1

CYTOKINES II

Khuong (Ken) B. Nguyen

November 1, 2000

QUESTIONS TO BE ADDRESSED ABOUT

CYTOKINES IN TWO LECTURES

•

WHY MIGHT CYTOKINES BE IMPORTANT? (WHY DO WE
CARE?)

•

WHAT ARE CYTOKINES?

•

WHAT ARE THE GENERAL CHARACTERISTICS OF
CYTOKINES? (HOW DO THEY WORK?)

•

WHERE DO THEY WORK?

•

WHEN DO/DON’T CYTOKINES WORK? (WHEN DO
CYTOKINES WORK FOR/AGAINST YOU?)

INTEGRAL INVOLVEMENT OF CYTOKINES

DURING AN IMMUNE RESPONSE

1. ALERT TO

INFECTION/TUMOR/ETC.

2. RECRUIT CELLS TO SITE

3. SPECIFY TYPE OF

IMMUNE RESPONSE

4. IMMUNE EFFECTOR

PHASE

5. IMMUNE DOWN-

REGULATION

6. IMMUNE MEMORY AND

RESETTING THE SYSTEM

1. EARLY MEDIATORS

(IFN-

α/β

)

2. CHEMOKINES (MIP-1

α

)

3. EARLY & LATE MEDIATORS

(IL-12, IFN-

γ

, IL-4, IL-5)

4. EARLY & LATE MEDIATORS

(IL-12, IFN-

γ

, IL-4, IL-5)

5. DOWN-REGULATORS

(IL-10, TGF-

γ

)

6. MAINTAINANCE CYTOKINES,

ETC. (GM-CSF, IL-3, IL-7, etc.)

2

WHEN DO/DON’T CYTOKINES

WORK? (WHEN DO CYTOKINES

WORK FOR/AGAINST YOU?)

STEPS TO STUDYING

CYTOKINE FUNCTION

• Is the cytokine induced?

• When is the cytokine induced?

• What is the cytokine doing when it is

induced?

Cytokine induction...

• Messenger RNA (mRNA)

– Northern Blot

– RT-PCR

– RPA (ribonuclease protection assay)

– Gene chip

• Protein

– Western Blot

– ELISA

– Intracellular Cytokine Staining

– Protein array

3

USING ELISA TO DETECT CYTOKINES

Y

Y

Y

Y

1° Ab

Cytokine/Sample

Enzyme-2° Ab

Substrate

Color Change

INDUCTION OF CYTOKINES AFTER

LPS CHALLENGE (BY ELISA)

0

500

1000

1500

2000

IF

N-

(

U/m

l)

0

6

12

18 24

0

1000

2000

3000

4000

5000

IF

N-

(

pg

/m

l)

0

6

12 18

24

0

10000

20000

30000

40000

50000

TN

F (

pg

/m

l)

0

6

12 18

24

Hours Following Stimulation

0

50000

100000

150000

200000

250000

300000

IL-

6 (

pg

/m

l)

0

6

12 18

24

0

100

200

300

400

500

IL-

12 p4

0 (

pg

/m

l)

0

6

12

18 24

METHODS TO STUDYING

CYTOKINE FUNCTION

• Add cytokine function

-To animals in vivo (IL-12 induction of IFN-gamma

)

-Transgenic mice (LT and lymph node generation)

-To cells in vitro (IFN and antiviral effects)

4

METHODS TO STUDYING

CYTOKINE FUNCTION

• Remove cytokine function

-Soluble Receptors

-Genetic Mutation (KO mice)

-Neutralizing Antibodies

-KO Cytokine

-KO Cytokine Receptor

-KO Specific Intracellular Signal Transducer

DICHOTOMIZED T CELL

CYTOKINE PRODUCTION

T

IL-2
IFN-

γ

TNF

TYPE 1

CELL-MEDIATED IR

(INTRACELLULAR

ORGANISMS)

IL-4
IL-5

TYPE 2

HUMORAL IR

(PARASITES)

IL-12

IL-4

IL-4

IFN-

γ

STUDYING DICHOTOMIZED T CELL

RESPONSES IN VITRO

M

ϕ

M

ϕ

M

ϕ

M

ϕ

M

ϕ

T

T

IL-12

IL-4

5

THE LEISHMANIA PUZZLE...

LEISHMANIA

(LM)

C3H

B6

BALB/c

RESISTANT

RESISTANT

SUSCEPTIBLE

WHY?

DICHOTOMIZED T CELL RESONSES IN VIVO

IFN-

γ

IL-4

6

IL-12 IS INDUCED AFTER

LEISHMANIA INFECTION

IFN-

γ

IL-12

Day 0

IL-12 INDUCED IFN- DRIVES A TYPE 1 T CELL

RESPONSE AFTER LEISHMANIA INFECTION

1. ANTI-IL-12

1. IL-4

2. ANTI-IFN-

γ

2. IL-4

T CELLS FROM SUSCEPTIBLE MICE MAKE IL-4

IL-4

7

IL-12 CAN INDUCE A TYPE 1 T CELL

RESPONSE IN SUSCEPTIBLE MICE

1. IL-12

1. IFN-

γ

2. ANTI-IL-4

2. IFN-

γ

TOO MUCH CYTOKINE?

LPS

DEATH

SHOCK

INDUCTION OF CYTOKINES AFTER

LPS CHALLENGE (BY ELISA)

0

500

1000

1500

2000

IF

N-

(

U/m

l)

0

6

12

18 24

0

1000

2000

3000

4000

5000

IF

N-

(

pg

/m

l)

0

6

12 18

24

0

10000

20000

30000

40000

50000

TN

F (

pg

/m

l)

0

6

12 18

24

Hours Following Stimulation

0

50000

100000

150000

200000

250000

300000

IL-

6 (

pg

/m

l)

0

6

12 18

24

0

100

200

300

400

500

IL-

12 p4

0 (

pg

/m

l)

0

6

12

18 24

8

CYTOKINE MEDIATED TOXIC SHOCK

SHOCK

ANTI-IL-12

ANTI-IFN-

γ

ANTI-TNF