玻尿酸藉由結合CD44活化中性白血球的分子機轉研究

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國立臺灣大學醫學院分子醫學所碩士論文

Institute of Molecular Medicine College of Medicine

National Taiwan University Master Thesis

玻尿酸藉由結合CD44活化中性白血球的分子機轉研究 Studies on the Molecular Basis of CD44 and

Hyaluronan in Neutrophils Activation

林佳慧 Chia-Hui Lin

指導教授：余家利博士 Advisor: Chia-Li Yu, M.D., Ph.D.

中華民國 102 年 7 月 July, 2013

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中文摘要

CD44 是一個具有多重功能且位於細胞表面的醣蛋白質，其功能涉及細胞之間的相互作用、細胞附著、遷移、增殖和血管新生，而且在大多數的哺乳動物細胞中皆有表現。玻尿酸（hyaluromic acid; HA）是 CD44 一個非常重要的配體，廣泛分佈在整個結締組織、上皮細胞或神經組織中。CD44 與玻尿酸結合會活化其下游訊息

傳遞路徑而引發許多作用。在之前的研究中已經證明CD44 具有誘導淋巴細胞活

化、再循環和吸引淋巴細胞的功能。一些研究報告更指出CD44 和玻尿酸的結合可

以調控發炎反應、拮抗組織損傷和修復由發炎所造成的細胞傷害、減少發炎因子

的釋放和增加幹細胞遷移。然而CD44 與玻尿酸結合後對於中性白血球的影響目前

仍不清楚。在我們初步的研究中，發現了一些有趣的結果。首先，脂多醣（LPS）

和anti-CD44 會誘導多核型白血球 (PMN)中 CD44 的表現量增加。而玻尿酸的刺

激可以增加多核型白血球的吞噬作用並誘導細胞骨架的重組。CD44 與玻尿酸的結

合能誘導IL-8 的產生。接下來我們更發現，玻尿酸可以誘導 MAP kinase (p38)、

ERK1/2 磷酸化增加。在未來的研究方面，我們將添加不同的促發炎因子，觀察多

核型白血球CD44 的表現量。並使用抑製劑阻斷訊息傳遞路徑研究其對多核型白血

球的吞噬作用和cytokine 產生的影響以及所扮演的角色。此外，我們也將使用玻

尿酸酶分解玻尿酸，以研究CD44-HA 的結合對於多核型白血球的影響。最後，我

們藉由降低CD44 的表現量確認玻尿酸與 CD44 結合對於發炎反應的調解具有重要

性。

關鍵字：CD44、玻尿酸、MAP kinase (p38)、ERK1/2、多核型白血球吞噬作用、

細胞骨架的重組

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Abstract

CD44 is a multifunctional cell-surface glycoprotein that is involved in cell-cell

interaction, cell adhesion, migration, proliferation and angiogenesis and is widely

expressed in a large number of mammalian cell types. Hyaluronic acid (HA), one of

important ligand of CD44, is distributed widely throughout connective, epithelial, and

neural tissues. After CD44-HA binding initiates CD44 downstream signaling pathways,

the signaling induces multiple functions. Previous studies have demonstrated that CD44

induces the lymphocyte activation, recirculation, and homing. Some paper have shown

that CD44-HA interaction could regulate inflammation, tissue injury and repair through

regulating inflammatory cell recruitment, release of inflammatory cytokines, and stem

cell migration. However, the effects of CD44-HA interaction on neutrophils remained

unclear. In our preliminary study, several interesting results were found. First, we found

that the LPS and anti-CD44 could increase the expression of CD44 on PMN. HA could

also enhance the phagocytosis activity and cytoskeleton rearrangement of PMN and its

binding to CD44 could induce the interlukin-8 (IL-8) production. Furthermore, we

found that HA could induce phosphorylation of MAP kinase (p38), and ERK1/2

signaling pathways. In the future, we will test the effect of different pro-inflammatory

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cytokines on the surface expression of CD44 on PMN. The signaling pathways will be

selectively blocked for the observation of their effects on phagocytosis and cytokines

production of PMN. In addition, the hyaluronidase will be used to evaluate the

CD44-HA interaction on PMN. Lastly, the knockdown of CD44 expression in PMN

will be carried out to define the CD44-independent mechanisms by which HA can

mediate inflammation.

keywords：CD44、hyaluronic acid、MAP kinase (p38)、ERK1/2、PMN phagocytosis、

cytoskeleton rearrangement

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目錄

口試委員會審定書………... i

中文摘要 ... ii

Abstract ... iii

Chapter 1. Introduction ... 4

Chapter 2. Materials and methods ... 7

2.1 Isolation of PMN and MNC from normal human peripheral blood 7 2.2 Cell culture ... 7

2.3 Detection of CD44 expression treated with inflammatory molecule by flow cytometry ... 8

2.4 Cytokine measurements ... 9

2.5 Detection of PMN phagocytosis-enhancing activity of HA by flow

cytometry ... 9

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2.6 Preparation of whole cell extraction... 10

2.7 Western blotting ... 10

2.8 Immunofluorescence microscopic observation of cytoskeleton change in activated-PMN ... 11

2.9 Statistical analysis ... 12

Chapter 3. Results ... 13

3.1 The expression of CD44 on MNC and PMN ... 13

3.2 HA induced IL-8 production ... 14

3.3 HA enhanced PMN phagocytosis activity ... 14

3.4 HA induces cytoskeleton rearrangement on PMN ... 15

3.5 HA induced phosphorylation of MAPK and ERK1/2 on PMN and MNC ... 16

3.6 HA abolished induced IL-8 production on differiated-HL60 ... 16

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Chapter 4. Discussion ... 18

Chapter 5. Conclusions and Future directions ... 21

Chapter 6. Figures ... 22

Chapter 7. References ... 38

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Chapter 1. Introduction

CD44 is a multifunctional cell-surface glycoprotein and widely expressed in a large

number of mammalian cell types. The most abundant standard isoform of human CD44

protein (CD44s) contains 363 amino acids and its molecular weight is approximately 37

kDa. The structure of CD44s consists of three regions, a C-terminal cytoplasmic domain,

to which numerous signaling molecules bind directly or indirectly on activation of the

ligand binding, a transmembrane domain, and an extracellular domain which is the

so-called link modules of hyaluronan (HA) binding proteins (1, 2) (Figure 1A). The

variant isoforms of CD44 (CD44v) inserts an alternatively spliced exons within the

extracellular domain, a membrane proximal domain, which can alter the binding affinity

for HA and confer interaction with alternative ligands. The CD44 isoform go through

the post-translational modification with a molecular weight of about 80 kDa (2). The

CD44s is widely expressed in a large nimber of mammalian cell types, and the

expression of CD44v is detectable in hematopoietic cells (3), particularly in MNCs (4)

and in reactive lymph node cells (3, 5). The principal and important ligand of CD44 is

hyaluronic acid (HA), a ubiquitous component of the extracellular matrix (ECM). But it

can also interact with other ligands, such as osteopontin (6), collagens (7, 8), and

fibronectin (9). HA is a linear, polymeric glycosaminoglycan composed of repeating

(10)

disaccharides D-glucuronic acid and N-acetyl-D-glucosamine linked by a glucuronidic β

(1→3) bond (10, 11) (Figure 1B). HA is widely distributed throughout connective,

epithelial, and neural tissues.

CD44-HA interactions mediate cell adhesion and migration in a variety of physiological

and pathophysiological processes, including tumour metastasis, wound healing and

leukocyte extravasation at sides of inflammation (11, 12, 13, 14). Previous studies have

demonstrated that CD44 induces the lymphocyte activation, recirculation, and homing

(15, 16). According to Paul W. et al. have indicated that the role of CD44 in regulating

HA interactions depends on the cell types, and the effect and mechanism of CD44-HA

interaction on macrophage have reported (11, 17). However, the effects and mechanism

of CD44 binding on neutrophils remain unclear.

CD45, as leukocyte common antigen (LCA), is glycoproteins uniquely expressed on the

surface of all leukocytes and their hemopoietic progenitor cells. CD44 is also a one of

common LCA on neutrophils. CD45 is a family of high molecular weight

transmembrane protein tyrosine phosphatase (PTPase) expressed on all nucleated

haematopoietic cells (18). Many authors have reported the possible roles of CD45 and

its isoforms in T and B cell differentiation (19), natural killer T cells and cytotoxic T

lymphocyte functions (20), cytokine production by MNC (21) and TCR-associated

signalling in T cells (22).

(11)

Previous studied have shown that the lower CD44 expression in immunodeficiency

animal model or disease patient. However, the expression of CD44 is higher in

inflammation condition (1, 2). CD44 has a vital role in involving in immune response.

So we want to know the role and mechanism of CD44-HA interaction on PMN. To

figure out the relationship of CD44 and CD45, and the effects and mechanism of CD44

binding to HA on neutrophils. In the present study, we found that anti-CD44 could

increase the expression of CD44 on PMN. CD44-HA interaction could induce IL-8

production through MAP kinase (p38), and ERK1/2 signaling pathways. In addition, it

could enhance phagocytosis and cytoskeleton rearrangement of the PMN.

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Chapter 2. Materials and methods

2.1 Isolation of PMN and MNC from normal human peripheral blood

Heparinized venous blood obtained from normal individuals was mixed with

one-quarter volume of 2% dextran solution in 37℃ (molecular wight: 425000-575000)

and incubated at room temperature for 30 minutes. The cell suspension was gently

layered over Ficoll-Hypaque density gradient solution (specific gravity 1.077; GE

Healthcare, Waukesha, Wisconsin, USA) and centrifuged at 500 x g for 30 minutes. The

MNC were aspirated from the interphase whereas the PMN were collected from the

bottom. The residual RBC in PMN was lysed in cold 0.85% ammonium chloride

solution. These cells were then rinsed twice with PBS and re-suspended in RPMI 1640

(Gibco/BRL, Grand Island, New York, USA) supplemented with 10% (v/v) fetal bovine

serum (FBS), 2 mM L-glutamine (hereafter referred to as complete medium). The

viability of PMN and MNC were detected by trypan blue dye and the cell concentration

was adjusted to 2×10⁶/ml in complete medium.

2.2 Cell culture

Human promyelocytic leukemia cell line (HL-60) was maintained in complete medium

at 37℃ in a humidified atmosphere containing 5% CO2. HL-60, MNC and PMN (2×10⁶

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cells/ml) were treated with LPS (20ng/ml), h-IgG (1μg/ml), anti-CD44 (1μg/ml),

anti-CD45 (1μg/ml), anti-CD3 (1μg/ml)/ anti-CD28 (1μg/ml), and hyaluronic acid (HA

1mg/ml and 2mg/ml) at 37℃, then cultured and harvested at the indicated time points.

Induction of differentiation was obtained by seeding the cells at a concentration of 5 x

10⁵/ml in the presence of DMSO (Microbiological Associates, Rockville, Md.) at a final

concentration of 1.3% (v/v) for 5 days. After exposure, the cells were resuspended in

DMSO-free medium. Next, differentiated-HL60 were treated with LPS (20ng/ml),

anti-CD44 (1μg/ml), and hyaluronic acid (HA 1mg/ml and 2mg/ml) at 37℃, then

cultured and harvested at the indicated time points.

2.3 Detection of CD44 expression treated with inflammatory molecule by flow

cytometry

MNC and PMN were treated with anti-CD3 (1μg/ml)/ anti-CD28 (1μg/ml) or LPS

(20ng/ml) at 37℃, before being cultured and harvested at the indicated time points. The

cells were washed twice with PBS. The cells were then fixed with 4%

paraformaldehyde for 30 minutes at room temperature and incubated with

anti-CD44-FITC overnight to detect the CD44 expression. The percentage (%) and

mean fiuorescence intensity (MFI, denoted by mean channel number) of CD44

expression were determined by FACSort flow cytometry (Becton Dickinson) at wave

(14)

length 488nm excitation.

2.4 Cytokine measurements

After treatment with HA and pro-inflammatory molecule at indicated time points, the

cells were centrifuged at 800 x g for 10 minutes for the detectection of cytokine

concentrations in cell culture supernatant. The concentrations of cytokines including

IL-8 were quantified using their respective ELISA kits (R&D Systems, Minneapolis,

Minnesota, USA).

2.5 Detection of PMN phagocytosis-enhancing activity of HA by flow cytometry

Fluoresbrit carboxylate microspheres (0.75 μm in diameter, Polyscience Inc.) were

washed with PBS in advance twice and opsonized by incubation with fresh human

serum at 37℃ for 2 hours. Fresh prepared PMN (2×10⁶ cells/ml) were treated with LPS

(20ng/ml), h-IgG (1μg/ml), anti-CD44 (1μg/ml), anti-CD45 (1μg/ml), and HA (1mg/ml

and 2mg/ml) at 37℃ for 1 hours. The mixture was let reacting with opsonized beads

(1×10⁸ beads/ml) at 37℃ for 1 hour in 5% CO2-95% air. After incubation and wash by

PBS twice, the percentage (%) and mean fluorescence intensity (MFI, denoted by mean

channel number) of PMN phagocytosis were determined by FACSort flow cytometry

(Becton Dickinson) at wave length of 488nm excitation.

(15)

2.6 Preparation of whole cell extraction

After treatment with HA or control medium at different time points at 37℃, the cells

were centrifuged at 800 x g for 10 minutes followed by wash with cold PBS. After

centrifugation at 800 x g for 10 minutes, the pelleted cells were lysed with cold RIPA

buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1%NP-40, 1% sodium deoxycholate,

0.1% SDS ) containing protease inhibitor cocktail and phosphatase inhibitor cocktail

(Roche) and kept on ice for 30 minutes. The cell lysates were centrifuged at 10,000 x g

at 4℃ for 20 minutes to remove the debris and the supernatants were applied for

Western blot. The protein concentration of the cell extraction was measured using the

BCA Protein Assay (Pierce).

2.7 Western blotting

Proteins were separated by 10% SDS-PAGE and transfer to polyvinylidene fluoride

(PVDF) membrane (Millipore Inc.) in a Mini Trans-Blot cell (Bio-Rad) for 2 hours at

350 mA. The PVDF membranes were blocked with Tris-buffered saline and Tween 20

(TBST, 50 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH 7.6) containing 1% BSA at

room temperature for 30 minutes, and probed with a specific antibody overnight at 4℃.

After washing the membrane three times with TBST buffer each for 5 minutes, the

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complexes were detected by HRP-conjucated-secondary antibody (Jackson ImmunoLab)

and ECL Western Blotting Substrate (Pierce) chemifluorescence detecting system.

2.8 Immunofluorescence microscopic observation of cytoskeleton change in

activated-PMN

After stimulation at 37℃ by HA or LPS for 2 hours, the activated-PMN were wash with

PBS and then fixed for 30 minutes at room temperature with a solution of 4%

paraformaldehyde followed by washing with PBS. Then mixture was centrifuged and

the supernatant was discarded. The cells were resuspended in 200 μL of PBS. 5 to 10

μL of the cell suspension was smeared above a gelatin-coated slide. Placed the slide on

a hot plate (low heat setting), and allowed the liquid to evaporate. The PMN were

permeabilized with 0.1% Triton X-100 for 15 minutes at room temperature, and washed

three times with PBS. The blocking solution (10% FBS in PBS) was added to inhibit the

non-specific binding. PMNs were stained with fluorescent phalloidin (1:200) for

visualization of actin filament at room temperature for 30 to 60 minutes. The slides

were washed three times with PBS and stained with DAPI

(4’,6-diamidino-2-phenylindole, 1:1000) at room temperature for 5 minutes. After three

times washes with PBS, the slides were mounted with glycerol and gently covered the

cover slip avoiding producing the air bubbles. The cells were visualized the

(17)

cytoskeleton was observed using a fluorescence microscope (Olympus, Japan).

2.9 Statistical analysis

The statistical analyses were performed using the analysis of variance (ANOVA).

Statistical significance was defined as p＜0.05.

(18)

Chapter 3. Results

3.1 The expression of CD44 on MNC and PMN

Previous investigations have shown that during inflammation, CD44 expression is

upregulated on hematopoietic and parenchymal cells (23, 24), and it plays a crucial role

in an variety of inflammatory diseases including murine models of inflammatory bowel

disease, collagen- and proteoglycan-induced arthritis, cutaneous inflammation,

experimental autoimmune encephalomyelitis, and IL-2-induced vascular leak syndrome

(25, 26, 27, 28). To understand whether the inflammatory molecules could enhance the

CD44 expression on MNC and PMN, we used pro-inflammatory molecules anti-CD3/

anti-CD28 on MNC. The results indicated that co-stimulated anti-CD3/ anti-CD28 as

well as LPS provided a strong pro-inflammatory signal on PMN (29, 30). As

determined and demonstrated by flow cytometry data, anti-CD3/ anti-CD28 had

significantly enhanced the percentage of CD44 expression on MNC compared to the

control (Figure 2A). On the other hand, LPS also slightly increased the CD44

expression on PMN (Figure 2B). Accordingly, we concluded that pro-inflammatory

cytokines could increase the CD44 expression on PMN.

(19)

3.2 HA induced IL-8 production on PMN

Next, we determined whether CD44-HA interaction could induce the PMN into the

activation condition and enhance inflammation. We used LPS, anti-CD44, anti-CD45

and HA to stimulate the PMN and then detect the interleukin-8 (IL-8) concentration,

which is a one of the neutrophil-specific CXC subfamily of chemokines. IL-8 is also a

potent neutrophil chemotactic and activating factor and is a primary pro-inflammatory

cytokine produced by many cells (31, 32, 33, 34). We could demonstrate that LPS

increases the IL-8 production and HA has significantly increased IL-8 concentration,

compared to the controls. However, anti-CD44 and anti-CD45 might have little

increasing effect on IL-8 production. Therefore, CD44-HA interaction could promote

PMN to go inyo the activated-form.

3.3 HA enhanced PMN phagocytosis activity

Previous studied have shown that the role of CD44 in binding, ingestion (phagocytosis),

and clearance of apoptotic cells (35) as well as microbial pathogens (36). A direct

antibody ligation of CD44 on macrophages enhances the subsequent uptake of apoptotic

cells (37, 38). To further confirm the CD44-HA interaction could enhance the PMN

phagocytosis, we used HA to stimulate PMN and Fluoresbrit carboxylate microspheres

(20)

to determine the PMN phagocytosis by flow cytometry. LPS could enhance the PMN

phagocytosis compared to the controls (49.23% to 75.57%) and so was taken as positive

controls. The CD44-HA interaction significantly increased the phagocytosis activity

(49.23% to 93.01%), to an extent of even greater than LPS stimulation. Anti-CD44

(49.23% to 62.87%) and anti-CD45 (49.23% to 63.57%) also stimulated the PMN

phagocytosis (Figure 4), but this increase was smaller than that exerted by LPS and HA.

These results of anti-CD45 were consistent with previous reports (39). It suggested that

CD44-HA interaction could enhance the PMN phagocytosis activity.

3.4 HA induces cytoskeleton rearrangement on PMN

Previous investigations have indicated that the cytoplasmic domain of CD44 has a

linker protein- ankyrin specific binding site (36, 40). Ankyrins proteins are a family of

adaptor proteins that mediate the attachment of integral membrane proteins to the

spectrin-actin based membrane cytoskeleton (41). To understand if CD44-HA

interaction on PMN could initiate the CD44 downstream signaling pathway and induce

cytoskeleton rearrangement, the cells were treated with HA. After treatment with HA,

we used phalloidin to stain actin filaments and observed the aggregation of actin

filaments. We found that not only HA could induce cytoskeleton rearrangement but also

anti-CD44 and anti-CD45 could partially induce the actin aggregation (Figure 5). These

(21)

results indicated that CD44 on PMN could mediate the cytoskeleton change and

CD44-HA interaction could enhance cytoskeleton rearrangement to achieve its

biological function.

3.5 HA induced phosphorylation of MAPK and ERK1/2 on PMN and MNC

Previous studied have demonstrated that CD44-HA interaction could activate several

signaling pathways (42, 43). To further understanded the molecule mechanism of the

CD44-HA interaction on activated-PMN and activated-MNC, we observed the

phosphorylation of signal transduction. In Fig 6A and 6B, you can see that HA could

increase the phosphorylation of MAPK (P38) and ERK1/2 on MNC. On the other hand,

CD44-HA interaction could also significantly increase the phosphorylation of MAPK

(P38) and ERK1/2 on PMN (Figure 6C and 6D). These results indicated that HA could

induce and activate MAPK (P38) and ERK1/2 signaling pathway.

3.6 HA abolished to induce IL-8 production on differiated-HL60 cells

According to Liu J. et al. have shown that down-regulation of CD44 contributes to the

differentiated-HL60 cells (44). To investigate the effect of CD44-HA interaction on the

differentiation of HL-60 cells, we treated with DMSO to induce cells differentiation and

detect IL-8 production after treatment with HA. We found that LPS increased the IL-8

(22)

production, however HA and anti-CD44 abolished to induce IL-8 production on

differiated-HL60 cells (Figure 7). Therefore, it indicated that HA is specific binding to

CD44 and induce downstream signaling pathway.

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Chapter 4. Discussion

1. The role of hyaluronic acid in tissue and cell

Hyaluronic acid, a ubiquitous component of the extracellular matrix (ECM), is widely

distributed throughout connective, epithelial, and neural tissues. CD44 is expressed in

numerous cell types. Previous papers have shown that CD44 is involved in and

mediated many biological functions. In normal condition, HA is enriched in ECM. How

the cell determines and regulates HA binding to CD44 further prevents HA induced

downstream signaling pathway from activating PMN or MNC is unknown. Otherwise,

many authors have reported that the CD44 molecule plays a central role in the

development of collagen- or proteoglycan (cartilage delivered)-induced arthritis (45, 46,

47, 48, 49). The expression of CD44 and extractable HA is increased in the arthritic

inflamed joint tissues, suggesting that they are associated with the inflammatory process

(50). Hyaluronic acid is primarily used to increase mobility of the joints, clear eye

vision, combat signs of aging such as wrinkles, relief fibromyalgia and help wound heal.

The phenomenon is obviously contrary to our current results showing that HA could

induce tissue inflammation. So, it might have another mechanism underlying the

mediation and regulation of the CD44-HA interaction. There have several possibilities

(24)

to affect the CD44-HA interaction: one is hyaluronan synthases (HAS). Previous papers

have indicated that inflammation molecules, TNFα, increases the transcription of HAS

and the expression on cell membrane (51). And another is hyaluronidase, it may be have

some mechanism to regulate it enzyme activity or concentration in cells to increase or

decrease the concentration of HA. There have the papers shown that the HA oligomer

compete for endogenous polymeric HA, thus replacing high affinity, multivalent and

cooperative interactions with low affinity, low valency receptor interactions (52). The

last is the expression of CD44, according to figure 7 that down-regulation of CD44

abolished CD44-HA activation. And these possibilities need further improve and

understand its regulation and detail mechanism.

(25)

2. The relationship of CD44 and CD45

CD45, as leukocyte common antigen, is a family of high molecular weight

transmembrane protein tyrosine phosphatase (PTPase) expressed on all nucleated

haematopoietic cells (17). Many authors have reported the possible roles of CD45 and

its isoforms in cell differentiation (18), cytokine production (20) and TCR-associated

signalling (21). Recent studied have shown that CD45, negatively regulatory role for

CD45 in CD44 signaling leading to actin rearrangement and cell spreading in activated

thymocytes and T cells. According to Yu C. L. et al. 2002 and our present data the

CD45 and CD44 had similar function on PMN. The relationship of CD44 and CD45

mediating and regulating PMN remains to be confirmed.

(26)

Chapter 5. Conclusions and Future directions

This study showed the effect of CD44-HA interaction on PMN. First, we found that

pro-inflammatory molecule (LPS and anti-CD3/anti-CD28) could increase the

expression of CD44. Next, we showed that HA could activate PMN and increase the

IL-8 production. We also demonstrated CD44-HA interaction not only could enhance

the phagocytosis activity but also induced cytoskeleton reorganization. We observed

that CD44-HA interaction could induce phosphorylation of MAPK and ERK1/2

signaling pathway. Last, we investigated that HA abolished induce the IL-8 production

on the differentiation of HL60 cells induced by DMSO. In the future, we are going to

use signaling transduction inhibitor to block the signaling pathway and to investigate

the effects of phagocytosis as well as cytokines production on PMN. In addition, we

will use the hyaluronidase to investigate the CD44-HA interaction on PMN. Last, we

will knock down the CD44 in PMN expression to define the CD44-independent

mechanisms by which HA can mediate inflammation.

(27)

Chapter 6. Figures

A

B

Figure 1 Domains of CD44 and the structure of hyaluronic acid (HA). (A) The

major domains of the standard isoform of human CD44 are shown. They consist of

three regions, a C-terminal cytoplasmic domain which signals molecules directly or

indirectly to binding region, a hydrophobic transmembrane domain, and a extracellular

(28)

domain, which is the so-called link modules of hyaluronan binding proteins. However,

the variant isoforms of CD44 inserts a alternatively spliced exons within the

extracellular domain, a membrane proximal domain, which can alter the binding affinity

for HA and confer interaction with alternative ligands. (B) Hyaluronic acid is composed

of repeating polymeric disaccharides D-glucuronic acid (GlcA) and

N-acetyl-D-glucosamine (GlcNAc) linked by a glucuronidic β (1→3) bond.

(29)

A

B

Figure 2 Pro-inflammatory molecule enhances the expression of CD44 on MNC

and PMN. (A) MNC added with anti-CD3 and anti-CD28, (B) PMN added with LPS and inflammatory molecule could enhance the expression of CD44.

(30)

Figure 3 HA induced IL-8 production. The detailed procedures were described in

“Materials and Mathods” LPS as positive control. HA could induce the IL-8 production

by PMN. However, anti-CD44 or anti-CD45 had little effect on IL-8 concentration.

(31)

(32)

Figure 4 HA enhanced PMN phagocytosis activity. LPS enhances the phagocytosis

and is used as positive control. HA had greater enhancement of phagocytosis compared

to control and LPS. And anti-CD44 and anti-CD45 induced increase in phagocytosis,

compared to the controls to an extent less them that by LPS.

(33)

A

(34)

B

(35)

C

(36)

D

(37)

E

(38)

F

(39)

Figure 5 HA induced cytoskeleton rearrangement on PMN. (A) medium, (B) IgG,

(C) LPS, (D) anti-CD44, (E) anti-CD45, (F) HA. LPS induce cytoskeleton

rearrangement and could enhance the actin aggregation phenomenon. In (F) HA could

clearly induce the aggregation of actin, compared to the control. (D) and (E) also show

the structure of actin aggregation in PMN. HA, anti-CD44 and anti-CD45 are presumed

to induce cytoskeleton rearrangement in PMN.

(40)

A

B

(41)

C

D

Figure 6 HA induced phosphorylation of MAPK and ERK1/2 on PMN and MNC.

(A) phosphor-ERK1/2, (B) phosphor-P38 on MNC. (C) phosphor-ERK1/2, (D)

phosphor-P38 in PMN. In (A) and (B) HA increase the phosphorylation of MAPK and

ERK1/2 in MNC. Anti-CD3 and anti-CD28 is positive control. In (C) and (D) HA

increase the phosphorylation of MAPK and ERK1/2 in PMN. LPS is positive control.

(42)

0 500 1000 1500 2000 2500

medium LPS

20 n g/ml

anti-CD44 hya

luron an 1m

g/m l

hyaluronan 2mg/ml

(pg/ml)

HL-60 (DMSO)

Figure 7 HA abolished to induced IL-8 production on differiated-HL60. The

detailed procedures were described in “Materials and Mathods” LPS as positive control.

The differentiation of HL60 cells were induced by DMSO contributes to decrease the

expression of CD44. HA could abolish to induce IL-8 production on differiated-HL60

as well as anti-CD44.

(43)

Chapter 7. References

1. Toole B. P., Slomiany M. G. Hyaluronan, CD44 and Emmprin: partners in cancer

cell chemoresistance. Drug Resist Updat. 11, 110-121. (2008)

2. Goodison S., Urquidi V., Tarin D. CD44 cell adhesion molecules. Mol Pathol. 52,

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