Cytokines and acute phase reactants

Toll-like receptors: adjuvant receptors play major roles in self discrimination Innate immune response (macrophage, dendritic

cells, neutrophils) is initiated upon TLR activation• Pathogen associated molecule pattern (PAMP):

Lipopolyssacharide, dsRNA,….etc.• Pattern recognition receptor (PRR): Toll like

receptors TLR) Adaptive immune response is controlled by innate

immune response.

Pathogen associated molecule pattern (PAMP): danger motif

Toll-Like Receptor (TLR) Family

Toll was first identified in 1997 in Drosophila.

Toll-like receptor exist in mammals as a family.

Basic structure: Toll/Il-1R (TIR) domain and leucine rich repeat (LRR)

LRR

TIR

TLRs and their ligands

TLR2 (heterodimerization with TLR1, TLR6) , TLR3, TLR4, TLR5 and TLR9 are well studied

Cell surface and endosome

Distribution of Toll-like receptors (TLRs)

TLR1TLR2

TLR3

TLR4 TLR5TLR6TLR10

TLR7

TLR8

TLR9

Toll/IL-1R (TIR)

NF-κBNF-κB IRF3IRF3

TNF- α, IFN- α, IL-1 β, IL-6, IL-12Chemokines, TLRs, co-stimulatory molecules

MyD88MyD88

EndosomessRNA

dsRNA

CpG dsDNA

LPS FlagellinCompounds of bacterial cell wall

TLR’s and their ligands

Immunological Reviews 2004 202: 250–265

Nat. Immunol. (2001)

TLR4 signaling1.MyD88-dependent 2.MyD88-independent pathways

Toll–like receptors Toll–like receptors : activating innate and adaptive immunity

PAMPs

Dendritic cell maturation

Nature immnol. Vol. 2 (2001)PAMP: pathogen-associated molecular patternsPAMP: pathogen-associated molecular patterns

Outline:

What is cytokine?

Property of cytokine and its receptor

TH1/TH2 Cytokine

Cytokine-related disease and therapy

Acute phase reactants

Cytokines Low-molecular-weight regulatory

proteins (<30KD), belong to hematopietin, interferon, chemokine or tumor necrosis factor family

Bind to the specific receptors on target cell membrane High affinity with dissociation constants

ranging from 10-10 to 10-12 M Regulate the function and

development of cells

The induction and function of cytokines

Properties of cytokines Pleiotropy: different functions on

different cells Redundancy: many cytokines

mediate similar functions Synergy: Antagonism: Cascade induction

Pleiotropy of cytokines

Cytokine cascade

Reduncancy, synergy and antagonism of cytokines

Classification of cytokine receptor

Subfamilies of class I cytokine receptors have signaling

subunits in common IL-2 receptor subfamily

IL-2, 15, 7, 9, 4 common subunit

IL-6 receptor subfamily IL-6, 11, CNTF, LIF/OSM common gp 130 subunit

GM-CSF receptor subfamily GM-CSF, IL-3, 5 Common subunit

Interactions between cytokine-specific subunits and a common signal-transducing subunit of cytokine receptor

CD25

Signal transduction mediated by most class I and II cytokines receptors

Cytokines from TH Cell

依照 T-help cells 所製造的淋巴激素，將其分為三類： TH0 、 TH1 、 TH2 。（ 1 ） TH 1 cells ： IL-2 、 IFNγ ， to

promote cell-mediated immune response ， augment IgM and IgG2a synthesis by B cells ， activity of macrophages 。

（ 2 ） TH 2 cells ： IL-4 、 IL-5 ， to promote antibody-mediated immune response ， lead to IgG1 and IgE responses ， lead to increased numbers of local and/or circulating eosinophils ， support allergic reaction 。

（ 3 ） TH 0 cells ： IL-2 、 IFNγ 、 IL-4 、 IL-5 。

Cytokine-mediated generation and cross-regulation of TH subsets

Cross-regulation at the intracellular level

Regulation of TH subsets by cytokines

Essential for TH2 cell development

Important for TH1 cells

Stimulatory effects

Inhibitory effects

TH1/TH2 balance determines disease outcome

Mycobacterium leprae, an intracellular pathogen

Cytokine-Related Diseases Bacterial septic shock (cytokine storm)

Cytokine overproduction Bacterial cell-wall endotoxins (lipopolysaccharide,

LPS)marcophage to overproduce IL-1 and TNF- Bacterial toxic shock and similar diseases

Bacteria produce toxins that act as superantigens The large number of T cells activated o result in

excessive production of cytokines Lymphoid and myeloid cancers

Cytokines and their receptors abnormally produced IL-6 overproduction plasmacytosis cancer

Sepsis or systemic inflammatory response syndrome (SIRS)

Pro-inflammatory mediators in sepsis

Cytokine cascade during LPS stimulation

Clinical trials in sepsis

Cytokine-based therapy: immunosuppression

Cytokine-based therapy

Purpose to detect cytokines

indication of immune rejection in organ rejection instead of organ failure.

monitoring course of disease such as TNF in septic shock and rheumatoid arthritis.

evaluation during immunotherapy such as IL-2 injection in HIV or IFN- in HBV carrier and HCV.

Methods to detect cytokines (in vitro and in vivo)

BioassaysImmunoassays: ELISART-PCR, Nouthern blotin situ hybridization, ELISPOTFlow cytometry to detect intracellular

cytokines

The frequency of cytokine-secreting T cells can be determined by the ELISPOT assay

Cytokine-secreting cells can be identified by intracellular cytokine staining

Cytokine in more detail

Mediators of innate immunity

Mediators of adaptive immunity

Mediator of innate immunity

•Tumor necrosis factor (TNF) (Interleukin-1 (IL-1)

•Interleukin-6 (IL-6)

•Interleukin-12 (IL-12)

•Interferon (IFN) Type1

Tumor necrosis factor (TNF) Interleukin-1 (IL-1)

主要是由 activated mononuclear phagocytes 所分泌的

主要刺激物為 lipopolysaccharide (LPS)

TNF 也是造成 Septic shock 和 Rheumatoid arthritis 的原因之一

•太高濃度的 TNF 會引起一連串的反應而導致Septic shock

•在 Rheumatoid arthritis 中 , TNF 等的 cytokine會刺激 synovial cell( 分泌滑液的細胞 ) 生成collagenase, 造成關節的 destruction

•而 IL-1 則是會啟動一些嚴重的 inflammation

•Treatment: anti-TNF,IL-1 antibody, IL-1 receptor antagonist (IL1Ra)

Interleukin-6 (IL-6)

•Produced by activated TH2 cells, macrophages, endothelial cells, fibroblasts

•It synergize with IL-1 and TNF to induce the acute-phase response in liver cells.

•B cell differentiation factor. ( 為 multiple myeloma 的主要 factor)

Interleukin-12 (IL-12)

•Produced by macrophage and dendritic cells

•Induce differentiation of TH1 subset

•Induce IFNγ production

Type1 interferon

•IFN-α 由 lymphocyte and macrophage 製造• IFN-β 由 fibroblasts and some epithelial cells 製造

Mediator of adaptive immunity

•Interleukin-2

•Interleukin-4

•Interleukin-13

•Interleukin-5

•Interferon (IFN) Type2

•Interleukin-10

•Transforming growth factor (TGF-)

Interleukin-2 (IL-2)

•Produced by activated TH1 cells and NK cells

•Activate NK cells into lymphokine-activated killer (LAK)

Interleukin-15 (IL-15)

•Produced by non-lymphoid cells, such as dendritic cells and cell of monocytic linage

•作用類似 IL-2

Lymphokine-activated killer cell (LAK)由 S. Rosenberg 所發現 , 用 IL-2 去和 lymphoid cell(90% 以上為 NK cell)

作用 , 結果發現這些 cell 對 tumor destruction 很有用 .

最重要的是 , 這種將 IL-2 和 lymphoid cell 作用的環境可以在 in vitro,

並對許多的 tumor 都可以作用 , 所以這些可以用來作 clinical trail

Interleukin-4 (IL-4)

•Produced by mast cells, TH2 cells and bone marrow stromal cells

•Enhance B cell proliferation, and induced class II MHC expression

•Promote heavy-chain class switch to IgE

Interleukin-13 (IL-13)

•Produced by mast cells, T cells and NK cells

•作用與 IL-4 類似

Interleukin-5 (IL-5)

•Produced by mast cells, TH2 cells and eosinophils

•Induce eosinophil formation and differentiation

Transforming growth factorβ(TGFβ)

A group of five cytokines released by many cell types

A potent inhibitor of IL-1-induced T-cell proliferation

Inhibit the proliferation and activation of lymphocytes and other leukocytes(T cell and macrophages)

Interleukin-10 (IL-10)

•Produced by activated subsets of TH2 cell

•Inhibit the production of IL-2 and IFN by TH1 cells

•Co-operate with TGFβ to stimulate IgA synthesis

Type2 InterferonIFNγ: 由 TH1 cells 和 NK cells 製造

Acute phase reactants(APR) Approximately 30 plasma proteins

produced rapidly by liver cells in response to TNF , IL-1 and IL-6 during inflammation such as infection trauma or neoplasia

Major APRs ： C-reactive protein(CRP)

APRs Metal-binding proteins: Ceruloplasmin Coagulation proteins: Fibrinogen Complement proteins: C2, C3, C4, C5, C9, Factor

B, C1 inhibitor Negative APRs ： Albumin Transferrin , ApoAl ,

ApoAll Functions ： modulating inflammation ， enzyme

inhibitor ， scavengers ， repair and resolution

C-reactive protein(CRP) 急性發熱性疾病患者的血清能和 pneumococus

的萃取物產生沉澱反應，早期稱為 Fraction C ，後又改稱為 C-polysaccharide ，能與 CPS 形成沉澱反應的物質就稱之為 C-precipitin 。

這種血清物質經常出現會出現在各種不同的發熱性疾病或細菌感染性疾病，或是在一些非感染性疾病，當時推測可能有一種不存在於正常人體血液的蛋白質所引起就稱為 C 反應蛋白 (C-reactive protein ； CRP)

CRP 一個完整的 CRP 分子是由五個 subunit ，

藉著非共價鍵結形成圓環狀的 pentamer (MW 120 KD)

Half life 19 h CRP 會和肺炎球菌的 C-polysaccharide

結合而形成沉澱，但是這種反應必須有鈣離子的存在

CRP 的生理功能 刺激 lymphocyte and monocyte

phagocytosis 阻止微生物感染 幫助分解消散被破壞的組織如染色質活化

補體典型途徑 從體內循環中清除不正常的物質

CRP 的臨床意義 CRP 在臨床上的運用，主要是在判斷是否發炎，發炎性

疾病的狀況，有否再復發，評估抗發炎藥物治療的效果，特別是對類風濕性關節炎的監視更是一個極好的指標

手術後，若患者血清居高不下，或呈現二次上升，表示傷口可能有併發感染

病毒性的感染， CRP 升高的程度會略低於細菌感染的增加

過去曾用 ESR 來作為測定 APR 的方法，但是由於使用ESR is a nonspecific indicator of disease ，會受到許多因素的干擾，所以後來都採用敏感度高的 CRP 。

區別診斷的依據 細菌性腦膜炎患者的血清 CRP 會顯著上升，

而病毒性腦膜炎的 CRP只少量上升大多數細菌性腦膿腫的患者， CRP 會顯著

增加膀胱炎的血清 CRP沒有顯著增加；但是在腎盂腎炎時，血清 CRP卻會增高

Major CRP increase Infections Hypersensitivity complications of infections

Rheumatic fever Erythema nodosum leprosum

Inflammatory disease Rheumatoid arthritis Ankylosing spondylitis Systemic vasculitis

Allograft rejection-Renal transplantation Malignancy-Lymphoma, Sarcoma Necrosis-Myocardial infarction, Tumour

embolisation, Acute pancreatitis Trauma, Burns, Fractures

Minor CRP increase (High sensitivity CRP,hs-CRP

<0.3 mg/dl Associated with cardiovascular disease such as atherosclerosis, stroke and myocardic infarction

Alpha1-antitrypsin (AAT)

Belong to “serpins” a group of proteins responsible for the inhibition of the serine proteases which trigger coagulation kinin release fibrinolysis and complement activation

AAT decrease in emphysema patients ( 肺氣腫 )

Can be detected by RID and nepherometry

Ceruloplasmin Major copper binding protein of

plasma Deficiency in Wilson’s disease Can be detected by RID and

nepherometry or ELISA

Haptoglobulin Bind to hemoglobulin the complex

which are removed in liver Can be detected by RID or functional

assays

Other immune cell products Neopterin

produced by activated macrophage, as a immune cell activation marker

beta 2 microglobulin MW 11,800 protein associated with HLA class I,

increase in renal disease, autoimmune diseases leukemia, lymphoma and multiple myeloma. serum normal range: 0.7-3.4 mg/l, urine: 5-154 mg/l.

soluble CD8 soluble IL2 receptor (Tac protein) shed adhesion molecules (sICAM-1, sVCAM-1,

sL-selectin, sE-selectin) induced by cytokines released from leukocytes,

enothelium, and other tissues