Glucocorticoids alter

fever and IL-6 responses

to psychological stress

and to

lipopolysaccharide.

Opracowanie: Michał Przemysław Pruchniak

Background

Glucocorticoids can modulate the febrile response to an

injection of endotoxin.

Cytokines such as Interleukin-1, Interleukin-6 and Tumor

Necrosis Factor cause the release of glucocorticoids from
adrenal cortex.

Concentration of IL-6 and TNF in plasma is rising after

injection of lipopolysaccharide.

Nevertheless the role that play these cytokines in

modulating the febrile response are not completly
understood.

Background

Injection of doses of TNF results in fever but there

are some evidences that endogenously produced TNF
lowers body temperature.

Hypothesis that IL-6 is a endogenous pyrogen.

Glucocorticoids inhibit the in vitro production of IL-6

and TNF but it is unclear to what extent it regulates the
in vivo production of these cytokines.

Materials and

methods

Experimental Animals

Adrenalectomized rats (ADX)

Shame-ADX rats

Normal rats (control)

Temperature Measurement

Body temperature was monitored using surgically

implanted transmitter into peritoneal cavity with was
connected with the Dataquest III system. Implantation

was made 7 days before experiments.

Materials and

methods

Intracerebroventricular Cannulas

The RU 38486 (20mg/kg) was added by the cannula

implanted into the third ventricle of a brain. At the end of
experiments, the locations of the cannula tracks

were

histologically verifield.

Lypopolysaccharide

The LPS was obtained from E. Coli endotoxin (Sigma lot
no. 97F-4089).

Corticosterone replacement peelets

25, 50, 100 mg and placebo peelets purchased from

Innovative Research.

Expermental Design:

Protocol1

The puropse: Test that the hypothesis that LPS injection
would lead to increased level of TNF-like and IL-6-like
activities and higher fevers in ADX rats compared to
shame-AXD rats.

1)

The body temperarature of 7 AXD and 5 shame AXD
rats with were injected with 50 mg/kg were mesure
at 5 min. intervals for 24 hours.

2)

Other group of 12 AXD and 12 shame-AXD rats were
used to colect plasma for cytokines assay. The half
of them were injected with 50mg/kg LPS and the
rest with saline. One hour postinjection three
animals from each group were killed and the rest
after 4 hours. The blood was collected for assays.

Results: Protocol 1

1)

Injection of LPS led to higer fever in the ADX group
than in shame AXD.

2)

IL-6 activity were not signficantly different 1h post
LPS injection but after 4h were higher in ADX than
shame-ADX.

3)

Activity of TNF were the same after 1 and 4 hours in
both groups

4)

Injection of saline has not influence at concentration
of IL-6 and TNF in ADX and shame-ADX rats.

Expermental Design:

Protocol2

The pourpose: Test that exposure to an open field
would result in higher circulating levels of IL-6-like
activityand higher fevers in ADX rats compared with
shame-ADX rats.

1)

13 ADX rats and 11 shame-ADX rats were exposed
to an open field to induce psychological stress.
Open field was a big cage with fluorescent lights
suspended from the celling.

2)

After 30 minuts of exposure the temperature were
messured and next animals were killd and their
blood was collected for assays.

Results: Protocol 2

1)

Exposure to an open field induced in higher fever in
the ADX-rats than in shame-ADX

2)

Also the activity of IL-6 were higher in ADX group

Expermental Design:

Protocol3

The pourpose: Test the hypothesis that administering
the glucocorticoid antagonist RU 38486 to rats before
injecting them with LPS would result in higher levels of
IL-6-like activity and fever simmilar to that observed in
ADX rats.

1)

6 rats were given 20mg/kg RU 38486 with corn oil,
6 rats were given corn oil and 6 rats were given
saline.

2)

21 rats were given 20mg/kg RU 38486 and 21 rats
were given corn oil. Afer temperature returned to
normal stage animals were injected with 50ug/g LPS
and body temperature were mesure at 5-min.
intervals.

Results: Protocol 3

1)

Administering of RU in corn oil, corn oil or saline did
not cause changes in body temperature.

2)

After LPS injection there were 2 rises in body
temperature. At the RU group the body temperature
were higher in both rises.

3)

The RU animals have higher amount of IL-6 in
serum than animals treated with corn oil.

Expermental Design:

Protocol4

The purpose: Test the hypothesis that administering RU
to rats before exposing them to an open field stress
would result in higher circulating levels of IL-6-like
activity and similar fever to ADX rats.

1)

5 rats were admininostered 20mg/kg RU with corn
oil and 5 wih only corn oil.

2)

Afer body temp. levels off animals were exposed to
an open field for 30 min. Body temperature was
monitored and blood were collected for assays.

Results: Protocol 4

1)

After exposure to an open field RU rats developed
higher temperature than corn-oil rats

2)

IL-6 activity also were higher at RU rats

Expermental Design:

Protocol5

The purpose: test the hypothesis that administering RU
intracerebroventriculary to rats before injecting them
with LPS would result in higher circulating levels of IL-6-
like activity and higher temperature.

1)

6 rats were injected intracerebroventriculary with
RU suspended in 2%ethanol-saline, 6 rats were
injected intracerebroventriculary only with
2%ethanol-saline

2)

All animals were next injected with LPS and body
temperature were mesured

Results: Protocol 5

1)

Body temperature were higher in RU treated rats
before and after LPS injection

2)

Concentration of IL-6 were the same in both groups

Expermental Design:

Protocol6

The purpose: test the hypothesis that ADX animals
given a replacement dose of corticosterone, with
mimics plasma concentration observed in stressed and
nonstressed animals, would develop fevers similar to
those observed in shame-ADX rats given placebo
pellets.

1)

AXD rats were given with placebo pellets, 100, 50,
25 mg corticosterone pellets and shame-ADX rats
were given only placebo pellets

2)

After 7 days animals were exposured to an open
field for 30 minuts. After 24h animals were injected
with LPS and next exposured to stress situation and
killed.

Results: Protocol 6

Discussion

I.

There were evidences that glucocorticoids
modulate the febrile response. This experiment
show that not only LPS-induced fever is higher in
ADX rats, but also plasma concentration of IL-6 is
increased.

II.

Open field stress causes rise in body temperature
that have similarities to LPS-induced fever and ADX
rats develop higher temp. than shame-ADX. The
plasma IL-6 activity of ADX rats were also higher
than shame-ADX.

Discussion

III.

Using orally glicocorticoids antagonist RU 38486
increased body temperature in response to LPS and
open field stress. IL-6 concentration was also
increased.

IV.

Intracerebrovntricular injection of RU led to
increased body temp in response to LPS. However
the IL-6 concentration was the same (It is possible
that examination group was to small).

V.

The data from sixth protocol sugest that elevated
level of glicocorticosteron is needed to supress
fever.