Immunoexpression of the CD30 ligand/CD30 and IL … · immunohistochemistry the expression of two...

Summary. To elucidate the role of Th2 cytokines inautoimmune thyroid diseases, we have studied byimmunohistochemistry the expression of two Th2ligand/receptor systems (CD30-L/CD30 and IL-6/IL-6R)in goitrous Graves’ disease (GD) and Hashimoto’sthyroiditis (HT).

A total number of 50 nodular goiters (NG), including10 GD showing a lymphoid aggregate grade I, 30 HT 8of which had a lymphoid aggregate of grade I, 12 ofgrade II and 10 grade III, and 10 colloid goiters havebeen evaluated. In addition, 5 normal thyroids wereincluded in the study as controls.

Reactivity of ligand and cognate receptor of bothCD30-L/CD30 and IL-6/IL-6R pathways was observedin a greater proportion of GD, compared to HT(P<0.005). In HT, the expression of CD30-L/CD30system was detected more frequently than IL-6/IL-6R(P<0.05) and showed an inverse correlation with thegrade of lymphoid aggregate, whereas IL-6/IL-6Rcorrelated positively with lymphocyte infiltration(P<0.05).

Based on our results concerning a dominance of Th2cytokines in GD, we postulate that CD30-L/CD30 andIL-6/IL-6R systems could play a major role in thepathogenesis of GD. However, the expression ofCD30L/CD30 and IL-6/IL-6R in HT suggests that Th2mechanisms are involved also in tissue damage of HT.The two systems could contribute to drive theautoimmune response skewing toward a Th2 phenotypeand this appears to be correlated with the lymphoidaggregate grade. Key words: CD30 ligand, IL-6, CD30, IL-6 receptor,Autoimmunity, Thyroid, Immunohistochemistry

Introduction

The CD30 ligand (CD30-L) and the cognate receptorCD30 belong to the tumor necrosis factor (TNF)/nervegrowth factor (NGF) ligand and receptor superfamilies,respectively (Durkop et al., 1992; Falini et al., 1992;1995; Smith et al., 1993). The interleukin 6 (IL-6) is apleiotropic cytokine; its receptor (IL-6R) includes an 80-kD IL-6-binding subunit called IL-6Rα and a gp130/IL-6ß subunit which is responsible for the signaltransduction and stabilization of the α-chain ligandcomplex (Hibi et al., 1990; Hirano, 1998). Both CD30-Land IL-6 are able to regulate cellular growth (Gruss etal., 1994; Blood, 1994; Yokomuro et al., 2000) uponinteraction with the corresponding receptor (Kishimotoet al., 1992; Smith et al., 1993) and their effects arecorrelated to the expression levels (Jones, 1994; Gruss etal., 1996). In addition, they are synthesized by Th2 cells,which play a pivotal role in the autoimmunecompartment (Hirano et al., 1998; Croft, 2003).

In thyroid tissue, we have previously reported thatthe expression of CD30-L/CD30 and IL-6/IL-6R isdifferent in the benign and malignant nodules,suggesting a different role of the two signaling systemsin benign and malignant cellular proliferation (Trovato etal., 2001, 2003; Ruggeri et al., 2002). However, the roleof these two systems in autoimmune thyroid diseases(AITD) has not been fully elucidated yet. IL-6expression has been previously evaluated in Graves’disease (GD) as well as Hashimoto thyroditis (HT)(Grubeck-Loebenstein et al., 1989; Zheng et al., 1991;Paschke et al., 1994; Watson et al., 1994; Kayser et al.,1995; Ajjan et al., 1996), but no data are currentlyavailable regarding the expression of these signals inAIDT associated with nodular goiters (NG). SinceCD30-L/CD30 and IL-6/IL-6R are involved inmechanisms of both cell proliferation and autoimmunity,we wished to evaluate the expression of these fourmolecules in goitrous GD and HT, compared to NG notassociated with AITD.

Immunoexpression of the CD30 ligand/CD30 and IL-6/IL-6R signals in thyroid autoimmune diseasesR.M. Ruggeri2, G. Barresi1, S. Sciacchitano3,4, F. Trimarchi2, S. Benvenga2,5 and M. Trovato11Department of Pathology, University of Messina, Italy, 2Section of Endocrinology, Clinical/Experimental Department of Medicine andPharmacology, University of Messina, Italy, 3S. Pietro Fatebenefratelli Hospital - AFaR, Rome, Italy, 4Department of Experimental Medicine and Pathology, University of Rome “La Sapienza”, Italy and 5Molecular Endocrinology Infradipartimental Project, AOU Policlinico “G. Martino”, Messina, Italy

Histol Histopathol (2006) 21: 249-256

Offprint requests to: Maria Trovato, MD, PhD, Dipartimento di PatologiaUmana, Policlinico Universitario G. Martino Padiglione D, 98125Messina, Italy. e-mail: [email protected]

DOI: 10.14670/HH-21.249

http://www.hh.um.es

Histology andHistopathologyCellular and Molecular Biology

Materials and methods

Tissue collection

Thyroid tissue specimens were retrieved from thearchives of the Department of Human Pathology,University of Messina, Italy. They included 5 normalthyroids harvested during autopsy and 50 thyroidsurgical samples taken from 50 patients who hadundergone thyroidectomy for large NG. The 50 patientswere recruited at the Endocrinology Unit of theUniversity of Messina, and included: 10 patients withNG associated with GD (6 females and 4 males; themean age ± SD at the time of thyroidectomy was43±12); 30 patients with NG associated with HT (25females and 5 males; mean age ± SD: 52±8); 10 patientswith NG and no clinical, laboratory or ultrasonographicevidence of AITD (9 females and 1 male; mean age ±SD: 55±13). All patients were euthyroid at the time ofthe thyroidectomy. The GD patients had been renderedeuthyroid with antithyroid drugs (thiamazole) prior tosurgery by a 6-12 months treatment course. The GD orHT lesions were classified at the histological diagnosisaccording to the criteria proposed by Doniach & Roittand Li Volsi (Doniach et al., 1976; Li Volsi, 1990). TheGD and HT lesions were studied paired with theassociated nodules.

Thyroid tissues were fixed in 4% formalin androutinely processed through graded alcohol and xyleneto paraffin wax. Haematoxylin-eosin (H&E) stainedsections of each specimen were performed prior toimmunohistochemistry. In each H&E section the intra-glandular inflammatory lymphoid aggregates have beenevaluated. A lymphoid aggregate was defined asincluding, at least, 150 lymphocytes and a variablenumber of plasma cells per high-power field. When thistype of lymphoid infiltration was arranged into well-developed follicular centers with central macrophage-like cells showing a large, clear cytoplasmic appearance,it was identified as a lymphoid aggregate with germinalcenter. The lymphoid aggregates were graded as follows:0 = no lymphoid aggregate or at most one single, smalllymphoid aggregate without germinal center in eachsection; I = occasional, usually small lymphoidaggregates with rare or absent germinal centers in each section; II = several, usually mixed, small and large lymphoid aggregates with some germinal center in each section; III = numerous, large lymphoid aggregates with frequent germinal centers in each section. Immunohistochemistry

Serial sections of the selected blocks were cut atfive-micrometers for the immunohistochemical studies.Immunohistochemistry was performed, separately, bymouse monoclonal antibodies (MAb) raised againsthuman CD30-L (h-CD30L-Fc type II, 1:100, Genzyme,Cambridge, MA, USA) and CD30 (or Ki-1 antigen)

(clone Ber-H2, 1:100, Dako, Carpinteria, CA, USA),respectively, and by goat MAb raised against human IL-6 and IL-6-Rα, respectively, (1:100, Sigma, St. Louis,MO, USA, respectively), using the biotin-streptavidin-peroxidase method (LSAB kit from Dako Corporation,Carpinteria, CA).

Antigen retrieval technique as described by Gown etal. (1993) was carried out. Tissue sections weredeparaffinized in xylene and rehydrated in alcohol.Then, the endogenous biotin was inactivated by additionof 0.05% (v/v) solution of streptavidin in phosphate-buffered saline (PBS) and the endogenous peroxidaseactivity was quenched by adding 0.3% (v/v) solution of3% H2O2/methanol for 30 min. The slides, placed in 10mM citrate buffer adjusted to pH 6.0 with 2 M sodiumhydroxide, were microwaved for 15 min (WhirlpoolAVM 300, power set at 500 watts). Microwave exposurewas broken into three equal time periods and, at the endof the first cycle, 50 ml of distilled water was added tothe slide holder to prevent loss of fluid from boiling.Staining was obtained with the LSAB system (kit fromDako). 3,3’-diaminobenzidine (DAB, Sigma) activatedwith 0.05% hydrogen peroxide was used to develop theend reactions. Sections were counterstained withMayer’s haematoxylin, dehydrated and mounted.Specificity of the binding was assessed either byomitting the primary antiserum or by replacing theprimary antiserum with normal mouse or goat serum. Ineach of these conditions, no staining was evident. Inaddition, an immunoabsorption test was performed toconfirm the specific immunoreactivity of each MAb.Specimens of Hodgkin lymphoma were used as positivecontrols for the CD30-L and CD30 immunoreaction,while specimens of colic mucosa showing inflammatorybowel disease were used as positive controls for the IL-6and IL-6R immunoreaction. For the evaluation of theresults, the following criteria were used: (i) number ofpositive cases; (ii) number of reactive epithelial, stromaland lymphoid cells per case: the count of the number ofreactive cells was based on evaluation of 1000cells/case, using 50x magnification; (iii) sub-cellularlocation of the staining: cytoplasm and/or membrane.Based on the proportion of stained cells, we scored thereaction value as low (1-10% stained cells), moderate(11-30%) and high (>31%).

Histological and immunohistochemical evaluationswere done twice and blindly by two differentpathologists (M.T., G.B.) with an inter-observerconcordance of nearly 100%. Where minimal inter-observer discrepancies were present, the mean value wasconsidered as the result.Statistical analysis

Differences between proportions were analyzed byχ2 test with Yates’ correction for continuity. Theassociation between two variables was analyzed by thelinear regression analysis. The level of statisticalsignificance was set at P<0.05.

250CD30 ligand / CD30 and IL-6 / IL-6R in thyroid autoimmune diseases

Table 2. Lymphoid aggregate grade and expression of the CD30-L/CD30 and IL-6/IL-6R systems in epithelial cells of Hashimoto’s thyroditis and colloidnodules.

Lymphoid aggregate grade and expression of the CD30-L/CD30 and IL-6/IL-6R systems in epithelial cells of Hashimoto’s thyroditis and colloid nodules.*: The proportion of positive cells was calculated based on evaluation of 1000 epithelial cells using 50x magnification. Semiquantitative grading ofimmunostained cells distribution was scored as low (1-10% stained cells), moderate (11-30%) and high (>31%), as specified in Materials and Methods.

Results

Histopathology

All 50 NG showed histological features of colloidnodules (CN). Most of them were characterized by largecolloid filled follicles, built of small flat or cuboidalfollicular cells with dark nucleus and eosinophiliccytoplasm, even associated with multicellularmicrofollicles. Others were collected in solid budsshowing few tiny lumina or, apparently dropping off theepithelial layer of cystically dilated follicles, resulted ina papilliferous structure called “Polster di Sanderson”. In

all 50 CN no intranodular lymphoid aggregates wereobserved (grade 0) (Tables 1 and 2).

The histomorphological hallmark of GD was amarked hyperplasia with well-developed papillaeassociated with a multifocal lymphocytic thyroditis. Thepapilla, without fibrous stromal axis, were lined byfollicular cells with a basally located dark nucleus andcolumnar or cuboidal/tall cytoplasm. All 10 GD showedlymphoid aggregates of grade I (Table 1).

The HT showed small follicles with scarce, dense,pink colloid, delimitated by cuboidal follicular cells withdark nucleus and eosinophilic cytoplasm. In most cases,oncocytic metaplasia was evident and consisted of


Table 1. Lymphoid aggregate grade and expression of the CD30-L/CD30 and IL-6/IL-6-R systems in epithelial cells* of normal thyroids, colloid nodulesand Graves’ disease.

Lymphoid aggregate grade and expression of the CD30-L/CD30 and IL-6/IL-6R systems in epithelial cells of normal thyroids, colloid nodules andGraves’ disease. *: The proportion of positive cells was calculated based on evaluation of 1000 epithelial cells using 50x magnification.Semiquantitative grading of immunostained cells distribution was scored as low (1-10% stained cells), moderate (11-30%) and high (>31%) as specifiedin Materials and Methods.

CD30-L/CD30 SIGNAL IL-6/IL-6-R SIGNALLigand Receptor Ligand Receptor

SPECIMENSLymphoidaggregate

gradePositivecases

Staining score Positivecases



Staining scoreLow Moderate High Low Moderate High Low Moderate High Low Moderate High

Normal thyroid (n = 5) 0 0/5 0 0 0 0/5 0 0 0 1/5 1 0 0 0/5 0 0 0

Colloid nodules (n = 10) 0 0/10 0 0 0 0/10 0 0 0 3/10 3 0 0 0/10 0 0 0

Grave's disease (n = 10) I 10/10 3 7 0 10/10 6 4 0 10/10 6 4 0 10/10 10 0 0Colloid nodules

associated with Grave'sdisease (n = 10)

0 0/10 0 0 0 0/10 0 0 0 3/10 3 0 0 0/10 0 0 0

CD30-L/CD30 SIGNAL IL-6/IL-6-R SIGNALLigand Receptor Ligand Receptor

SPECIMENSLymphoidaggregate

gradePositivecases




Staining scoreLow Moderate High Low Moderate High Low Moderate High Low Moderate High

Hashimoto’s thyroditis(n= 30)

12/30 3 6 3 12/30 7 5 0 30/30 13 11 6 4/30 4 0 0

I (n= 8) 6/8 1 3 2 6/8 3 3 0 8/8 4 3 1 0/8 0 0 0

II (n= 12) 4/12 1 2 1 4/12 3 1 0 12/12 7 3 2 2/12 2 0 0

III (n= 10) 2/10 1 1 0 2/10 1 1 0 10/10 2 5 3 2/10 2 0 0Colloid nodulesassociated with

Hashimoto’s thyroditis (n = 30)

0 0/30 0 0 0 0/30 0 0 0 10/30 10 0 0 0/30 0 0 0

follicular cells with oxyphil cytoplasm while,occasionally, clear metaplasia was seen and consisting offollicular cells with clear nucleus and eosinophiliccytoplasm. Lymphoid aggregates were graded as I (8/30or 27%), II (12/30 or 40%) or III (10/30 or 33%)(χ2=16.2, P<0.001 vs GD) (Table 2). Immunohistochemistry

No expression of CD30-L/CD30 could be detectedin any normal thyroids and CN (Tables 1 and 2). IL-6expression was observed in 20% of the normal thyroidsand in 30% of the CN, but all cases were IL-6R,negative, confirming our previous data (Ruggeri et al.,2002). In both normal thyroids and in CN, IL-6+immunoreaction was always low and it was detectedonly in the cytoplasm of epithelial cells. No differencesin the expression of the two signals were observedbetween CN associated or not associated to AIDT.

All GD expressed the CD30-L/CD30/IL-6/IL-6R(Table 1), while the contemporary expression of thesefour proteins was seen in only 4/30 (13%) of the HT(χ2=21.1, P<0.001 vs GD) (Table 2).

When comparison between GD and HT is limited tothe grade I lymphoid infiltration (because so were allGD), then the proportion of CD30-L+/CD30+/IL-6+/IL-6R+ cases was 100% in GD and 0% in HT (χ2=14.2,P<0.001). The simultaneous expression of the twoligand/receptor systems was observed in HT with agreater extent of lymphoid infiltration, and precisely in17% of HT cases with grade II and in 20% with grade IIIinfiltration.

As mentioned above, in GD, the immunoexpressionof each ligand was invariably associated with therespective receptor. In contrast, only 40% of the HT co-expressed CD30-L and CD30 (χ2=8.62, P=0.003 vs GD)and even fewer (13%) co-expressed IL-6 and IL-6R(χ2=21.09, P<0.001 vs GD). In HT, the 40% rate wasgreater than the 13% rate. The four HT expressing IL-6and IL-6R were invariably CD30-L+/CD30+.Interestingly, in HT the extent of lymphoid infiltrate wasrelated inversely with the proportion of CD30-L+/CD30+cases but directly with the proportion of IL-6+/IL-6R+(Fig. 1).

In both GD and HT lesions, CD30-L+, CD30+, IL-6+or IL-6R+ immunoreaction was similar and concernedprimarily the epithelial cells. In particular, CD30-L+immunoreaction was moderate, CD30+ was low as wellas IL-6+ and IL-6R+ (Tables 1 and 2). In the reactive GDand HT epithelial cells, CD30-L+ and CD30+immunostaining was detected in the membrane andcytoplasm (Panel A and B of Figures 2 and 3,respectively) and IL-6+ and IL-6R+ immunostaining wasconfined to the cytoplasm (Panel C and D of Figures 2and 3, respectively).

In all GD and HT showing CD30-L+/CD30+/IL-6+/IL-6R+ immunostaining, low immunoreaction wasobserved for CD30, IL-6 and IL-6R in the cytoplasm ofstromal cells (data not shown). In HT with a lymphoid

aggregate grade III, low immunoreaction was recognizedfor IL-6 in the cytoplasm of lymphocytes and for CD30-L and CD30 in the cytoplasm of lymphoid elements withlarge-clear cytoplasm (macrophages-like cells) includedin germinal centers (Panel A and B of Figure 3). Discussion

In the present study, we report that the members oftwo signaling pathways, namely CD30-L/CD30 and IL-6/IL-6R are expressed in GD and HT lesions, suggestinga role of these pathways in these two diseases. On thecontrary, CN arising in the context of AIDT don’texpress CD30-L, CD30 and IL-6R and this pattern isalso visible in CN not associated with AIDT. These dataindicate that CD30-L/CD30 and IL-6/IL-6R signalingpathways are not involved in the growth of CNs, evenwhen CNs are associated with AIDT.

Rather, the expression of these signals in AIDTappears to be related with the mechanisms of the


Fig. 1. Hashimoto’s thyroiditis. Panel A. Inverse correlation between thegrade of lymphoid aggregate and CD30-L/CD30 expression. Data arefrom the Table 1. Panel B. positive correlation between the grade oflymphoid aggregate and IL-6/IL-6R expression. Data are from the Table1.

autoimmune response. It should be considered that, evenif both CD30-L and IL-6 belong to Th2 cytokines, theyproduce different effects on autoimmune processes.CD30-L/CD30 signal protects the organs againstautoimmunity through the regulation of the balanceTh1/Th2 response. This system induces a positiveregulation of T cells by expansion of the Th2 cell subsetand suppression of Th1 response (Croft, 2003). Instead,the deregulated production of IL-6 and its receptor is

implicated in the pathogenesis of autoimmune diseasesby inhibition of autoreactive T-cell apoptosis (Kallen,2002).

The difference in the relative amounts of Th1/Th2cytokines triggers the onset of GD or HT. In particular,GD is induced by the TSH receptor stimulatingautoantibodies, whose production is likely to depend onTh2 cells function (Burman et al., 1985; Weetman,2003). Th1 cytokines, on the other hand, facilitate cell-


Fig. 2. Graves’ disease. Immunoreactive CD30-L (A) and CD30 (B) were detected in tissues from Graves’ disease. Representative positive cases areshown: positive thyrocytes appear brown. The CD30-L and CD30 immunostaining is located on the membrane and cytoplasm of follicular cells (arrow).Note the absence of CD30-L and CD30 immunoreactions in adjacent CN. IL-6 (C) and IL-6R (D) immunoreactions were detected in epithelial cells(brown deposits in cytoplasm) from GD lesions. The IL-6 and IL-6R reactivity is located in the cytoplasm of epithelial cells (arrow). A, x 130; B, x 300; C,x 150; D, x 250

mediated immunity and delayed-type hypersensitivityresponse in destructive processes of HT (Phenekos et al.,2004). More recently, Harii et al. (2005) reported thatTLR-3 protein (a Toll-Like Receptor involved in innateimmune responses) is overexpressed in HT, but not inGD, and TLR-3 downstream signals “may be importantin the pathogenesis of Hashimoto’s thyroiditis and in theimmune cells infiltrates”. TLR-3 preferentially actsthrough INF regulatory factor (IRF)-3 and causes thesynthesis and release of type I IFNs (Th1-relatedcytokines), but it also signals through nuclear factor kB(NK-kB), that involves MAPK and produces variouscytokines (e.g. TNF-alfa and IL-6, Th2-relatedcytokines). So, Th2 response seems to play a role also inthe pathophysiology of HT, in line with previous datafrom Chiovato et al showing that anti-microsomal

autoantibodies are able to induce damage of follicularcells (Chiovato et al., 1993).

Our study indicates that CD30-L, CD30, IL-6 andIL-6R are expressed in AIDT, but the expression of thetwo signaling systems differs in GD and HT. Further, inHT we have observed that the lymphoid aggregate gradeis correlated inversely with the expression of CD30-L/CD30 and positively with IL-6/IL-6R.

The expression of CD30-L/CD30/IL-6/IL-6R occursin a greater proportion of GD with respect to HT. Thisfinding is consistent with the specific characteristic ofGD to be prominently a Th2 autoimmune disease whileHT is referred to as a Th1 disease. In line with otherstudies (Chiovato et al., 1993; Okumura et al., 1997), theexpression of the two Th2-related systems observed inHT suggests that not only Th1 but also Th2 responses


Fig. 3. Hashimoto’s thyroiditis. A and B: CD30-L (A) and CD30 (B) immunoreactions in HT lesions. Representative positive cases are shown. TheCD30-L and CD30 immunostaining is located on the membrane and cytoplasm of follicular cells (arrow). Positive thyrocytes appear brown. Note aCD30-L and CD30 reactivity even in lymphoid aggregates defined as grade III (arrows). C and D: IL-6 (C) and IL-6R (D) immunoreactions in HTlesions. The IL-6 and IL-6R immunostaining is detected in the cytoplasm of follicular cells (brown deposits in cytoplasm) as indicated by the arrow. NoteIL-6 reactivity (indicated by arrows) in lymphoid aggregates grade III. A, B, x 130; C, x 150; D, x 250

could be involved in immunological mechanisms leadingto the disease. Moreover, the major expression of theCD30/CD30-L signal with respect to IL-6/IL-6R leadsus to suppose that it is the CD30-L/CD30 signal thatmay contribute to skewing of the immune responsetoward a Th2 phenotype in HT. This type ofconsideration is supported by the counterregulatoryactivity of CD30+ cells as part of a homeostatic responsethat attempts to control inflammation and tissue damagein Th1-driven diseases (Gerli et al., 2001).

All GD expressing CD30-L/CD30/IL-6/IL-6Rshowed a lymphoid aggregate grade I. The HT reactiveto CD30-L/CD30/IL-6/IL-6R had a lymphoid aggregategrade II or III, while in HT with a lymphoid aggregategrade I the expression of IL-6R was absent. This findingleads us to hypothesize that the lack of expression of IL-6R in HT with a lymphoid aggregate grade I could favora Th1 environment, while the co-expression of both IL-6and IL-6R in GD with lymphoid aggregate grade I mightcontribute to Th2 response.

The possible explanation of the striking difference inIL-6 and IL-6R in HT with respect to GD could begiven, in our opinion by the different lymphoidaggregate in the two AIDTs, based on the tenuous butsignificant correlation.

Another result of our study is the correlation of thetwo signalling systems expressions with the degree oflymphocytic infiltration. In fact, the CD30-L/CD30immunoreaction decreases in HT cases showing alymphoid aggregate grade II or III with respect to gradeI, while the IL-6/IL-6R signal is more frequentlyexpressed in HT cases with lymphoid aggregate grade IIor III. These data induce us to consider that in HT thelymphocytes infiltrate could down-regulate the epithelialexpression of CD30-L/CD30 and up-regulate that of IL-6/IL-6R.

In conclusion, we postulate that CD30-L/CD30 andIL-6/IL-6R systems could play a major role in thepathogenesis of GD. However, the two systems areexpressed also in HT, suggesting that Th2 mechanismsare involved in tissue damage of HT. The two systemscould contribute to drive the autoimmune responseskewing toward a Th2 phenotype and this appears to becorrelated with the lymphoid aggregate grade.

Recently, CD30 has been proposed as a target in theimmunotherapy of some malignancies (Falini et al.,1995; Schnell et al., 2002; Matthey et al., 2004).Moreover, anti-CD30L antibodies have been tested, ableto block the effects of CD30L/CD30 interaction (DelPrete et al., 1995). In AIDT, CD30 and CD30-L may bean unexplored therapeutic potential aimed at modulatingthe Th1/Th2 balance driving the immune system towardthe development of a protective anti-autoimmunityresponse.References

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Accepted October 26. 2005


Immunoexpression of the CD30 ligand/CD30 and IL … · immunohistochemistry the expression of two...

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