assessment of t cell activation in a mouse model of ... · including nerve injury[1, 2]. interest...

Journal of Otology 2010 Vol. 5 No. 2

Correspondence author: GAO Zhiqiang, Department ofOtorhinolaryngology, Peking Union Medical CollegeHospital, Chinese Academy of Medical Sciences andPeking Union Medical College, Beijing 100730, ChinaEmail: [email protected]

Original ArticleAssessment of T Cell Activation in a Mouse Model of Traumatic

Facial Nerve InjuryQUAN Shi-ming1, PENG Ben-gang1*, GAO Zhi-qiang2*

1 Department of Otorhinolaryngology, Beijing Ji Shui Tan Hospital, Beijing 100035, China;2 Department of Otorhinolaryngology, Peking Union Medical College Hospital, Chinese Academy of Medical

Sciences and Peking Union Medical College, Beijing 100730, China

Abstract Objective To investigate T cell activation following facial nerve axotomization and latentneuroimmunologic mechanisms in traumatic facial paralysis. Methods A murine model of facial nervetransaction was used. Lymphocytes from cervical and mesenteric lymph nodes in BABL/c mice atspecific times were collected and expression rates of CD69 on T cells were assessed by flowcytometry. Results Infiltrating T cells were detected around the facial neurons in the facial nervenucleus in mice whose facial nerve was transected. Immunofluorescent staining showed recruitment ofactivated T cells. Three days post-facial nerve transection, the expression rate of CD69 on T cells fromcervical draining lymphoid nodes(CDLNs) was significantly different from that on T cells frommesenteric lymph nodes(MLNs) (P＝0.0457), whereas the latter was similar to that in animalsundergoing sham surgeries and that in blank control animals(p= 0.2817 and 0.2724, respectively). Twoweeks post-nerve transection, the T cell CD69 expression rate from CDLNs remained at a higher leveland than that in the sham-operation animals(p= 0.0007). At two weeks, CD69 expression rate on Tcells from MLNs was also up-regulated and different compared with the sham-operation animals andwith itself at three days postoperation(p= 0.0082 and 0.0133, respectively ). Conclusion T cells appearto be activated and up-regulated in CDLNs following facial nerve transection. There is even evidence ofT cell activation in MLNs at 2 weeks post-nerve transection. This suggestes an alteration of immuneresponse from local to general immunity in the acute stage of facial nerve trauma, which may helpcoordinating and controlling the scales and orientation of the neuroimmune response during thepathogenesis and progression of facial nerve trauma.

Key words facial nerve; T cell; CD69; injury; Neuroimmunomodulation

1 IntroductionT cell activation plays an important role in

triggering immune response in many diseases,including nerve injury[1, 2]. Interest in immune

activation in facial nerve injury is increasingbecause studies have shown that irreversibleaxonal damage occurs earlier than previouslythought[3]. The classic viewpoint of facial nerveinjury pathology focuses on demyelinating lesionscharacterized by the presence of T cells, B cellsand phagocytic macrophages[4]. Disease activity isprobably initiated by systemic activation ofmyelin-reactive T cells, followed by activated Tcell migration into the central nerve system

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(CNS), including facial nucleus, and the sequentialrecruitment of additional inflammatory cells tofacial motoneurons [5]. Cells primarily accumulatein the perivascular space and subsequentlymigrate into the CNS parenchyma. Therecruitment and migration of leukocytes into theCNS are orchestrated by chemokines. Andactivated T cells that express specificcombi-nations of adhesion molecules andchemokine receptors are preferentially attractedto CNS by chemokines produced within thetarget tissue. Hence, in patients with facial nerveinjury, the expression of adhesion molecules andchemoki-nes on the surface of activated T cellsgoverns the recruitment of immune cells to theCNS [6, 7]. Moreover the expression of otheractivation and differentiation molecules reflectsthe function of recruited T cells. However, thestatus of T cells and definitive molecular andcellular immuno-pathological mechanisms infacial nerve injury are unknown.

CD69 is the earliest cell surface marker ex⁃pressed in activated immune cells. Generallyspeaking, in static state (without being stimulat⁃ed), the immune cell does not express CD69, al⁃though it can be rapidly spurred to expressing bystimulating signals[8]. Therefore, CD69 is the idealindex to appraise immune cell’s state offun-ction [9-12]. Its stimulation leads to IL-2 pro⁃duction and increases CD25 expression, finallycausing T cell proliferation. The present studywas consequently undertaken to detect the pres⁃ence of activated T cells in a mouse model withfacial nerve transection. To this point, T lympho⁃cytes collected from cervical draining lymphnodes (CDLNs) and the mesenteric lymphnodes(MLNs) were used to represent local and

“remote”(systemic) immune responses respec⁃tively in the current study. Early activation antigenCD69 on T cells was examined by flow cytometryso as to provide preliminary reference data on la⁃tent neuroimmunologic mechanisms of traumatic

facial paralysis.

2 Materials and methods

2.1 Animals and surgical proceduresAll BABL/c mice used in the present study

were specific pathogen-free(SPF) animals. Sev⁃en-week-old male wild-type mice were ob⁃tained from Institute of Laboratory Animal Sci⁃ence, Chinese Academy of Medical Science (Bei⁃jing, China). All mice were provided autoclavedpellets and water ad libitum. Mice were permit⁃ted 1 week to acclimate to their environment be⁃fore manipulation and used at 8 weeks of age inall experiments. All mice were housed under a12-h light/dark cycle in microisolater cages con⁃tained within a laminar flow system to maintain apathogen-free environment. All surgical proce⁃dures were completed in accordance with ChinaAcademic of Medical Science guidelines on thecare and use of laboratory animals for researchpurposes. Mice were anesthetized with 10%Chloral Hydrate for all surgical procedures. Usingaseptic techniques, the right facial nerve of ani⁃mals in the facial nerve transection group was ex⁃posed at its exit from the stylomastoid foramenand completely transected. The proximal and thedistal nerve stump were reset without suture. Be⁃havioral observations were used to assess recon⁃nection of the facial nerve(i.e., if the animal recov⁃ered from unilateral facial paralysis). For animalsin the sham operation group, the right facialnerve was exposed, but not transected. For bothgroups, the left side was untreated and served asan internal control in comparison to the right side.A separate group of untreated BABL/c mice wereincluded as blank control. Six animals from eachgroup were examined and tested at 3 (DPO3) and14 (DPO14) days post operation, respectively.2.2 Light microscopic immunohistochemistry stain⁃ing with CD32.2.1 Brain stem slice preparation

After the animals were killed with overdose

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10% Chloral Hydrate, they were first perfused in⁃tracardially(30 ml/min) with 200 ml of phosphatebuffer solution (PBS) (10 mM Na2HPO4 , 0.84%NaCl, pH 7.4), followed by 200 ml of 4% for⁃ma-ldehyde (FA) in PBS (4% FA/PBS), and thebrain stem was removed and post-fixed by 2 hrimmersion in 1% FA/PBS at 4° C on a rotator (8rpm). The tissue was cryoprotected by an over⁃night rotating immersion in sucrose (30% su⁃crose, 10 mM Na2HPO4, pH 7.4, 4°C), frozen ondry ice, and then cut in a cryostat at the level ofthe facial motor nucleus. Sections(30 μm) werecollected on warm, 0.5% gelatin-dipped slides.2.2.2 Immunohistochemistry staining with CD3

Tissue sections were incubated with the prima⁃ry anti-mouse CD3 antibodies(PharMingen) over⁃night, then with biotinylated goat anti-rat second⁃ary antibody and avidin-biotin peroxidase com⁃plex (ABC, Vector), completed with visualizationwith diaminobenzidine/H2O2(DAB, Sigma). Sec⁃tions were washed in PBS following each incuba⁃tion step.2.2.3 Light microscope examination of facial nu⁃cleus

Examination of the facial nerve nucleus wascompleted at the central lab of Peking Union Med⁃ical College Hospital with a Nikon microscope(Eclipse 80i). Sections of facial nucleus were ex⁃amined for distribution and recruitment of T cellswith positive CD3 staining, identified in Buffy col⁃or under the microscope.2.3 Double-color immunofluorescent staining withantibodies and flow cytometry

Anti-CD3 mAb (monoclonal antibodies) -FITC,anti-CD69 mAb-PE and isotype-specific controlantibodies were obtained from eBioscience (SanDiego, CA) Lymphocytes harvesting and fluores⁃cence antibody staining have been described pre⁃viously in detail [11, 12]. In brief, the lymphocytes sep⁃arated from CDLNs and MLNs at DPO3 andDPO14 were suspended in RPMI1640 with 10%fetal bovine serum, with the concentration adjust⁃

ed to 109 cell/L. After counting survival percent⁃age with trypan blue, the suspension waswashed twice with PBS and condensed to 50 μL.The suspension was incubated for 30 min in thedark at room temperature with the appropriate di⁃lution of anti-CD3 mAb-FITC and anti-CD69mAb-PE, washed twice with cold PBS, and final⁃ly fixed with 4% paraformaldehyde to ready fortesting. Drops containing targeting cells fromCDLNs were examined and photographed underfluorescence microscope with double-color im⁃munofluorescent staining. A EPICS XL flow cy⁃tometer (Beckman-Coulter) was used for flow cy⁃tometry analysis. Three samples were collectedand tested on each set test day. Data are present⁃ed as mean±s, and differences between groupswere compared using t-test. P value <0.05 is setas statistically significant.

3 Results

None of the animals in the present studyshowed signs of recovering from unilateral facialparalysis after facial nerve transection.3.1 CD3 in facial nucleus

CD3 is one of the specific cell surface markerson T cells. Its expression on T cells in the facialnucleus in this study is depicted in Figure 1 aspositive staining under the microscope.3.2 Immunofluorescent staining of T cells fromCDLNs in mice with facial nerve transection

Expression of T cell surface marker CD69 wasvisible under oil immersion lens followingCD3-FITC, CD69-PE double-color immunofluo⁃rescent staining, indicating T cell activation (Fig2).3.3 Expression of CD69 on T cells three days afteroperation.

CD69 antigen was minimally or not expressedon CD3+ T cells from lymph nodes in blank con⁃trol group mice in the state of quiescence. Threedays after the operation, CD69 expression wasseen on CD3 + T cells from CDLNs in mice that

R

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had undergone facial nerve transection orsham-operation (Fig. 3A and B), indicating T cellactivation. This was significantly different com⁃pared with T cells from MLNs (P=0.0457 and0.0226, respectively). CD69 expression was posi⁃tive on 0.81 ± 0.21% of T cells from CDLNs inmice with facial nerve transection, compared to0.52 ± 0.16% in those in the sham-operationgroup (P=0.2759). CD69 expression rates in Tcells from MLNs were similar in the nerve tran⁃section and sham-operation groups (P=0.4700)(Fig. 3C and 3D).

3.4 Expression of CD69 on T cells 14 days after op⁃eration.

Two weeks after facial nerve transection,CD69 expression on T cells from CDLNs re⁃mained positive (0.62±0.10) % and not significant⁃ly different from 3 days after operation (P=0.2294), indicating continuous T cell activation.However, CD69 expression on CDLN T cells insham-operation mice had dropped to normal lev⁃els at 0.14 ± 0.01% (Fig. 3F), lower than that atDPO3 (P=0.0212). A significant difference in ex⁃pression of CD69 on T cells from CDLNs wasseen between mice with transected facial nerveand those in the sham-operation group(P=0.0007). At the same time, CD69 expression on Tcells from MLNs increased in mice with facialnerve transection to 0.49±0.05% (Fig. 3G), higherthan sham-operation mice and than at DPO3(P=0.0082 and P=0.0133, respectively) (Fig. 3H).

4 Dｉｓｃｕｓｓｉｏｎ

At present, Neuritis and demyelization are gen⁃erally acknowledged as common pathophysiologi⁃cal foundation of peripheral facial paralysis (Bell’spalsy, traumatic, infectious and others) by schol⁃ars home and abroad[4,13 ]. Cellular immune re⁃sponse and humoral immune response play a keyrole in the outbreak and development of peripher⁃

Figure 1 CD3 expression in facial nucleus. A: Lack of CD3 expression in facial nucleus on the left (non-operative)side with a clean background. DAB×400. B: CD3 expression in facial nucleus on the right (operative) side 3 dayspost operation in the same mouse. Positive CD3 staining is visible against a clean background, representing T cellinfiltration around facial motoneurons. DAB×400）

Figure 2 Immunofluorescent staining of T cells fromCDLNs in a mouse 14 days after facial nerve transec⁃tion. Green-CD3-FITC staining; red-CD69-PEstain⁃ing. Double-color CD3-FITC and CD69-PE immunoflu⁃orescent staining is seen in orange color (CD3+CD69+cells), indicating activated T cells (×1000).

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al facial paralysis[14,15]. However, precise cellularand molecular immune mechanisms of peripheralfacial paralysis have not been discovered so far,leading to a weak foundation of theories to thecontemporary treatment. With currently availabletreatments (conservative medical treatments, sur⁃geries, and complimentary treatments includingtraditional Chinese herbal medicines and acupunc⁃ture and moxibustion), a significant number of pa⁃tients are left with such sequelae as facial paraly⁃sis and permanent facial deformity, causing greatagony and psychological burden to them [16]. It canbe inferred from our previous research that therepresentative immune cell activation and recruit⁃

ment of T cell and monocyte/macrophage may bethe cellular immunity foundations of peripheral fa⁃cial nerve demyelization and facial motoneurondeath[17, 18]. The pathophysiological meanings of Tcell activation and CD69 expression in a mousemodel of traumatic peripheral facial paralysiswere probed here with fluorescence-activatedcell sorting.

T cell activation is a result of complicated ac⁃tion between multiple ligand-receptors, whichare offered by T cells and antigen-presentingcells (APC) including the formation of immunologi⁃cal synapse(IS)[19,20]. The interactive integration trig⁃gers biochemistry events in T cells that lead to

Figure 3 Expression of CD69 on T cells at DPO3 (A-D) and, DPO14(E-H). A, B, E and F: T cells from CDLNs inthe facial nerve transection (A and E) and sham-operation (B and F) groups, respectively. C, D, G and H: T cellsfrom MLNs in the facial nerve transection (C and G) and sham-operation (D and H) groups, respectively.

Group

Facial nerve transection

Sham-operation

P2

DPO3

CDLN

0.81 ± 0.21

0.52 ± 0.12

0.2759

MLN

0.18 ± 0.00

0.17 ± 0.08

0.4700

P1

0.0457

0.0226

DPO14

CDLN

0.62 ± 0.10

0.14 ± 0.01

0.0007**

MLN

0.49 ± 0.05

0.10 ± 0.00

0.0082**

P1

0.1397

0.0386

Tab 1 Expression of CD69 on T cells ( x± s，%)

NOTE: (1) P1, CDLN vs MLN; P2, Facial nerve transected group vs Sham-operation group（2）*P <0.05, (**)P <0.01

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cellular immune response. Such effects as accel⁃eration or inhibition on activation, proliferation, dif⁃ferentiation and effector action are carried outthrough cytokines secretion and cell-cell contact.T cell activation plays a key role either in normalor in pathological immune response. The degreeand direction of immune response are deter⁃mined by the upshot of T cell activation.

Three days post the operation, CD69 expres⁃sion ascended in CD3+ T cells from CDLN in ani⁃mals with facial nerves transection and sham-op⁃eration(Fig. 3 A and B) compared to T cells fromMLNs, which reflected the systemic immune sta⁃tus, as in a“far site” local immune response.This suggest that the organism starts or increas⁃es local cellular immune response in response toa combined acute stage effect by trauma andnerve injury with activated T cells, although thesystemic immune response is yet to start. Thesimilarly high levels of CD69 expression in CD3+T cells from the CDLN two weeks after facialnerve transection suggests continuous T cell acti⁃vation. At this time, CD69 expression on CDLN Tcells in animals with sham-operation haddropped to a normal level, similar to that at 3days post operation, suggesting that the organ⁃ism had recovered from unsophisticated trauma⁃tism (no-nerve-transected) with the normal levelimmunity response. Neural traumatism effect in⁃cludes antigen exposure, continuous repair andgeneration based on low-level autoimmune re⁃sponse. A moderate upregulation of immune reg⁃ulation network is needed to exert the function ofimmune elimination and immune surveillance.The status with appropriate T cell activation is aprimary step to triggering the adjustment and con⁃trol of the network.

The results of light microscopic immunohisto⁃chemistry and immunofluorescent staining indi⁃cate distribution and recruitment of T cells in thefacial nucleus (Fig 1-2), confirming that, mediat⁃ed by T cell’s antigen receptor, CD4+ T cells are

activated by distinguishing peptides including au⁃toantigens such as myelin basic protein (MBP)and proteolipid proteins (PLP), presented by anti⁃gen presenting cells (APC) such as stelliform glialcells, glial cell (GC) and Schwann cells. Then, acti⁃vated CD4+ T cells overshoot blood brain barrier(BBB) and infiltrate facial nucleus [21]. Activated Tcells, small GCs and stelliform GCs produce vari⁃ous kinds of chemokine, cytokines and adhesivemolecules which recruit more T cells and mono⁃cytes / macrophages around the peripheral injuryarea and facial nucleus. Two effects are broughtabout: first, clearing and phagocytization ofnecr-otic metamorphic motorneurons, myelinsheath and axon, etc; second, secondary allergyand experimental autoimmune encephalomyeli⁃tis(EAE) induced by conglomeration of inflamma⁃tory cells dominated by T cells and macrophages[22, 23]. There is a dynamic course for immune regu⁃lation in the immune system.

CD69 expression upregulation in T cells fromMLNs is probably through the mechanism of sys⁃temic immune regulation networks started by lo⁃cal immune response. Two weeks after nerve in⁃jury, the upregulation of CD69 expression in Tcells of lymph nodes is simply the result of thesurveillance mechanisms of the immune regula⁃tion network, which is started by the key processimmune response with appropriate T cell activa⁃tion. Abnormal T cell activation, too high or toolow, means nerve pathological emergence for theorganism(autoimmunity / immune deficiency) [24].

The restorative effect in early stage of facialnerve injury (in two weeks) determines the resultof final nerve repair. The change of micro-envi⁃ronmental in damaged nerve cells and axons, es⁃pecially the expressions of various kinds of im⁃mune cells and molecules together with the dy⁃namic equilibrium of their regulating networks, isthe key incidents of nerve repair and regenera⁃tion. They impenetrate from the start to the endof rehabilitation and regeneration of nerve’s inju⁃

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ry. The conception of ‘immune microenviron⁃ment for repair and regeneration of facial nerve’,as we have previously introduced [17,18, 25], mainlyconcerns with the change of expression and regu⁃lation network with various immune cells and im⁃mune molecules in the process of facial nerverenovation. It is a dynamic equilibrium for its main⁃tenance of the normal immune microenviron⁃ment. It is the activated T cells that target to re⁃cruiting such immune activated cells as mono⁃cytes/macrophages, such cytokines as neuro⁃trophic factors, which regulate Schwann cells pro⁃liferation and continuous axon regeneration [26].Studies have showed that specific T cell sub⁃groups have a relationship with the survival of fa⁃cial motorneurons[18, 27]. Many questions remain:what are the roles of dialogue mechanisms of Tcell-axon / neuron-Schwann cells / glial cell?Which factors, and through which concrete signalpathways, play a key role for T cells and mono⁃cytes/macrophages system activation and recruit⁃ment? What are the roles of real molecular mech⁃anisms of the action by specific T cells on facialmotor neurons survival? We believe that they willbe well explained with the progress of systemat⁃ic studies on immune microenvironment for re⁃pair and regeneration of facial nerve.

Acknowledgements

This work was supported by grants from theNational“Tenth-Five”Scientific & TechnologicalProblem-Tackling Project (№:2004BA720A18-01) and Social Welfare Founda⁃tion of Scientific Research Institutes of ChineseMinistry of Science & Technology (№:2002DB40097). We are grateful to LiYumei andZhang Biao for their consummate flow cytometrytechnique support.

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（Received December 23, 2010）

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assessment of t cell activation in a mouse model of ... · including nerve injury[1, 2]. interest...

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