Blood, Vol. 92 No. 8 (October 15), 1998:
pp. 2971-2972
CORRESPONDENCE
Neural Regulation of Bone Marrow
 |
LETTER |
To the Editor:
Benestad et al1 recently published a paper in BLOOD
that presented data showing no effect of interfering with neural input to the bone marrow in mice. Specifically, they found no effect of
cutting the sciatic nerve or femoral nerve, of neonatal sympathectomy, or of electrical stimulation of the nerve. In addition, they found no
effect on blood flow to the marrow of any of these procedures but
note that the overlying muscle vasculature did respond. Thus, they
concluded that they could not ascribe any function to the innervation
of the marrow and that this finding "supports the physiologic
relevance of ex vivo experiments on bone marrow."
There are a number of methodological issues arising in the experiments
reported in Benestad et al1 that may have led to the data
described that are contrary to those we have
reported.2 We showed a marked effect of femoral denervation
on cell egress and cellular retention in the femoral bone marrow as
well as on the movement of immature and mature cells between the marrow
and peripheral circulation. Perhaps significantly, we have identified the followed methodological differences between the reports.
(1) The sciatic nerve was cut at the level of the sciatic notch by
Benestad et al.1 However, we found that cutting the nerve
at these low levels produced no effect on the femoral marrow. Although
the investigators found loss of innervation, as demonstrated by
glyoxylic acid staining, on blood vessels near the tibia, it is still
possible that crucial nerve projections in the other nerves and
branches that project down the leg to the tibial region, including the
subbranches of the sciatic and femoral nerve, were left intact. In
tracer studies, we injected horseradish peroxidase into the femur and
obtained stained profiles in the obturator and obdurator nerves, among
other nerves projecting down the leg (unpublished data). The
sciatic nerve is complex and composed of fibres emanating
from at least 5 nerve roots of the spinal cord. In our experiments, we
cut all of these nerve roots at their exit from the spine.
(2) In the electrical stimulation studies, the investigators first cut
the sciatic nerves and then stimulated the cut end on one side of the
animal. They used chloral hydrate and pentobarbitone anaesthesia
throughout these experiments. If the nerve input is indeed important,
it is difficult to imagine that cutting the nerve and then stimulating
it will produce a clear result as much of the effect of nerve
cutting is unlikely to be recovered by nonspecific stimulation of all
the fibers entering the system. Furthermore, we have also found that
pentobarbitone anaesthetic has the same effect as cutting the nerve, so
that recovery from the nerve cut, or any significant effect of nerve
stimulation, is highly unlikely in these experiments.
(3) The investigators used neonatal sympathectomy to investigate the
role of adrenergic input to the marrow. If, as we and others have
proposed,3 the nervous system has an executive role in
coordinating and regulating host defence, then permanent deletion of a
pathway of control (particularly in early development of the control
system) will not show the significance of that pathway to normal, adult
physiology. Of course, the response to a challenge may well expose this
missing coordination pathway. We found some recovery from the effects
of whole nerve denervation after 14 days and during sympathetic
blockade in adults. Given this recovery from denervation in adult
animals, it is unlikely that neonatal deletion will show significant
effects. Benestad et al1 acknowledge this possibility in
their discussion.
An interesting aspect of the data is the finding that denervation did
not affect the vascular volume of the bone, suggesting that the
innervation is not involved in vasomotor control as in other tissues
such as muscle. Thus, one must ask the question of what is the role for
the innervation to the bone marrow? Yamazaki and Allen4
showed clear synaptic connections between nerve terminals and
perivascular/stromal cells. It is inconceivable that such a
relationship is without function, and our experiments have demonstrated
that this innervation probably controls the blood-marrow interface,
presumably through control of the perivascular cells. Furthermore, our
data implicate adrenergic input in this control and suggest that other
transmitters are involved in the retention of cells within the marrow
itself. This pattern of control through accessory and stromal cells has
been found in other lymphoid tissues, including the thymus and lymph
nodes.
In conclusion, aspects of the methodology used in the study by Benestad
et al1 may have led to the negative data produced. With a
complicated system such as host defence and immunity, a reductionist
approach (ie, ex vivo or in vitro) has many advantages in the
dissection of the system. However, to exclude the influence of other
body systems, notably the neuroendocrine system that has been shown to
have major influences on both immunity and host defence, denies an
integration into the physiology of the whole body.
J.A. Miyan
C.S. Broome
A.D. Whetton
Department of Biomolecular
Sciences
UMIST
Manchester, UK
| |
REFERENCES |
1.
Benestad HB,
Gundersen IS,
Iversen PO,
Haug E,
Nja A:
No neuronal regulation of murine bone marrow function.
Blood
91:1280,
1998[Abstract/Free Full Text]
2.
Afan AM,
Broome CS,
Nicholls SE,
Whetton AD,
Miyan JA:
Bone marrow innervation regulates cellular retention in the murine haemopoietic system.
Brit J Haematol
98:569,
1997[Medline]
[Order article via Infotrieve]
3. Downing JEG, Kendall MD: Peripheral and central neural mechanisms
for immune regulation through the innervation of immune effector sites,
in The Physiology of Immunity. JA Marsh, MD Kendal, editors. CRC Press,
1996
4.
Yamazaki K,
Allen TD:
Ultrastructural morphometric study of efferent nerve terminals on murine bone marrow stromal cells, and the recognition of a novel anatomical unit: the "neuro-reticular complex."
Am J Anat
187:261,
1990[Medline]
[Order article via Infotrieve]
| |
RESPONSE |
Nerves to Murine Bone Marrow: Roles in Cell Production
or Cell Release?
To Dr Maestroni's specific points, we make the following comments.
(1) The recent publication that he claims was ignored by us (Afan
et al1) is indeed interesting. It was received by the library of our National Hospital 1 month after we submitted our manuscript2 to BLOOD, so we could not discuss its
findings. However, it is commented on by us now (see below).
(2) We wrote, "It is possible that effects, which may be both
nonspecific and not related to the innervation of bone marrow, may take
place after treatment of adult, nonadrenalectomized animals with
6-OH-DA. The same kind of objection could be raised against the
interpretation of the prazosin results." We cannot see that any of
the data or arguments in Dr Maestroni's letter invalidate this
statement. His claim that "A time-course study showed that this
effect was exerted directly on hematopoietic progenitors in the bone
marrow" is curious. In the study cited, a response was first
recorded 6 hours after initiation of treatment of the mice, a time
period long enough for indirect effects to take place, such as induced
secretion of cytokines or other agents that might be the ultimate
effectors. In fact, the Discussion of the quoted report also contains
the following: "This finding suggests that the mechanism of the
norepinephrine-induced rescue is indirect, perhaps acting via
production of cytokines... . More comprehensive studies are needed
to elucidate norepinephrine's mechanism of action; ... ."
(3) Although interesting, the summary of Dr Maestroni et al's data on
bone marrow catecholamines does not convince us about a functional role
for the bone marrow innervation. It is impossible to judge the validity
of these data without access to the full report, which is unpublished.
It is puzzling that, on the one hand, norepinephrine can inhibit
myelopoiesis, but, on the other hand, showed a positive association
with the proportion of bone marrow cells in non-G1/G0 phases of the
cell cycle. However, of greater relevance may be their claim that
endogenous catecholamines in the bone marrow may have a nonneural
origin, thus opening for the possibility that adrenergic effects might
take place without the involvement of bone marrow nerves.
To the points raised by Miyan et al, we make the following comments.
(1) The points raised about surgical denervation miss the target. Like
Miyan et al, we are aware that nerve section at the level of the
sciatic notch will not effectively denervate femoral marrow. For this
reason, we used the tibia, not the femur, for all these experiments. We
checked by mononamine histochemistry the extent of denervation, not
only by staining small vessels close to the tibia, but also by
examination of marrow plugs expelled from the tibia, and included a
statement about this in the report: "Two to 3 days after
sciatic nerve section, this system of nerve fibers was either
completely absent or reduced to occasional patches."
(2) Miyan et al suggest that the sympathetic effects on bone marrow are
prevented (A) by pentobarbitone and (B) by nonspecific electrical
stimulation of all the axons in the nerve. Although none of these
hypotheses can explain our results after nerve section, either of
them could explain our results after nerve stimulation. In any case,
smooth muscles in other tissues do contract in response to nerve
stimulation in the presence of pentobarbitone,3 and denervated marrow vasculature relaxed in response to a humoral signal.4 Effects of nerve stimulation were shown, as
mentioned in our report, by the skeletal muscular contraction evoked by a test stimulus, before a neuromuscular blocking agent was applied, and
by the contractile response of muscular blood vessels in the electrically stimulated limb.
(3) The possible effects of surgical denervation were searched for 2 to
7 days after the operation, so here at least there was not much time
for adaptation. With the neonatally performed chemical sympathectomy,
which we consider less specific, the situation is different. We
discussed the possibility of nerve regeneration (which was not
found in the iris examinations) and compensation by the adrenal glands
and other hormonal systems (which was not very likely, because we found
no qualitatively different effects of sympathectomy or surgical
denervation between mice with and without adrenal glands).
We did not measure "the vascular volume of the bone," but rather
the blood flow to the bone marrow. Considering the histological data
published by others, we too had anticipated a constrictive response
of bone marrow vessels to electrical stimulation, even though we would
not go so far as Miyan et al and claim that "It is inconceivable
that such a relationship is without function" (see below). However,
blood vessels to leg muscle contracted; marrow vessels did not.
Furthermore, as mentioned in our report, this finding is consistent
with previous findings with either surgically denervated or chemically
sympathectomized rats. Similarly, electrical stimulation of the
sympathetic trunk in the rat decreased blood flow to the hind limb skin
and muscle, but not to the bone marrow, as we also mentioned in our
report.
Miyan et al suggest that denervation lifts a "control on the
blood-marrow interface, presumably through control of the perivascular cells." However, taken at face value, their data showing a loss of
approximately 50% of femoral progenitor cells in the denervated limbs
and 100% increase in intact, contralateral marrow of splenectomized mice on day 4 after surgery do not make sense to us. According to
our own unpublished measurements, which agree well with those of
others, about 12% of the total mouse bone marrow is present in a
denervated hind limb. Presumably, the intact contralateral femoral
marrow is representative of the remaining, innervated 88%. Thus, the
recorded progenitor cell loss from the denervated marrow cannot make up
for the gain in the innervated marrow. Moreover, their chemical
sympathectomy of adult mice had no effect on the number of
colony-forming cells in the femur on day 4.
Being physiologists, we would certainly not "exclude the influence
of other body systems, notably the neuroendocrine system" on
bone marrow function or deny "an integration into the physiology of
the whole body." Our main point is that, by paired comparisons between surgically denervated and sham denervated mouse hind limbs, we
could not substantiate any neuronal effects; we did not examine most of
the endocrine system.
Nerve effects on bone marrow found by others do not fit into a
consistent picture. For example, most published results indicate that
transmitters stimulate cell formation or cell release, whereas Maestroni et al claim that adrenergic agents inhibit myelopoiesis and
Miyan et al claim that nerves promote cellular retention in the
femoral bone marrow. On this background it is important to validate all
procedures, sham as well as test ones, and with positive results of the
experimental procedures to minimize the possibilities that the results
may be due to, or influenced by, for example, a stress response (eg,
corticosterone analyses) or endotoxin exposure.
All publications we are aware of in this field point to a functional
role of the bone marrow innervation, as referred to and discussed in
our report. There are nerve fibers in the bone marrow containing
transmitters for which bone marrow stromal and parenchymal cells
possess receptors, and such transmitters have definite functional effects on the relevant cells in vitro. Even then, with all odds pointing to a functional role for the innervation, we could find no
significant differences between innervated (and in some cases electrically stimulated) and denervated contralateral bone marrow in
our mice, concerning either cell generation or cell mobilization. This
was a surprise. In such a situation it may be prudent to consider what
Prof Lewis Wolpert writes in his book The Unnatural Nature of
Science: "... many of the misunderstandings about the nature
of science might be corrected once it is realized just how
`unnatural' science is. I will argue that science involves a special
mode of thought and is unnatural for two main reasons, ... Firstly, the world just is not constructed on a common-sensical basis.
This means that `natural' thinking
ordinary, day-to-day common
sensewill never give an understanding about the nature of science.
Scientific ideas are, with rare exceptions, counter-intuitive: they
cannot be acquired by simple inspection of phenomena and are often
outside everyday experience. Secondly, doing science requires a
conscious awareness of the pitfalls of `natural' thinking. For common
sense is prone to error when applied to problems requiring rigorous and
quantitative thinking; lay theories are highly unreliable... . For
example, the creationist view in the middle of the nineteenth century
held that species were fixed and all the animals were made perfectly
adapted to their environment. But this was clearly not true of some
animals: some ducks with webbed feet did not swim and why should blind
animals that lived in caves have
eyes? ..."5
H.B. Benestad
P.O. Iversen
A. Njå
Department of Physiology
Institute of Basic
Medical Sciences
University of Oslo
Oslo, Norway
| |
REFERENCES |
1.
Afan AM,
Broome CS,
Nicholls SE,
Whetton AD,
Miyan JA:
Bone marrow innervation regulates cellular retention in the murine haemopoietic system.
Br J Haematol
98:569,
1997
2.
Benestad HB,
Strøm-Gundersen I,
Iversen PO,
Haug E,
Njå A:
No neuronal regulation of murine bone marrow function.
Blood
91:1280,
1998
3.
Maehlen J,
Njå A:
Selective synapse formation during sprouting after partial denervation of the guinea-pig superior cervical ganglion.
J Physiol (London)
319:555,
1981[Abstract/Free Full Text]
4.
Iversen PO,
Nicolaysen G,
Benestad HB:
Blood flow to bone marrow during development of anemia or polycythemia in the rat.
Blood
79:594,
1992[Abstract/Free Full Text]
5.
Wolpert L:
The unnatural nature of science.
London, UK, Faber and Faber Ltd
, 1993
