Chemokine and complement

Chemokine classification Chemokine function Chemokine receptor Colony stimulating factor (CSF) Complement

Chemokines participate in leukocyte recruitment during inflammation

chemokine

Chemokines Chemotactic cytokine A large family of structurally

homologous cytokines stimulate transendothelial leukocyte

movement from the blood to tissue site of infection (Extravasation)

Cause rapid shape changes in leukocytes(pseudopod formation)

Classification of chemokine at least 43 members, posses 4 conserved

cysteine residules, and based on the position of two of the four invariant cyctein residuels, almost all fall into one of two subgroup, CC and CXC chemokines

One receptor bind more than one chemokines and one chemokine binds to more than one receptor (redundancy)

Chemokine and chemokine receptor CC chemokine: first two of the four

cysteine residues are adjacent. CXC chemokine: a single amino acid

residue interposed between the first two cysteines. ELR+: chemotactic for neutrophil, angiogenesis ELR-: chemotactic for lymphocyte, restrain

angiogenesis

CX3C chemokine: fractalkine(CX3CL1) C chemokine: lymphotactin(XCL1)

Chemokines signal through receptors coupled with heterotrimeric large G proteins

Chemokine inhibits HIV entry to cells

Colony-Stimulating Factors CSFs are cytokines that stimulate

stem cells or their progeny to produce large number of blood cells

Variety of CSFs are made such as G-CSF and GM-CSF

CSFs are potentially important in the treatment of human disease(GM-CSF 用在一些 immunocompromised condition)

CSFs GM-CSF G-CSF M-CSF Erythropoietin

(EPO)

IL-3(multi-CSF) IL-7 IL-9 IL-11

CSFs GM-CSF

lymphocytes,monocytes,fibroblast,endothelial cells to stimulate granulocytes and macrophage precursors

to mature and proliferate G-CSF

G-CSF,GM-CSF have been injected into patients to increase stem cell (CD34+)for peripheral blood stem cell (PBSC)transplantation

M-CSF lymphocytes,monocytes,fibroblast,endothelial cells to stimulate macrophage precursors to mature and

proliferate

Erythropoietin(EPO) produced by kidney to stimulate RBC to

mature and proliferate. EPO have been injected into patients with

anemia

Complement outline Complement activation pathways Complement receptors Complement regulation Complement functions Complement abnormality Complement detection Complement fixation assay

The complement system Sequential activation (cascade) of

zymogens to generate proteolytic enzymes

Activated complement components bind to surface or be degraded.

Complement activation is inhibited by regulatory proteins on host cell surface.

Overview of complement system

The classical pathway is initiated by activation of the C1 complex

C1q hexamer bindingC1r cleaves C1sC1s cleaves C4, C2

classical pathway generates C3b

The mannan-binding lectin pathway is homologous to the classical pathway

Mannan binding protein associated serine protease (MASP) like C1r, C1s

Complement activation is largely confined to the surface on which it is initiated

C3 is the same

Hydrolysis of C3 causes initiation of the alternative pathway of complement

C3b bind to microbial surface but not host cell can activate factor B

Classical and lectin pathways can also activate alternative pathway

Surface-bound C3 convertase deposits large numbers of C3b fragments on pathogen surfaces and generates C5 convertase activity

Complement receptors CR1 promote phagocytosis of C3b, C4b

coated immunocomplex CR2 bind C3d or EBV stimulate B cells and

FDC CR3 (Mac 1 or CD11b/CD18), integrin

bind iC3b, ICAM-1, cause phagocytosis CR4 (CD11c/CD18) like Mac-1 with

different chain

Complement receptors

Small fragments of some complement proteins can initiated a local inflammatory response

The terminal complement proteins polymerize to form pores in membranes that can kill certain pathogens

Complement control proteins regulate all three pathways of complement activation and protect the host from its destructive effects

Factor I cut C3b on host surface

Complement–mediated functions

Cell lysis Chemotaxis(C5a>C3a>C4a) Opsonization(C3b)

Elevated complement levels

Inflammatory condition Trauma Acute illness(C3)

Decreased complement levels

Excessively activated recently Currently being consumed A single complement component is

absent because of a genetic defect

C2 -- the most common complement deficiency

C3 -- an acute phase protein C4 -- the most sensitive indicator of

disease activity

C1 inhibitor (C1INH) deficiencty causes angioedema

Hereditary angioedma (HAE): genetic defect cause C1 inhibitor (C1INH) deficiencty

Autosomal dominant, occurs in 1 in 106.

C4 and C2 degrade to produce C4a anaphylatoxin and C2a-kinin like fragment.

Treat with steroid to induce liver to produce C1INH and other proteins

Hereditary and acquired C1 inhibitor (C1INH) deficiencty

CD59 inhibit C9 bind to C5b, 6, 7, 8

Decay-accelerating factor (DAF) displace Bb from C3b and C2b from C4b

Both CD59 and DAF are defect in PNH because enzyme to synthesize phosphoinositol glycolipid is missing in PNH

paroxysmal nocturnal hemoglobinuria (PNH)

Complement Assay

Component Assays

Functional Assays

Component AssaysRadial immunodiffusion ( 免疫擴散法 ,RID)NephelometryRadioimmunoassayTurbidimetryLatex agglutination photometric assay

Functional assay: CH50 目的：藉由測病人血清使 50%50%紅血球溶血紅血球溶血 所需

的體積，可得血清中的補體在典型途徑典型途徑中的功能。

原理： Patient’s serum （含補體） sensitized SRBC （含 Ab-Ag ）

　　 SRBC hemolysis （典型途徑活化）

CH50 方法：病人血清（ 1:50 or 1:60 ）＋ SRBC

(EA) 37℃ 水浴槽　　離心　　 O.D添加物 1 2 3 4 100%

TBS-G 0.9 0.7 0.6 0.5

血清 ml 0.2 0.3 0.4

EA ml 0.6 0.6 0.6 0.6 0.6

H2O ml 0.9

O.D 0.008 0.102 0.185 0.322 0.664

CH50

CH50Von-Krogh transformation ：

X 軸： log (y/1-y) Y 軸： log (milliliter of serum) y= 各管校正過的吸光值／完全溶　

　　　　　 血的校正吸光值 溶血百分比溶血百分比

　　 當 y/1-y=1 時　　有 50% SRBC 　　　 溶血

CH50

CH50缺點：

SRBC 脆性不一　　

SRBC 上免子的 Ab Affinity 不同　黏上 SRBC 量不同

檢體的採集和保存

Complement Fixation 目的：測定待測血清 ( 需去補體 ) 中是否含

有特定抗體或抗原。 原理： test system 和 indicator system

會互相競爭 complement 。Test system:未知 Ab 和已知 Ag or 未知 Ag 和已知 Ab

Indicator system ： sensitized SRBC

Complement Fixation

Complement Fixation

Complement Fixation

Complement Fixation: the importance of different controls

Complement Fixation