Endocrine control of circadian rhythms

1
Endocrine control of circadian rhythms (Conn
Freeman, chs. 22 and 23)
2
Zeitgebers Biological circadian behaviors are
defined as conditions or processes that occur in
phase with the daily light-dark
cycles. Evolution favored the development of
organisms with intrinsic circuits generating an
internal timing that roughly corresponds to the
daylight-darkness cycles whose exact time tuning
is reset more or less directly by light. Such
mechanisms are often called Zeitgebers From
the German Time Givers
3
Most living beings display at least some
behaviors in phase with the circadian
rhythms Plants Algae Bacteria Invertebrate Verte
brate Mammals For most of them endogenous
zeitgebermechanisms have been found or at lest
proposed
4
Activities with circadian rhythms Wake-sleep Fee
ding-digestion Stress-corticosteroid level Body
temperature
5
Circadian activity in rodents Rodents display a
clear circadian activity pattern. Many rodents
are active during the dark part of the day and
sleep during daylight hours. In the presence
of constant darkness they still exhibit an
endogenously-generated circadian motor activity
pattern that typically tends to shorten
progressively. The presence of 24 hour
light-dark cycles synchronizes the endogenous
rhythm.
6
Circadian activity in humans
Similar arguments hold for humans, except that
most people, left without external daylight clues
would settle for circadian rhythms longer than 24
hours (typically 25-26 hours).
Retraining to an externally imposed rhythm
(re-setting the phase between the internal
oscillator and the external oscillator) takes
usually about one day per each hour difference
between the two oscillators.
7
Multiple intrinsic oscillators Different
activities that are normally in phase with
circadian rhythms may be controlled by
independent oscillators. In the continuous
presence of light, hamsters may exhibit activity
daily patterns that may be split in two different
superimposed oscillations. In humans, the
oscillator regulating daily levels of motor
activity and body temperature may split in
daylight clueless conditions
8
Role of the suprachiasmatic nucleus The
bilateral SCN of the hypothalamus is the main
circadian oscillator in mammals. In rat each SCN
is made up by a highly interconnected network of
about 8000 neurons Ablation of the SCN results
in loss of circadian rhythmicity An isolated SCN
from a mammal body continues to fire with
circadian pattern Fetal SCN implanted in an
animal that previously got the SCN removed
restores the circadian rhythmicity
9
Mechanisms of rhythmicity Slice recording
experiments show that an explanted SCN cell
retains the ability to fire in circadian rhythms
once removed from The body, for up to three
cycles. Cultures of SCN retain the ability to
release VP for up to 4 cycles. Embryonal cells
of SCN can cycle for several weeks in
culture. Single SCN neurons seem then to be the
unit of circadian oscillation Despite intense
investigation the mechanisms underlying single
cell and SCN tissue circadian rhythmicity are not
well understood
10
Mechanism of synchronization of the intrinsic
circadian oscillator
11
SCN entrainment by other factors than light In
the same way that the presence of light during
the subjective night-time can re-train (re-set
the phase of) the SCN oscillator, other input may
do the same. Input from several neurotransmitter
and hormonal system can influence circadian
rhythms. Glutamatergic input is most powerful in
resetting the oscillator. Glutamate-induced
circadian resetting is probably mediated by
activation of NMDA receptors, a inflow, NO
release, increase in intracellular Ca, and
activation of the CREB (cyclic AMP regulatory
element binding factor- a transcription factor)
are related to it
12

• Intrinsic molecular mechanisms or rhythmicity
• Per, Timeless and Clock genes
• The generation of intrinsic circadian rhythms
  appear to involve gene transcription and
  translation, and is different from other types of
  cellular oscillations, based on membrane
  properties (V-gated channels, release of Ca from
  intracellular stores).
• Studies on Drosophila have identified a series of
  genes that show the following properties
• When they are mutated the fly looses circadian
  rhythmicity
• they interact with each other to generate a
  rhythm with a similar period to the circadian one
• their expression and cycle time is
  light-sensitive

13
Role of PER, TIM, CLK, and CYC in circadian
rhythms
14

• Cyclic gene activation-repression
• In darkness (Lowcase gene, CAPITAL protein)
• Clk and Cyc are constitutvely expressed and CLK
  and CYC bind to each other in the nucleus
• the dimer activates the expression of Per and
  Tim which start to be expressed in the cytosol
• 6 hours later PER and TIM start to accumulate,
  bind to each other and enter the nucleus near
  dusk and accumulate in the nucleus peaking about
  4 hours before dawn.
• elevated levels of the PER-TIM dimers in the
  nucleus inhibit the CLK-CYC ability to activate
  transcription.
• transcription of Per and Tim decreases and Per
  and Tim RNA levels falls during the next few
  hours
• with lower levels of Per and Tim the cycle
  starts over again

15
How does light synchronize the cycle? Lights
degrades TIM protein. Since only the dimer TIM
PER enters the nucleus, light prevents the entry
of the complex in the nucleus, blocking the
progression of the cycle. At night TIM is present
again, binds PER, the complex enters the nucleus
and inhibits the dimer CLK-CYC transcription
ability. In mammals similar genes have been
found, but the whole process is mediated by the
release of melatonine by melanocytes of the
pineal gland and by the direct retinohypothalamic
pathway from the retina. In spite of the fact
that most hormonal circadian rhythms are directly
controlled by the SCN, the pineal gland is
relevant in the modulation of body activity and
sleep onset.
16
Pineal gland It is an umpaired glan, also called
epiphysis, it is very small and light (a fraction
of a gram), located at the opposite extreme of
the hypothalamus of the cerebral acqueduct . Not
all mammalian species have it. Species endemic of
equator or tropical regions have a small or
absent pineal gland, while high latitude endemic
species have a larger one, suggesting a season
related function. In some fish, amphibians and
reptiles it is connected to a third eye a
structure with photoreceptors located on the apex
of the skull. In mammals it receives photic
information only indirectly through a complex
pathway.
17
Pineal gland The pineal gland receives
sympathetic afferents from postganglionic fibers
of the autonomic system. Is highly
vascularized. Most pineal cells are secretory
parenchymal cells called pinealocytes and secrete
also Ca3PO4 (hydroxyapatite)which partially
calcify the pineal gland during puberty and does
not affect secretory ability of pinealocytes.
18
Secretory function of the pineal gland The
pineal gland synthesizes many molecules
including VP Renin-angiotensin ACTH and other
POMC related peptides The distinctive molecule
synthsized by pinealocytes is MELATONIN, which is
synthesized from triptophan and then serotonin.
The concentration of serotonin is high during the
day (photophase) and falls during the night time
(scotophase) because serotonin is converted to
melatonin during the night time. Melatonine goes
in the circulation and is metabolized by the
liver in about 4 hours. Melatonine low affinity
(200 pM) receptors are ubiquitous. High affinity
(gt1nM) receptors are located in the SCN, through
which melatonin regulates circadian rhythms in
the hypothalamus and in the rest of the brain.
19
Function of melatonin Cardiovascular Thermoregul
ation Photoperiodic regulation of prolactin
release Expression of melatonin binding sites in
the pituitary Reproductive functions (high
affinity receptors are found in granulosa cells
of follicles or prostate epipthelial cells) In
the absence of photic regulation, other stimuli
may act as zeitgebers (social activity, food
availability, etc.)
20
Melatonin secretion Melatonin is synthesized
during night time regardless whether the species
is diurnal, nocturnal, or crepuscular. Melatonin
release persists even in the absence of
photoperiodic signals. The duration of melatonin
release is associated with the duration of the
dark phase (horizontal bar below graps). (Do not
confuse melatonin with melanin, which is the
molecule that pigments the skin, synthesized from
tyrosine)
Long photoperiod short photoperiod
Melatonin is open circles Sleepiness is black
circles
21
Light effects on melatonin levels
The duration of the dark period determines the
production of melatonin
Light has a suppressive effect on the production
of melatonin
22
Young Old
Age decreases the photic modulation of melatonin
synthesis and release
Pinealocytes are able to generate an intrinsic
circadian signal (intrinsic circadian oscillator)
23
Mammal regulation of photic input to the pineal
gland (retino-hypothalamic tract

• Different from lower cold blooded vertebrates,
  the photic input pathway in mammals comes from
• retina
• suprachiasmatic nucleus
• paraventricular nucleus
• spinal cord
• superior cervical ganglion (NE release)
• pineal gland (pinealocytes have NE ß receptors)
• activation of NAT (synthesis of melatonin

24
SP short period, LP long period Left control,
right, melatonin
Melatonin concentration inversely correlates with
body temperature
Reproductive influence of melatonin Melatonin
greatly increases the synthesis and release of
sex hormones in some species.
25
Neuroendocrine immunology
Conn and Freeman, Ch.29
26
Neuroendocrine and immune system share the
capability to act specifically, send signals at
large distance inside the body, and been able to
induce systemic activation through the autonomic
system The endocrine system can initiate
distance signaling potentially mobilizing the
whole organism. Thats how Thyroid
hormones Growth factors Stress hormones produce
their effect. The immune system has the
function of defending the organism from external
invasion and from intracellular non-functioning
cells and molecular debris (misfolded or worn out
proteins, fragments of lipid and other organic
molecule turnover). They have in common the
ability to initiate specific responses
communicated at large distance from the
initiation of the process.
27
Function of the immune system Is to survey the
state of the body organs and respond to invaders
exogenous, or body cells or molecule became
harmful (for ex. Cancer) by activating a complex
cascade of chemical and cellular processes whose
end result is to eliminate the invaders and
recover organs function. Like a police or army,
which are in charge of using violence in a
controlled manner, the immune system is in
continuous risk of falling into either of two
dangerous extremes Inactivity overactivity
risks associated Ineffectiveness autoimmune
diseases Exogenous agent take over inflammation
damage allergic reaction Responses at the
extreme in either direction include death or
irreversible damage
28
Role of the neuroendocrine system in the
regulation of the immune system activation
The neuroendocrine system, particularly the HPA
(adrenal axis) and prolactin (from the pituitary)
but also other parts of the endocrine system,
overview the function of the immune system in an
equilibrium that is subject to all the factors
that affect stress (physical and psychologic) and
immune system activation.
29
Energetics From the energetic point of view,
both the neuroendocrine and the immune system can
trigger extremely costly biological processes

• Endocrine system
• - Increase (or decrease) metabolism
• - Tissue growth
• - Inititate Fight or flight response
• Initiate motor activity for different purposes
• Initiate stress response

Immune system - Leukocyte activation (cell
cycle and DNA duplication) - Fagocytosis and cell
lysis - Antibody production - Increase in
connective tissue extracellular proteins - Fever
Given the limited maximum power (in physical
terms amount of energy that can be produced in
a unit time), from an energetic point of view it
is indispensible that the two system have a tight
cross-talk
30
Overview of the immune system
We share our world with scores of other
microscopic and macroscopic forms of life. As
multicellular organisms we are at peace with most
of them, and even greatly benefit from living
together with some of them, but some others are
just extremely annoying and we just do not want
them inside our body, whether in the blood, in
our GI tract, or anywhere else.
31
Common external invaders
Viruses Very small. Mostly DNA or RNA. Have few
proteins. Need a host to reproduce
Protozoa and parasites Eukariotic (have
nucleus) Can be extremely complex Biochemically
resistant and smart
Bacteria have no nucleus, typically smaller than
eukariotic cell, biochemically smart
32
Immune organs and circulation
Immune cells in the uninfected health organism
rest in a limited number of organs, and only send
scout cells to probe the environment. A lymphatic
vessel system tied to the blood circulation
guarantees the access of immune cells to all
parts of the body. In case of infection or
disease, the immune system activates first the
innate response, and then if necessary- the
adaptive response
33
Innate and Adaptive immune systems
The fight against harmful pathogens, exogenous or
endogenous is tremendously complicated by the
fact that it is only overseen biochemically, and
the body presents a huge number of endogenous
molecules that need to be recognized as self, and
not been fought, and the number of potential
invaders, or molecules produced by them, is even
larger. For this reason, the complexity of the
immune system, in a precise manner, displays the
uncommon feature to be larger than the complexity
of the mammalian genome itself (apparently a
paradox). There are two levels of immune defense

• Innate immune system fast response to common
  harmful antigens
• Adaptive immune system slower response to
  specific harmful antigens

34
Roles of innate and adaptive immune systems Some
cells of the immune system are more involved in
the generalized immune response (1) Some of
these cells supply information to specialized
immune cells that initiate a delayed, specific
response (2) to the particular invader, or
associated molecules (can be parts of the
invaders or its fragments or toxins) All immune
cells can proliferate after infectious insult,
but two types of immune cells can respond with
life saving multiplication (clonal expansion) in
response to large amounts of invading bodies. B
cells and T cells ca undergo clonal expansion,
particularly if they have been previously trained
(immunization) to recognize a particular antigen
(a part of the invaders or one of its chemical
distinctive features).
35
Types of immune cells and erythrocytes
Lymphocytes T and B cells are capable of clonal
expansion in response to specific antigens.
Dendritic cells mature Monocytes and
Neutrophils Are all MACROPHAGES, phagocyte
and digest foreign bodies and present the
digested remnants (antigens) of the invaders to T
and B cells for initiating the adaptive response
36
Immune role of bone marrow and thymus
The bone marrow is the source for all blood
cells, including T and B cells. T cells migrate
to the thymus where they mature and then migrate
to peripheral organs. B cells mature in the bone
marrow and then migrate to the immune organs
37
Function of different types of blood cells
38
Role of antigen-presenting cells
Dendritic cells, mature Monocytes, and
Neutrophils, they all serve a common function to
present antigens to T and B cells
39

• Function of B and T cells
• Each B cells, once activated, proliferates and
  can produce antibodies against specific antigens
  one B cellone type of antigen (humoral
  response).
• Antibodies have multiple functions
• Neutralize a toxin or a whole invader (virus or
  bacterium)
• tag an invading body (virus or bacteria) for
  distruction by macrophages and T cells
• Each T cell, once activated, can also proliferate
  and produce two different types
• T cytotoxic (recognize invader or infected cell
  and phagocyte it or trigger its distruction)
• T-helper (produce a biochemical cascade that
  increase B-cell activation or greatly enhance
  phagocytic and cytotoxic responses)

40
Clonal selection
The body produces a large variety but a small
number of non activated B cells. At their first
encounter with their specific antigen, the
specific B cell that found its antigen and no
other, undergo clonal expansion. A similar
process underlies clonal expansion of T-cells.
41

• Clonal expansion
• B and T cells, once they recognize the presence
  of a pathogen, can undergo
• clonal expansion (allowing to effectively fight a
  numerous pathogen)
• production of memory cells (production of a
  small number of cells that will not subside at
  the end of the infection, and whose long-term
  guarantees a stronger and faster immune response
  immunity- at the next encounter with the
  pathogen, or its associated antigen)

The full immune response to a new antigen may
require up to 2 weeks, which is most often longer
than what it takes to the pathogen to take over
the host. A previous exposure to the antigen
produces memory cells, which in turn induce the
faster response to the known antigen.
42
Function of antibodies One function of
antibodies is promoting phagocytosis and
eventually digestion (destruction) of foreign body
43
T and B cells
The nuclei of leukocytes is larger than the
nucleus of most other cells, indicating a large
transcriptional activity. Activated B cells
display huge Golgi apparatus, indicative of
intense protein (antibody) synthesis. An
activated B cell can release around 2000
antibodies per second (huge metabolic demand).
44
Major histocompatibility (MHC) groups
Immune recognition by adaptive immune cells is
performed using MHC proteins. MHC proteins
expose in the external membrane digested
fragments of pathogens to B and T cells for
inducing their activation MHC I are present in
all nucleated cells and tells a T cytotoxic cell
that the presenting body cell has been invaded by
pathogen MHC II are present only in antigen
presenting cells, and tell T helper cells that
macrophages recognized a pathogen
45
Signaling molecules in the immune system

• Activation of adaptive immune cells is
  accompanied by signals that increase the
  permeability of the infected tissue to the
  leukocytes. In particular, the vascular
  epithelium needs to
• Attract leukocytes on its surface, and
• to open gaps to let leukocytes and other immune
  cells access the infected areas.

Altogether, many dozen molecules carry immune
system signals (the interleukin biochemical
network), including interfernon (IFN), and tumor
necrosis factor (TNF)
46
Bidirectional communication between
neuroendocrine and immune system
Regulation and control of systemic infection
cytokines released because of septic stress alert
the HPA axis which regulates the strength of the
immune response during the infection, and
terminate the response when immune activation is
not needed any more.
Too much immuno response may be directed against
self-antigens (autoimmune disease)
Too little immune response may lead to infection,
sepsis, and death
47
Stress and immune system Stress and depression
are associated with immunosuppression. The
probability to contract a cold is correlated with
the subjective level of stress like - Life
changes (job loss, bankrupt, marital or familiar
losses) - Strenuous academic examination - Sleep
deprivation - Problems with the law Which also
affect Cytokine level Immune cell
proliferation Non-immune factors to host
resistance can be affected by stress Peristalsis
Mucosa secretion Coughing Gastic acid
release For instance, stress might decrease acid
secretion in the stomach, which normally kills
most bacteria, opening the possibility to GI
infections.
48
Similarities between immune and nervous system
1) Hormones and cytokines have specific receptors
and work at distance 2) Both the immune and the
nervous system can learn (display self regulated
capability to adaptive long-term changes)

• Hormones released by the neuroendocrine system
  alter immune function by stimulating or
  suppressing
• - lymphocyte and macrophage proliferation
• cell-mediated cytotoxicity
• cytokine and antibody production

49
Major determinants of neuro-immune link
1) HPA axis 2) Autonomic nervous
system Glucocorticoids from the adrenal gland
are the main effector of the HPA axis and target
pretty much all organs, by diffusion through the
blood. The autonomic system targets immune
structures by direct innervation. ß receptors on
T and B cells decrease lymphocyte proliferation,
cytotoxicity and cytokine production. The Para
Ventricular Nucleus of the hypothalamus (PVN)
controls both the release of glucocorticoids ,
but also sympathetic preganglionic nuclei,
besides various central limbic structures
associated with stress
50
Effect of glucocorticoids on immune function

• Glucocorticoids induce
• Thymic involution, due to glucocorticoid-induced
  T-cell precursor apoptosis
• Increased susceptibility to infections, in
  patients with chronic stress
• decrease in the density of all immune cell
  types, except neutrophils
• Infections can lead to septic shock, which is an
  over-reaction of the immune system to infection
  that can be more detrimental than the infection
  itself. Cytokine cascades induced by endotoxins
  can lead to circulatory collapse.
• As cytokines are induced by the presence of a
  pathogen host, adrenal steroids concomitantly
  reduce their immune effectiveness to avoid
  self-destructive effects of cytokines

51
Animal models
Animal research supports the idea that a low
activation of the stress axis is associated with
docile subjects and autoimmune disease, and
vice-versa, high stress is associated with
aggression

• Lewis rat have a genetic defect inducing low
  level corticosteroids in response to acute
  stressor. They are also
• -docile, serene
• Non aggressive
• sensitive to rheumatoid arthritis and multiple
  sclerosis

• Fisher 344 have higher than normal corticosteroid
  response to stress. They are
• -aggressive
• Nervous
• Insensitive to autoimmune disease

52

• Immune effects of stress hormones other than
  corticosteroids
• Corticosteroids were considered classically the
  only stress hormones to affect immune response.
• Evidence is gathering showing that also other
  hormones produced along the HPA axis are released
  and sensed by lymphocytes. In particular
• CRH
• ß endorfins
• POMC
• The immune function of these peptides has not
  been fully investigated

53
Sex hormones effects and dimorphism in immune
function Marked differences are documented
between male and female immune responses. In
general, the female immune system is more active
and effective in ridding infections than the
males immune system. The other side of the coin
is that female are more prone to autoimmune
disease The occurrence of thyroid autoimmune
disease is 50-fold higher in women. The
occurrence of Lupus is 10-fold higher in
women. Organs for the maturation of T and B
cells have sex hormone receptors. Gonadectomy
induces hyperfunction of the thymus Androgens or
estrogens induces thymus withdrawal and Block
bone marrow B cell lymphopoiesis (generation of B
cells) Sex hormones stimulate cytokine
production from macrophages and induce integrin
(surface attractant molecules) on endothelial
cells. Estrogens increase the ratio Th/Tc
(CD4/CD8) cells, and B-cell and interferon-?
production. All this evidence supports a stronger
female immune system
54

• Pregnancy and immune response
• Major immune changes arrive with pregnancy
• Cell-mediated immune response is inhibited
• Humoral (antibody) immune response is preserved
  or enhanced
• Cytokines level declines.
• These changes are due to a large increase in
  estrogen and progesteron, but also of
  corticosteroids and other yet unknown substances.
• In pregnancy there is an inverse relationship
  between gonadal steroids and immunoresponsiveness
  (pretty much like with stress hormones)
• Autoimmune diseases associated with T cell
  cytoxicity decrease
• Autoimmune disease associated with B cell
  response (antibody) can worsen

55
Immune role of prolactin
Prolactin regulates lactation in mammals and
reproductive function in non mammals. Only
relatively recently has been discovered that
prolactin administration could recover most (70)
of the immunosuppressant effect of hypophysectomy.
Prolactin has a immunogen role that is inhibited
by bromocriptine, DA agonist that inhibits
prolactin release. Growth hormone is responsible
for another 20 of the immunogenic response of
the pituitary
56
Cellular immune role of prolactin
Co-activation of prolactin receptors on
lymphocytes is necessary together with antigen or
cytokine activation to initiate proliferation
(prolactin is a mitogen activator required
co-factor.
57
Convergence of glucocorticoids and prolactin
molecular pathways
Multiple intracellular cascades are activated by
prolactin. 1) Bag-1-RAF-MAPK pathway leads to
transcription factor activation Jak-induced
dimerization of Stat 5 after stat5
phosphorylation by Jak kinase also leads to
transcription factor activation
The dimerized Stat 5 complex interacts with the
activated glucocorticoid receptors bound to
cortisol) increasing transcription of Stat 5 and
decreasing the activity of glucocorticoid
activated promoters.
58
Illness response
Infection produces a series of changes at
different levels Behavioral Psychological Physio
logical Neural That work in concert to promote
survival. Changes include Fever Increased slow
wave sleep Decreased activity and social
interaction Decreased food ingestion and
digestion Taste aversion for novel
food Hyperalgesia At the same time,
glucocorticoid release increases energy
mobilization. The CNS is required for evoking
illness response which is provoked by IL1, IL6,
and TNF
59

• Global effects of cytokines
• Different types of IL1, IL6, interferon and TNF
  induce
• Sleep
• food aversion
• anorexia
• fever
• acute phase response (alteration of protein
  production by the liver associated with pathogen
  response)
• release of prostaglandins (derivatives of
  arachidoic acid associated with inflammation ,
  decreased by aspirin, tylenol or other NSAID non
  steroidal anti-inflammatory drugs) causing
  hyperalgesia
• It is not clear whether central or peripheral
  targets are responsible for these effects
• IL1 and TNF activate vagal afferents to NTS
  projecting to the Raphe n., which in turn
  projects back releasing
• subP
• CCK
• Glutamate
• to the spinal cord enhancing pain sensitivity.

60
Cytokines activate the HPA axis at the pituitary
level
Several cytokines can directly target pituicytes
and alter the release of pituitary hormones in
addition and in synergism to the effects
previously described
Besides these effects, cytokines can centrally
activate CRF release and stress response
providing a global mechanism to modulate behavior
in a purposeful and integrated manner. This also
serves as negative feedback for immune response,
as IL1 also causes a centrally mediated
immunosuppressive response. Role of cytokines in
neurodegeneration A hyperactive immune system
may play a role in neurodegeneration,
particularly following brain inflammation or
ischemic trauma. Cytokine glial responses are
particularly relevant in this field, which is
still under intense investigation
61
"The human mind is not capable of grasping the
Universe. We are like a little child entering a
huge library. The walls are covered to the
ceilings with books in many different tongues.
The child knows that someone must have written
these books. It does not know who or how. It does
not understand the languages in which they are
written. But the child notes a definite plan in
the arrangement of the books---a mysterious order
which it does not comprehend, but only dimly
suspects. Albert Einstein
"The first precept of the rule is that everything
that surrounds us is an unfathomable mystery. 
The second precept is that we must try to unravel
these mysteries without ever hoping to accomplish
this.   The third is that a warrior, aware of the
unfathomable mystery that surrounds him and aware
of his duty to try to unravel it, takes his
rightful place among mysteries and regards
himself as one.  Consequently, for a warrior
there is no end to the mystery of being, whether
being means a pebble, an ant or oneself.  That is
a warrior's humbleness.  One is equal to
everything. The Eagle's Gift, Carlos
Castaneda