (1927). Observations on the contractility of lacteals. Part II. Journal of Physiology 63, 1-18. GIRON, L. T., CRUCHER, K. A. & DAVIS, J. N. (1980). Lymph nodes - possible site for sympathetic neuronal regulation of immune responses. Annals of Neurology 8, 520-525. HEATH, T. J. & SPALDING, H. J. (1987). Pathways of lymph flow and from the medulla of lymph nodes in the sheep. Journal ofAnatomy 155, 177-188. HODGETTS, V. E. (1961). The dynamic red cell storage function of the spleen in the sheep. Australian Journal ofExperimental Biology 39, 187-196. HOLMAN, M. E., KASBY, C. B., SUTHERS, M. B. & WILSON, J. A. F. (1968). Some properties of the smooth muscle of rabbit portal vein. Journal of Physiology 196, 111-132. HOLMAN, M. E. & MCLEAN, A. (1967). The innervation of sheep mesenteric veins. Journal of Physiology 190, 55-69. JOHANNSEN, B. & LJUNG, B. (1967). Sympathetic control of rhythmically active vascular smooth muscle as studied by a nerve-muscle preparation of portal veins. Acta physiologica scandinavica 70,299-311. LJUNG, B., BEVAN, J. A., PEGRAM, B. L., PURDY, R. E. & Su, M. (1975). Vasomotor nerve control of isolated arteries and veins. Acta physiologica scandinavica 94, 506-516. MCHALE, N. G., RODDIE, I. C. & THORNBURY, K. D. (1980). Nervous modulation of spontaneous contractions in bovine mesenteric lymphatics. Journal of Physiology 309, 461-472. MCHALE, N. G. & THORNBURY, K. D. (1989). Increased leucocyte output from the popliteal node in response to stimulation of the sympathetic chain in the anaesthetized sheep. Journal of Physiology 418, 176P. MARTIN, H. E. (1932). Physiological leucocytosis. Journal of Physiology 75, 113-129. PASSANTINO. G. (1940). Osservationi sulla presenta di fibre muscolari liscie e di fibre elastiche nella capsule e trabecole di alcune linfoghiandole di vari Mammiferi (nell' Equus caballus, nel Bos taurus, nel Bos bubalus, nell' Ovis aries, nella Capra hircus, nel Sus scrofa domesticus, nel Canis familiaris, nel Felis catus e nel Lepus cuniculus). Archivio Italiano Anatomia e di Embriologia 43, 146-164. POPPER P., MANTYH, C. R., VIGNA, S. R., MAGGIO, J. E. & MANTYH, P. W. (1988). The localization of sensory nerve fibres and receptor binding sites for sensory neuropeptides in canine mesenteric lymph nodes. Peptides 9, 257-267. SHANNON, A. D., QUIN, J. W. & JONES, M. A. S. (1976). Responses of regional lymphatic system of the sheep to acute stress and adrenaline. Quarterly Journal of Experimental Physiology 61, 169-184. TUMER, A., OZTURK-DEMIR, N., BASAR-EROGLU, C. & NOYAN, A. (1983). Spontaneous contractions and stretch-evoked responses of isolated lymph nodes. Journal of Muscle Research and Cell Motility 4, 103-113.
Journal of Physiology (1990), 422, pp. 513-522 With 7figures Printed in Great Britain
NERVE-MEDIATED CONTRACTIONS OF SHEEP MESENTERIC LYMPH NODE CAPSULES
BY K. D. THORNBURY, N. G. McHALE, J. M. ALLEN* AND GWEN HUGHES* From the Department of Physiology, Queen's University ofBelfast, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland, and the *Biomedical Sciences Research Centre, University of Ulster, Newtownabbey BT37 OQB, Northern Ireland
(Received 11 September 1989) SUMMARY
1. Isometric tension was recorded in vitro from strips cut from the capsules of mesenteric lymph nodes of sheep. 2. One minute periods of field stimulation at frequencies of 1, 2, 4, 8 and 16 Hz (pulse duration, 0 3 ms) elicited tonic contractions of increasing force and duration. The stimulus frequency-response relationship began to flatten out at frequencies > 4 Hz, where the response was already 72 % of that at 16 Hz. 3. The response to field stimulation was abolished by tetrodotoxin (1 ,UM). 4. Phentolamine, rauwolscine and prazosin (all 1 IM) reduced the response to field stimulation, while desipramine (1 ,tM), potentiated it. 5. Atropine (1 /M) was without effect on the response. 6. These results suggest that sheep mesenteric lymph node capsules have a noradrenergic innervation which modulates their tone via an action on a-
adrenoceptors. INTRODUCTION
Morphological studies indicate that lymph nodes are innervated by a variety of fibre types including adrenergic (Giron, Crucher & Davis, 1980; Felten, Livnat, Felten, Carlsen, Bellinger & Yeh, 1984), substance P-containing and calcitonin generelated peptide-containing (Popper, Mantyh, Vigna, Maggio & Mantyh, 1988) types. It has been suggested that these nerves might modulate the immune system since sympathetic denervation of lymph nodes or the spleen alters the immune responsiveness of these organs (Besedovsky, Del Rey, Sorkin, Da Prada & Keller, 1979; Felten et al. 1984; Popper et al. 1988). Smooth muscle is also present in the capsules and trabeculae of lymph nodes in many species including cats, dogs, cattle, sheep and goats (Passantino, 1940). Adrenergic nerves appear to be associated with the trabeculae and there is evidence of a dense subcapsular adrenergic plexus which could conceivably innervate the capsular smooth muscle (Felten et al. 1984). At present the role of this muscle remains a matter for speculation, although one potentially important function is suggested by the recent demonstration that stimulation of the lumbar sympathetic chain in the sheep increases the output of MS 7933 17
PH Y 422
514
K. D. THORNBURY AND OTHERS
lymphocytes from the popliteal node (McHale & Thornbury, 1989). While the mechanism of this response has yet to be confirmed it may be mediated by contraction of the node capsule and/or trabeculae. A similar mechanism has been well described in the splenic capsules of dogs and sheep where contraction in response to stimulation by sympathetic nerves and circulating adrenaline results in expulsion of erythrocytes and a raised haematocrit (Barcroft, Harris, Orahovats & Weiss, 1925; Hodgetts, 1961). At present, however, there has been no functional study describing the effect of nerve stimulation on lymph node capsular smooth muscle; indeed it has not yet been confirmed that this tissue is even innervated. In the experiments described below we have used the technique of in vitro transmural field stimulation to study nervemediated contractions of capsular strips taken from sheep mesenteric lymph nodes. The evidence suggests that the smooth muscle of these node capsules possesses an adrenergic innervation which evokes contractions by an action on oc-adrenoceptors.
METHODS
Mesenteric lymph nodes were removed immediately after euthanasia with pentobarbitone (80 mg/kg, I.v.) from five sheep which had been previously anaesthetized for mesenteric lymph flow studies (anaesthesia induced with pentobarbitone 20-30 mg/kg, i.v. and maintained by breathing 1-3 % halothane in 02). Transversely orientated strips (approximately 15 mm long, 3 mm wide, 1-2 mm thick) were cut and stored until use at room temperature in Krebs solution composition (mM): NaCl, 120; NaHCO3, 25; KCl, 5 9; NaH2P04, 1-2; CaCl2, 2-5; MgCl2, 1-2, glucose, 11, gassed with 95% 02, 5% CO2. They were then mounted in water-jacketed organ baths (volume, 1 ml) maintained at 37 °C and perfused with Krebs solution. Initial resting tension was adjusted to 4 mN and a period of at least 30 min was allowed for equilibration. Tension changes were measured with Statham UC3 and Dynamometer UFI isometric transducers the outputs of which were written on Gould 2400S and Lectromed MX 216 chart recorders. Field stimulation was applied via platinum ring electrodes at each end of the organ bath. Pulses of 0 3 ms duration were delivered in trains at constant frequencies of 1, 2, 4, 8 or 16 Hz from Grass S88 and SI1 stimulators set at nominal output voltages of 50 V. Stimulation periods always lasted for 1 min and the recovery time between stimuli was usually 8-10 min. The drugs used were as follows: tetrodotoxin (Sigma); phentolamine mesylate (CIBA); rauwolscine hydrochloride (Carl Roth); prazosin hydrochloride (Pfizer); desipramine hydrochloride (Sigma); atropine sulphate (Sigma). All of these were made up to their final concentrations in Krebs solution. Summarized results are expressed throughout as the mean response+s.E. of the mean, and statistical comparisons of means were made using Student's paired t test.
RESULTS
Of the fifty-two preparations observed in the present study forty-eight showed spontaneous activity. This was mostly (forty-one preparations) of the type described by Tumer, Ozturk-Demir, Basar-Eroglu & Noyan (1983) and consisted of irregular contractions of 0-1-0-5 mN force (e.g. see small fluctuations in baseline tension of Figs 2 and 5). This activity was rarely present throughout the experiment, usually appearing in bursts lasting from several minutes to 05 h. In seven preparations the activity was more regular in force and frequency (0-5-1 beats/min) and lasted for over 30 min, while in four preparations there was no evidence of any spontaneous activity.
LYMPH NODE INNER VATION
515
Effect offield stimulation The typical effect of 1 min periods of field stimulation at frequencies of 1, 2, 4, 8 and 16 Hz is shown in the upper panel of Fig. 1. In contrast to the rather small spontaneous contractions described above, transmural field stimulation produced I 4mN
2min 2Hz
1 Hz
4Hz
8Hz
16Hz
6 5E 4 H
2
-
1
00
16 12 4 8 Stimulus frequency (Hz) Fig. 1. Upper panel: effect of stimulus frequency on isometric tension in a sheep mesenteric lymph node capsular strip. The preparation was stimulated for 1 min periods indicated by arrows. Lower panel: stimulus frequency-response relationship summarized for six preparations. Circles, mean responses; vertical bars, + standard error of mean.
relatively large increases in tone, ranging from 1P7 mN at 1 Hz to 5-9 mN at 16 Hz. These responses consisted of a rapid contraction phase which occurred during the first 10-15 s of stimulation, followed by a slower increase in tension to a plateau during the next 30-45 s. After the stimulus ceased, the muscle relaxed slowly, taking several minutes to reach its previous resting tension after stimulation at frequencies of 2 Hz or greater. A summary of the effect of stimulation on peak isometric tension (mN) in six preparations is presented in the lower panel of Fig. 1. The curve shows that the tension response increased steeply over the 1-4 Hz frequency range, but thereafter increases in stimulus frequency produced only slight improvement. Moreover, it was found that after stimulation at 16 Hz, subsequent responses to 4 Hz were reduced to approximately 60% of those obtained previously at this frequency. In contrast, consistent responses to repeated 4 Hz stimulations could be obtained for 1-2 h. For these reasons 4 Hz was chosen as the frequency with which to examine the effects of the various blocking drugs described below.
Effect of tetrodotoxin The effect of tetrodotoxin (1 /M) on the response to field stimulation at 4 Hz was tested to establish if the response was mediated by nerves. In the example shown in the upper panel of Fig. 2, two separate 1 min periods of 4 Hz stimulation elicited 17-2
K. D. THORNBUR Y AND OTHERS
516
TTX (1 uM)
t\
~
X
4Hz 4 Hz
#
I
~~
f' 4z 4 Hz
A
,
4z 4 Hz
mN ~~~~~4
2 min
4z 4 Hz
3-
E
2-
0 ._ C
H
U
Control
E
TTX (1 uM)
1-
0-
4 Hz
4 Hz
4 Hz
4 Hz
Fig. 2. Upper panel: effect of tetrodotoxin (TTX, 1 ,IM) on the response to stimulation at 4 Hz. Lower panel: summary of the effect of TTX in five preparations. f, mean response to a 1 min period of stimulation at 4 Hz in the absence of TTX; 0, responses after exposure to TTX; vertical bars, + standard error of mean.
Phentolamine (1
gM) 4 mN
5 min 4 Hz
4 Hz
4 Hz
4 Hz
41 3-
LII Control
2-
El
E
a)
Phentolamine (1
gM)
1-
0-
4 z 4 Hz
4 Hz
Fig. 3. Upper panel: effect of phentolamine (1 /iM) on the response to stimulation at 4 Hz. Lower panel: summary of the effect of phentolamine (n = 7). The format is similar to Fig. 2.
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similar responses. After infusion of tetrodotoxin for 10 min the response was almost completely abolished. This is confirmed in the summary of five such experiments represented by the column graph in the lower panel of Fig. 2. The open columns on the left refer to two successive stimulus periods before tetrodotoxin was infused, Rauwolscine (1 pM) 3 mN t7 4 Hz
4 Hz
4 Hz
+% 4 Hz
2 min
T
2-
zControl E
T
,0 1 X ;
Rauwolscine (1
pM)
CA
4Hz 4 Hz 4 Hz Fig. 4. Upper panel: effect of rauwolscine (1 /M) on the response to stimulation at 4 Hz. Lower panel: summary of the effect of rauwolscine (n = 5). The format is similar to Fig. 2. 4 Hz
while the two hatched columns represent two stimulus periods after infusion of the drug. Before tetrodotoxin the mean responses were not significantly different (P > 0-2), while in the presence of tetrodotoxin they were markedly depressed (P < 0 03). In contrast to stimulus-evoked contractions, spontaneous contractions persisted in the presence of tetrodotoxin (see small fluctuations in baseline, Fig. 2, upper panel).
Effect ofphentolamine, rauwolscine and prazosin In order to determine whether or not the above responses could have been mediated by adrenergic nerves acting on a-adrenoceptors the effects of the aadrenoceptor blocking agents phentolamine, rauwolscine and prazosin were tested. Of these phentolamine is thought to be non-selective with regard to oc-adrenoceptor subtype, while rauwolscine and prazosin are thought to be relatively selective for a2 and a, subtypes, respectively. Figure 3 (upper panel) shows a typical example of the action of phentolamine (1/UM) on the response to 4 Hz stimulation. Two 1 min periods of 4 Hz stimulation produced similar responses before phentolamine was perfused, but at 15 and 25 min after exposure to the blocker the contractions evoked by 4 Hz were markedly reduced. The effect of phentolamine on seven preparations is summarized in the column diagram in the lower panel of Fig. 3, the format of which is similar to that of Fig. 2. The open columns represent the mean responses of two stimulus periods, 10 min apart, in the absence of blocker, while the hatched columns refer to stimulus
K. D. THORNBUR Y AND OTHERS
518
Prazosin (1 PM)
i
(I
I--, j~
C-4
% 4 Hz
% 4 Hz
IL_ ¼
¼
¼
4 Hz
2 X 4mN
2min
4 Hz
2-
2
-I Control
E
c
m E Prazosin (1
1-
uM)
0
CD
4 Hz
4 Hz
4 Hz
4 Hz
Fig. 5. Upper panel: effect of prazosin (1 /M) on the response to stimulation at 4 Hz. Lower panel: summary of the effect of prazosin (n = 9). The format is similar to Fig. 2.
Desipramine (1 pM) 3 mN 1 Hz
2 Hz
¼1 Hz
¼ 2 Hz
2 min
T E 2-
Control
0 C c aD
H
E Desipramine
(1 pM)
-
0-
1 Hz
2 Hz
Fig. 6. Upper panel: effect of desipramine (1 #M) on the responses to stimulation at 1 and 2 Hz. Lower panel: summary of the effect of desipramine (n = 9). The format is similar to Fig. 2.
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519
periods 15 and 25 min after infusion of blocker. Phentolamine reduced the responses (P < 0-006 and P < 0-002, when compared with the second control period). Figure 4 shows that rauwolscine (1 /SM) had an effect similar to that of phentolamine when tested using a similar protocol. In the example shown in the upper panel two 1 min periods of 4 Hz stimulation elicited responses which were reduced after exposure to rauwolscine for 15 and 25 min. A summary of five experiments is shown in the lower panel of Fig. 4, confirming that rauwolscine reduced the responses (P < 0 03 at 15 min and P < 0003 at 25 min). Atropine (1
PiM) 4 mN 2 min
4 Hz
4 Hz
E c
4 Hz
4 Hz
3Z ~~~~~~~~~~~~]Control 2
Atropine (1
0
pM)
c
01
4 Hz
4 Hz
4 Hz
4 Hz
Fig. 7. Upper panel: effect of atropine (1 M) on the response to stimulation at 4 Hz. Lower panel: summary of the effect of atropine (n = 5). The format is similar to Fig. 2.
In comparison to phentolamine and rauwolscine, an equimolar concentration of the a,l-antagonist prazosin was less effective at blocking the effect of 4 Hz stimulation. Nevertheless, that the responses were partially blocked is evident from both the typical record and summary diagram (n = 9) shown in Fig. 5 (P < 003 after 15 min and P < 0-02 after 25 min of exposure to prazosin, respectively).
Effect of desipramine The above results with a-adrenoceptor antagonists suggest that the responses to field stimulation were mediated by adrenergic nerves. If this is the case, then one would also expect desipramine to potentiate the response, since this substance is known to block reuptake of noradrenaline into adrenergic nerve terminals. That the response was indeed enhanced in this way can be seen in Fig. 6. In these experiments responses to 1 and 2 Hz, rather than 4 Hz, were tested since these fell on the steep part of the stimulus frequency-response curve (see Fig. 1), and were thought more likely to show potentiation. In the upper panel of Fig. 6 an example is shown where, after exposure to desipramine (1 ,sM), the tension responses were both greater and more prolonged. Nine such experiments are summarized in the lower panel of Fig. 6
520
K. D. THORNBUR Y AND OTHERS
where it can be seen that the mean potentiation was more marked at 2 Hz (a 37 % increase, P < 0 02).
Effect of atropine Figure 7 (upper panel) shows the effect of atropine (1 /M) in a preparation where 4 Hz stimulation produced contractions of around 3 mN. Atropine made no obvious difference to the strength or duration of the response, suggesting that there was little or no cholinergic component. This conclusion is further supported by the five experiments summarized in the lower panel where responses in the presence of atropine were not significantly different (P > 0 56) from those obtained in the control period. DISCUSSION
Several investigators have previously examined the contractility of lymph nodes in vitro. There are early reports of isolated whole mesenteric nodes from cats (Florey, 1927) and dogs (Martin, 1932) contracting when adrenaline was added to their bathing medium. More recently Tumer et al. (1983) have described spontaneous activity in intact node preparations from the prescapular region in sheep and goats, of the guinea-pig mesentery, and of strips cut from calf prescapular nodes. Of these only the cattle nodes displayed regular, long-lasting activity characterized by discrete contractions and relaxations. The records of sheep nodes obtained by these authors were fairly similar to those obtained in the present study, despite a number of differences in the preparation (e.g. whole prescapular nodes versus strips of mesenteric node capsule). The fact that spontaneous activity was observed in the presence of tetrodotoxin in the present experiments suggests that it was of myogenic origin. So far as we are aware, this is the first study that has attempted to demonstrate the action of nerves on lymph node capsular smooth muscle. Transmural field stimulation with short pulses produced contractions which were effectively blocked by tetrodotoxin, suggesting that they were mediated by nerves. The stimulus frequency-response relationship was similar to those obtained for a variety of vascular smooth muscle preparations, with the greatest increases in response occurring in the 1-4 Hz range, and a levelling off at frequencies of 4-16 Hz (Johannsen & Ljung, 1967; Ljung, Bevan, Pegram, Purdy & Su, 1975). That the nerves concerned were adrenergic is suggested by two pieces of evidence. Firstly, the response to 2 Hz stimulation was potentiated by desipramine which blocks reuptake of noradrenaline into adrenergic nerve terminals and secondly, three different oadrenoceptor blocking agents reduced the effect of 4 Hz stimulation. Of these, phentolamine and rauwolscine, both of which block x2-receptors, were apparently more effective under the experimental conditions than was prazosin, a selective aclantagonist. This might suggest that the main postsynaptic receptor involved is of the °2 subtype. However, it is not possible to make this point confidently at this stage since all the blockers were used in a single dose, and the time dependence of the blockade exhibited in Figs 3, 4 and 5 suggests that equilibrium conditions may not been achieved by the end of the experiment (although the prazosin block appears to
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have come closer to this than the others, adding weight to the notion that it was less effective). Characterization of the a-receptor subtypes will therefore be the object of further study. It is interesting to note that the responses of lymph node capsules to adrenergic nerve stimulation more closely resemble those of mesenteric and portal veins (Holman & McLean, 1967; Johannsen & Ljung, 1967; Holman, Kasby, Suthers & Wilson, 1968) than those of mesenteric lymphatic vessels (McHale, Roddie & Thornbury, 1980). While the former characteristically take the form of relatively large increases in tone that dwarf the spontaneous activity inherent in these tissues, lymphatics show little increase in tone at stimulus frequencies of up to 4 Hz, but rather, shorten the period of their phasic contraction-relaxation cycle in a manner conducive to increased fluid propulsion. This suggests that the nerves of lymphatic vessels and lymph nodes serve different functions. While the former appear to modulate pumping activity of the vessels in the same way that sympathetic nerves do in the heart, the latter appear to produce increases in tone. The older workers thought that this might play a part in expelling lymphocytes from lymph nodes (Florey, 1927; Martin, 1932). Martin (1932) showed that intravenous adrenaline produced a lymphocytosis in man and, more recently, Shannon, Quin & Jones (1976) confirmed that emotional stimulation and intravenous adrenaline caused an increased efflux of lymphocytes from sheep prefemoral nodes. MeHale & Thornbury (1989) demonstrated a threefold increase in lymphocyte output from the sheep popliteal node when lumbar sympathetic chain was stimulated at 4 Hz. The effect of chain stimulation was almost abolished by phentolamine, suggesting that it was mediated by x-receptors. Taken together with the present results, it is conceivable that contraction of the lymph node capsule may play a part in this effect, either by simply squeezing out the node's contents, or perhaps by directing the incoming afferent lymph through the body of the node where it would pick up lymphocytes, rather than allowing it to skirt around the periphery via a low-resistance pathway provided by the subeapsular sinus (Heath & Spalding, 1987). In this way autonomic nerves may modulate the recirculation of lymphocytes and ultimately the immune response itself, thus providing one possible mechanism through which the nervous system and immune system interact (Besedovsky et al. 1979; Ader & Cohen, 1981; Felten et al. 1984). The authors wish to thank the Department of Health and Social Services (Northern Ireland) for providing financial support, and Pfizer for supplying prazosin free of charge. REFERENCES
ADER, R. & COHEN. N. (1982). Behaviourally conditioned immunosuppression and murine systemic lupus erythematosus. Science 215, 1534-1536. BARCROFT, J., HARRIS, H. A., ORAHOVATS, D. & WEISS, R. (1925). A contribution to the physiology of the spleen. Journal of Physiology 60, 443-456. BESEDOVSKY, H. O., DEL REY, A., SORKIN, E., DA PRADA, M. & KELLER, H. H. (1979). Immunoregulation mediated by the sympathetic nervous system. Cellular Immunology 48, 346-355.
FELTEN, D. L., LIVNAT, S., FELTEN, S. Y., CARLSEN, S. A., BELLINGER, D. L. & YEH, P. (1984). Sympathetic innervation of lymph nodes in mice. Brain Research Bulletin 13, 693-699.
K. D. THORNBUR Y AND OTHERS 522 FLOREY, H. (1927). Observations on the contractility of lacteals. Part II. Journal of Physiology 63, 1-18. GIRON, L. T., CRUCHER, K. A. & DAVIS, J. N. (1980). Lymph nodes - possible site for sympathetic neuronal regulation of immune responses. Annals of Neurology 8, 520-525. HEATH, T. J. & SPALDING, H. J. (1987). Pathways of lymph flow and from the medulla of lymph nodes in the sheep. Journal ofAnatomy 155, 177-188. HODGETTS, V. E. (1961). The dynamic red cell storage function of the spleen in the sheep. Australian Journal ofExperimental Biology 39, 187-196. HOLMAN, M. E., KASBY, C. B., SUTHERS, M. B. & WILSON, J. A. F. (1968). Some properties of the smooth muscle of rabbit portal vein. Journal of Physiology 196, 111-132. HOLMAN, M. E. & MCLEAN, A. (1967). The innervation of sheep mesenteric veins. Journal of Physiology 190, 55-69. JOHANNSEN, B. & LJUNG, B. (1967). Sympathetic control of rhythmically active vascular smooth muscle as studied by a nerve-muscle preparation of portal veins. Acta physiologica scandinavica 70,299-311. LJUNG, B., BEVAN, J. A., PEGRAM, B. L., PURDY, R. E. & Su, M. (1975). Vasomotor nerve control of isolated arteries and veins. Acta physiologica scandinavica 94, 506-516. MCHALE, N. G., RODDIE, I. C. & THORNBURY, K. D. (1980). Nervous modulation of spontaneous contractions in bovine mesenteric lymphatics. Journal of Physiology 309, 461-472. MCHALE, N. G. & THORNBURY, K. D. (1989). Increased leucocyte output from the popliteal node in response to stimulation of the sympathetic chain in the anaesthetized sheep. Journal of Physiology 418, 176P. MARTIN, H. E. (1932). Physiological leucocytosis. Journal of Physiology 75, 113-129. PASSANTINO. G. (1940). Osservationi sulla presenta di fibre muscolari liscie e di fibre elastiche nella capsule e trabecole di alcune linfoghiandole di vari Mammiferi (nell' Equus caballus, nel Bos taurus, nel Bos bubalus, nell' Ovis aries, nella Capra hircus, nel Sus scrofa domesticus, nel Canis familiaris, nel Felis catus e nel Lepus cuniculus). Archivio Italiano Anatomia e di Embriologia 43, 146-164. POPPER P., MANTYH, C. R., VIGNA, S. R., MAGGIO, J. E. & MANTYH, P. W. (1988). The localization of sensory nerve fibres and receptor binding sites for sensory neuropeptides in canine mesenteric lymph nodes. Peptides 9, 257-267. SHANNON, A. D., QUIN, J. W. & JONES, M. A. S. (1976). Responses of regional lymphatic system of the sheep to acute stress and adrenaline. Quarterly Journal of Experimental Physiology 61, 169-184. TUMER, A., OZTURK-DEMIR, N., BASAR-EROGLU, C. & NOYAN, A. (1983). Spontaneous contractions and stretch-evoked responses of isolated lymph nodes. Journal of Muscle Research and Cell Motility 4, 103-113.
