expression of major histocompatibility class ii antigens on polymorphonuclear neutrophils in...

Kidney International, Vol. 55 (1999), pp. 1811–1818

Expression of major histocompatibility class II antigenson polymorphonuclear neutrophils in patients withWegener’s granulomatosis

GERTRUD M. HANSCH, MARKUS RADSAK, CHRISTOF WAGNER, BETTINA REIS, ARMIN KOCH,ANDREAS BREITBART, and KONRAD ANDRASSY

Institut fur Immunologie, Abteilung Medizinische Biometrie, Medizinische Klinik und Poliklinik der Universitat Heidelberg,Heidelberg, Germany

Expression of major histocompatibility class II antigens on toms and prevents relapses [6]. Moreover, peripheralpolymorphonuclear neutrophils in patients with Wegener’s blood lymphocytes appear to be activated, as seen bygranulomatosis. increased expression of the interleukin 2 receptor

Background. Wegener’s granulomatosis is a systemic in-(CD25), b1 integrin (CD29), and other adhesion mole-flammatory disease of unknown etiology. Many studies suggestcules (ICAM-1 and LFA-3) [7–9]. Furthermore, an in-that autoimmune reactions are involved, and there is good evi-

dence for the participation of immunocompetent cells. In that creased proliferative response of patient-derived lym-context, we examined the activation of polymorphonuclear neu- phocytes to proteinase 3 (PR3) or to polymorphonucleartrophils (PMNs) of patients with Wegener’s granulomatosis. neutrophil (PMN) extracts, respectively, was described

Methods. In a prospective study, the expression on the sur-recently [10–12]. Also, PMNs appear to be activated [13]face of PMNs of CD64 and of the major histocompatibilityas indicated by enhanced surface expression of lysosomalclass II (MHC II) antigen was measured by cytofluorometry

in whole blood. The expression of those antigens was correlated enzymes, particularly PR3 and elastase [14, 15].to disease activity. In this prospective study, we analyzed PMNs of pa-

Results. Up to 15% of the peripheral PMNs of patients with tients suffering from Wegener’s granulomatosis with re-active disease expressed MHC II. Follow-up studies showed

spect to the expression of activation markers, includingthat expression correlated closely with disease activity and thatthe high-affinity receptor for IgG, CD64, and major his-it decreased rapidly under immunosuppressive therapy. Ex-

pression of CD64 was seen in approximately 50% of the pa- tocompatibility complex class II (MHC II) antigenstients, regardless of disease activity. (HLA-DP, HLA-DQ, HLA-DR). Currently, PMNs are

Conclusion. MHC II expression on PMNs might serve as a considered to be end-stage–differentiated cells unablenovel diagnostic marker for active disease and appears to be

to synthesize and express MHC II. Only one report de-suitable for monitoring immunotherapy. Moreover, our datascribes the induction on isolated human PMNs of MHCprovide evidence that PMNs, which are normally MHC II nega-II expression in response to interferon g or colony-stimu-tive, acquire MHC II antigens in the course of disease and

may be an unrecognized function within the afferent limb of lating factor (GM-CSF) [16]. In our study MHC II ex-the immune response. pression was found on the peripheral PMNs of patients

with active Wegener’s granulomatosis but not on PMNsof patients with inactive disease.

Wegener’s granulomatosis is a systemic disease of un-known etiology characterized by necrotizing granuloma,

METHODSvasculitis, and necrotizing glomerulonephritis [1–5]. Thereis strong evidence for there to be an autoimmune process Patientsinvolved, as immunosuppressive therapy with cyclophos- After having obtained informed consent, patients withphamide and corticosteroids efficiently relieves the symp- Wegener’s granulomatosis and other forms of vasculitis

attending the renal unit of the Heidelberg UniversityHospital were included in a prospective study analyzingKey words: vasculitis, MHC class II antigen, PMN, inflammation, nec-

rotizing granuloma, T-cell activation. the expression of MHC II and of CD64 on PMNs of theperipheral blood.Received for publication August 25, 1998

Wegener’s granulomatosis, microscopic polyangiitis,and in revised form December 18, 1998Accepted for publication December 21, 1998 and Churg–Strauss syndrome were diagnosed according

to the definition of the Chapel Hill conference [17] and 1999 by the International Society of Nephrology

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Hansch et al: Expression of MHC II antigens on PMNs1812

to the American College of Rheumatology criteria [18]. (Hamburg, Germany). For comparison, isotype-matchedmouse IgGs (Dianova) were used in comparable proteinDisease activity was determined with the “Birmingham

vasculitis activity score” (BVAS) [19]. In all patients, anti- concentrations. To avoid changes in surface expressioncaused by handling procedures, antibody labeling wasneutrophil cytoplasmic antibody (ANCA) titers, PR3

ANCA, and myeloperoxidase (MPO) ANCA were mea- performed in whole blood within two hours after with-drawal as follows: to 100 ml whole blood, 5 ml of thesured as reported elsewhere [20, 21]. Laboratory tests

were performed by routine methods (Hitachi autoana- respective antibodies were given. After incubation for30 minutes at room temperature, the erythrocytes werelyzer, blood cell counts by Coulter counter). Urine analy-

sis was done by test strips (Combur 8 kit; Boehringer lyzed by FACS lysing solution (Becton Dickinson,Mountain View, CA, USA) and the residual cells wereMannheim, Mannheim, Germany), Addis Count, and

measurement of 24-hour protein excretion were done centrifuged and resuspended in phosphate-buffered sa-line containing 1% bovine serum albumin (BSA) andwith Biuret, and urinary sediment was analyzed by phase

contrast microscopy. 0.1% sodium azide and 1% paraformaldehyde. Fluores-cence was measured by FACSCalibur (Becton Dickin-Patients with vasculitis and additional complications,

including acute infections, as detected by x-ray, blood son) and was analyzed by CellQuest. Results are ex-pressed as a percentage of gated cells in the respectivecultures, antiviral IgM titers, or measurement of procal-

citonin levels of more than 1 ng/ml [22], were excluded quadrant. CD64 expression on CD66b-positive PMNswas measured with the same method. A monoclonalfrom the study, as were patients with chronic inflamma-

tory disease (cholecystolithiasis, inflammatory bowel dis- antibody to CD64 was obtained from Dianova.ease, bronchitis, osteomyelitis), diabetes, or patients with

Determination of surface expression on T-cellskidney transplants.The first patients entered the study in September 1996, The CD3, CD4/CD8 ratio, and expression of CD25 as

and the study was closed in April 1998. A number of activation marker on T cells were measured by cytofluo-patients were seen repeatedly, usually in 8- to 10-week rometry using the respective antibodies in a Simultestintervals or when the disease became active again or kit (all obtained from Becton Dickinson).complications arose. According to the first clinical pre-

Statistical analysissentation, three groups of patients were formed: (a) pa-tients with active disease (BVAS of more than 1), (b) The results for MHC II and CD64 for the three groupspatients with inactive disease (BVAS 1 or 0) under im- of patients and for the healthy donors as the fourth groupmunosuppressive therapy, and (c) patients with inactive are presented as median with first and third quartiles.disease and no therapy. Nonparametric tests (Kruskall–Wallis test and Wilcoxon

Patients with systemic bacterial infections. Twenty- test) were used to compare the results. As pair-wise com-four patients with systemic bacterial infections were in- parisons were intended, a closure testing procedure wascluded in the study. Diagnosis was based on bacterial used to test first the global null hypothesis of no differ-culture, high plasma levels of C reactive protein, procal- ence between the four groups, followed by three groupcitonin levels higher then 1 ng/ml, and fever. comparison and finally the pair-wise comparison in case

Patients with rheumatoid arthritis. Twenty-four patients the previous null hypothesis could be rejected. P valueswith rheumatoid arthritis, diagnosed according to the have to be interpreted descriptively. All analyses wereAmerican Rheumatism Association criteria [23], were performed with SAS, version 6.12.also included in the study. The group was heterogenouswith regard to activity or severity of disease according to

RESULTSa house score. Most patients with active disease receivedPolymorphonuclear neutrophils of healthy donors ex-low-dose corticoids.

pressed little or no MHC II antigens (median 0.95%, inter-Healthy individuals. For comparison, PMNs of healthyquartile range 0.48 to 1.99%, MHC II-positive PMNs).individuals (N 5 54) of the same age and with the sameIn patients with active Wegener’s granulomatosis, thesex distribution as the patients of groups A and B wereexpression of MHC II was found on PMNs. In some pa-analyzed.tients, up to 15% of the PMNs were positive for MHC II

Determination of surface expression on (Fig. 1).polymorphonuclear neutrophils by cytofluorometry To assess whether MHC II expression on PMNs was

linked to disease activity, a prospective study with 58The respective surface molecules were detected bypatients with the diagnosis Wegener’s granulomatosisdouble labeling with fluorescein (FITC)-conjugated anti-was performed. When entering the study, 16 patients hadbody to CD66b as marker for PMNs and phycoerythrinactive disease (BVAS of more than 1). Forty-two pa-(PE)-conjugated antibodies to MHC II (anti-DP, -DQ,

-DR). The antibodies were purchased from Dianova tients with known disease were in remission either under

Hansch et al: Expression of MHC II antigens on PMNs 1813

Fig. 1. Expression of MHC II or of CD64 on PMNs of patients with Wegener’s granulomatosis. By cytofluorometry using a FITC-labeled antibodyto CD66b as marker for PMNs and a PE-labeled antibody to HLA-DR, a shift of the PMN population toward higher fluorescence intensity wasnoted with cells of a patients with active disease (upper right, B). PMNs of a healthy donor remain in the lower quadrant, indicating that PMNsare positive for CD66b, but not for MHC II (A). The lower panels show data for CD64 expression on PMNs. A FITC-labeled antibody to CD66bwas used together with a PE-labeled anti-CD64. A patient with active disease is shown in (D), and a patient with inactive disease is shown in (C ).

immunosuppressive therapy (N 5 21) or without therapy tients showed low expression of MHC II on PMNs. Theothers were negative (Fig. 2).(N 5 21). Of the 16 patients with active disease, 13

expressed MHC II on the peripheral PMNs (median All of the 16 patients who had active disease whenentering the study went into remission. Twelve remained6.5%; Fig. 2). Of the patients who entered the study

when under immunosuppressive therapy (BVAS 1 or 0), clinically inactive, but four developed a relapse, whichagain was associated with the expression of MHC IIonly 2 of 21 had MHC II-positive PMNs. Of the patients

with inactive disease and no therapy (N 5 21), five pa- antigens on PMNs (Table 1).

Hansch et al: Expression of MHC II antigens on PMNs1814

Fig. 2. Expression of MHC II antigens onPMNs. The data obtained with PMNs of pa-tients with Wegener’s granulomatosis, patientswith sepsis (N 5 24), and rheumatoid arthritis(RA; N 5 24), and of healthy donors (N 554) are summarized as box charts. Patientswith active disease when entering the study(N 5 16) showed higher MHC II expressionthan patients with inactive disease under im-munosuppressive therapy (N 5 21) or patientswith inactive disease and no therapy (N 5 21)or the patients with sepsis or RA, respectively.

Table 1. MHC class II expression and expression of CD64 on PMN of patients with Wegener’s disease

Disease activity when entering the study

Inactive disease and Inactive disease andActive disease immunosuppressive therapy no therapy

Total number of patients 16 21 21MHC class II positive PMN (,3% of PMN)a 13/16 2/21 5/21CD64 positive PMN (,10% of PMN)a 8/16 10/21 11/21Positive ANCA/PR3 titer (.1:20)a 11/16 11/21 9/21

Patients developing relapseTotal number 4 6 (7)b 2MHC class II positive PMN 4/4 5/6 (6/7)b 1/2CD64 positive PMN (,10%) 0/4 3/6 (4/7)b 1/2Rise in ANCA/PR titer (.1:20) 2/4 5/6 (5/7)b 0/2a Analyzing the data of the healthy donors, the third quartile of the level of MHC class II expression was used as the thresholdb Including the patient with two relapses

Within the observation time (20 months), a majority 21 with no therapy), eight suffered a relapse, which insix was associated with MHC II expression on PMNsof the patients came repeatedly to the hospital for a

routine check-up (6- to 8-week intervals) or when compli- (Table 1). One patient had two episodes of disease activ-ity, both occurring concomitantly with enhanced MHCcations arose. At each time, disease activity and MHC II

expression were determined, allowing correlation of II expression (Fig. 3).For statistical analysis, two situations were considered.these two parameters in the same patient.

The follow-up studies (in some patients up to 18 First, based on the clinical presentation when individualsentered the study, four groups were formed: patientsmonths) showed that MHC II expression remained low

or was unmeasurable when the disease remained inactive. with active disease (N 5 16), patients with inactive dis-ease and immunosuppressive therapy (N 5 21), and pa-Of the patients who had entered the study with inac-

tive disease (N 5 21 under immunosuppressive therapy; tients with inactive disease and no therapy (N 5 21),

Hansch et al: Expression of MHC II antigens on PMNs 1815

Fig. 3. A follow-up study of a patient withWegener’s granulomatosis. The patient en-tered the study with inactive disease. He de-veloped a relapse accompanied by a surfaceexpression of MHC II on PMNs and a rise inANCA titer. Under immunosuppressive ther-apy, MHC II expression declined, as did theANCA titer. In December 1997, the patientdiscontinued the immunosuppressive therapy.He developed a second relapse, again associ-ated with MHC II expression and a rise inANCA titer.

and healthy controls (N 5 54). Each patient was consid- PR3 antibody. Of the patients negative for MHC classII (N 5 5), two had a high autoantibody titer. One hadered only once, namely, when entering the study. The

data obtained during the follow-up studies were disre- only a low level of ANCA (1:40), and two had no autoan-tibodies at all. On the other hand, patients who nevergarded. Under these premises, MHC II expression was

statistically different in the four groups (P , 0.001) and had any ANCA were MHC II positive. In patients havingboth autoantibody and MHC II, the respective levels didalso in the three-group comparison (largest P value 0.01).

By pair-wise comparison, MHC II expression of the pa- not correlate with each other; moreover, disease activitymeasured as BVAS did not correlate with the autoanti-tients’ PMNs was different from MHC II expression of

the control group (largest P value 0.01), and MHC II body titer nor with the extent of MHC II expression.Under immunosuppressive therapy, the portion of theexpression of patients with active disease differed mark-

edly from that of both groups of patients with inactive MHC II-positive PMNs decreased within days. TheMHC II reduction paralleled the reduction of CD25 ex-disease (P , 0.01). There was, however, no difference

between patients with inactive disease and immunosup- pression on peripheral T cells and, as expected, precededthe decline of the ANCA titers (Fig. 4).pressive therapy and patients with inactive disease with-

out therapy and healthy controls (P 5 0.93). As a further parameter for leukocyte activation, theabsolute leukocyte count was considered. The medianIn the second analysis, whether or not a change in

disease activity coincided with a change in MHC II ex- values, 6.1 for patients with active disease, 6.7 for patientswith inactive disease and immunosuppressive therapy,pression in the same patient was calculated. In 14 of 19

events (74%, 95% CI, 0.49 to 0.91) the changes were and 5.95 for patients with inactive disease and no ther-apy, did not differ. Moreover, there was no correlationsimultaneous.

Eleven of the 16 patients (69%) who at the beginning between leukocyte count and MHC II expression (r 50.31 for the group of patients with active disease).of the study presented with active disease had ANCA

(titers ranging from 1:40 to 1:640) and autoantibodies to As another indicator of PMN activation, the expres-sion of CD64, the inducible high-affinity receptor forPR3 (ranging from 11.5 to 200), as had 7 of the 12 patients

with a relapse (75%; Table 1). MHC II expression, how- IgG (FcgRI) on PMNs, was measured in all patients aswell as in the healthy donors. PMNs of healthy donorsever, was not linked to the presence of ANCA or the

Hansch et al: Expression of MHC II antigens on PMNs1816

Fig. 4. A follow-up study of a patients withactive Wegener’ granulomatosis. The patiententered the study with active disease, MHCII on PMNs, ANCA titer of 1:640 and 78%of interleukin 2 receptor (CD25)-positiveT-cells. Under immunosuppressive therapy,the number of MHC II-positive cells declined,as did the ANCA titer and the percentage ofCD25-positive T cells.

either expressed no CD64 or very little CD64 on only alow percentage of PMNs. In approximately 50% of thepatients with active Wegener’s disease, a high expressionof CD64 was seen in up to 80% of the PMNs (Fig. 1).There was no difference between patients with activedisease and patients taking immunosuppressive therapyor patients with inactive disease and no therapy (Fig. 5).

The expression of CD64 was not linked to the expres-sion of MHC II. Of the patients with active disease andMHC II-positive PMNs (N 5 24), only 11 were alsopositive for CD64.

Major histocompatibility class II expression appearsnot to be restricted to Wegener’s disease, but was alsoseen in three of nine patients with active microscopicpolyangiitis. Also, PMNs of one patient with a newlydiagnosed Churg–Strauss syndrome showed a high ex-pression of MHC II (27.9% MHC II-positive PMNs),whereas two other patients with inactive Churg–Strausssyndrome had PMNs negative for MHC II.

To determine whether or not MHC II expression wasFig. 5. Expression of CD64 on PMN of patients with Wegener’s granu-also associated with other forms of inflammatory disease,lomatosis (WG). PMNs of normal donors did not express CD64, butPMNs of patients with various systemic bacterial infec-approximately 50% of patients with WG have CD64-positive PMN.

tions were tested for MHC II expression. Only 2 of 24 The median values obtained from patients with active disease or patientswith inactive disease were not different from each other but differedpatients had PMNs expressing a low amount of MHC IIfrom values obtained from healthy controls (P , 0.05).in 3 to 4% of the cells. The median value (1.17) was not

different from that seen for healthy controls (Fig. 2).As an example of chronic inflammatory disease, pa-

tients with rheumatoid arthritis were tested. Here, a me-our patients’ expression of CD25 as an indicator of T-celldian value of MHC II expression of 1.7% was deter-activation, as well as the ratio of CD4-positive to CD8-mined. None of these patients had a MHC II expressionpositive T cells. As already shown by others in almostabove 3% (Fig. 2).all patients, whether with active or inactive disease, theAn ongoing study with patients suffering from lupusproportion of CD8-positive cells was enhanced relativeerythematosus suggests that active disease is not corre-to CD4-positive cells [7]. Moreover, CD25-positive Tlated to MHC II expression on PMNs.cells ranging from 20 to 60% were found in all patients,Because recent publications point to the participation

of T cells in Wegener’s disease [7–9], we measured in again irrespective of disease activity (Table 2). In follow-

Hansch et al: Expression of MHC II antigens on PMNs 1817

Table 2. Analysis of peripheral T-cells of patients with lated PMNs of healthy donors [16]. Because activated TWegener’s granulomatosus

cells are found in vasculitic disease (unpublished obser-CD4/CD8 % CD3/CD25 vations) [7], it is feasible that T-cell–derived cytokines

ratio positive cells such as interferon g induce expression of MHC II onPatients with Wegener’s granulomatosis PMNs. The assumption is supported by the observation

Range 0.4–1.1 15%–68%that MHC II expression decreases rapidly after immuno-Median 0.86 28%

Healthy donors suppressive therapy in parallel with the decline of CD25-Range 1/9–2.4 0%–6% positive T cells. In that MHC II expression on PMNsMedian 2.02 2.3%

might be interpreted as an indicator of an ongoing im-mune response, the interpretation is also in line with thefinding that renewed increase of MHC II coincided withrelapse.up studies, a decrease in CD25-positive T cells was noted

In approximately 50% of the patients with active dis-in those undergoing immunosuppressive therapy (Fig. 4).ease, aside from MHC II, expression of CD64 was alsofound on PMNs. Expression, however, was not restricted

DISCUSSIONto patients with active disease but was seen in 53% of

Constitutive MHC II expression is restricted to profes- patients with inactive disease. Although not ruling outsional antigen-presenting cells, including dendritic cells, a role for the high-affinity Fc receptor in the course ofB cells, and cell of the monocyte/macrophage lineage. the disease, the expression of CD64 does not appear toPMNs of healthy individuals normally do not express be a suitable marker for the diagnosis of active disease orMHC II antigens. Expression, however, can be induced relapse. Apparently, the induction of CD64 expression isby culturing the isolated cells in the presence of g inter- regulated independently of MHC II.feron or granulocyte CSF [16]. We have now also found The consequences of MHC II expression on PMNsMHC II-positive PMNs in vivo. PMNs of the peripheral are still a matter of speculation. So far, the only knownblood of patients with Wegener’s granulomatosis ex- function of MHC II molecules is the presentation ofpressed MHC II antigens as detected by cytofluorometry. antigenic peptides to T lymphocytes. Whether or notMHC II expression was closely associated with active MHC II-positive PMN might fulfill such a function isdisease and declined rapidly under immunosuppressive under investigation. There are data showing that PMNtherapy. are able to function as accessory cells for staphylococcus

Thus, in addition to autoantibody titers [24–27], MHC enterotoxin-dependent T-cell proliferation [28], an ob-II expression on PMNs might be a useful marker for

servation that is particularly interesting with regards toactive disease, particularly for the recognition of a re-

a possible involvement of staphylococcus enterotoxinslapse. MHC II expression of PMNs coincided more fre-in the pathogenesis of vasculitis (abstract; Cohen Ter-quently with disease activity than the rise in PR3/ANCAvaert et al, J Am Soc Nephrol 8:533A, 1997) [29].titers, most probably because of the rapid up-regulation

In conclusion, (a) MHC II expression on PMNs mightof MHC II and the rapid turnover of PMNs.serve as a novel diagnostic marker for active vasculiticMajor histocompatibility class II expression is not up-disease and appears to be suitable for monitoring theregulated in patients with severe bacterial infection, sug-efficiency of immunotherapy. Extended long-term obser-gesting that MHC II expression is not imperativelyvations might tell whether MHC II expression precedeslinked to PMN activation as it occurs in infectious dis-relapse or progression of the disease [2]. (b) MHC II onease. Thus, MHC II expression of PMNs might be helpfulPMNs may result from T-cell activation occurring into discriminate between relapses and acute infection.active vasculitic disease, and points to an as yet unrecog-Furthermore, in patients suffering from rheumatoid ar-nized function of PMNs within the afferent limb of thethritis, no MHC II expression was seen, indicating thatimmune response.up-regulation of MHC II on PMNs is not necessarily

linked to all chronic inflammatory processes. Reprint requests to Gertrud Maria Hansch, Ph.D., Institut fur Immu-Although not yet studied systematically, MHC II ex- nologie, Ruprecht-Karls-Universitat Heidelberg, Im Neuenheimer Feld

305, 69120 Heidelberg, Germany.pression does not appear to be restricted to Wegener’sEmail: n50@ix.urz.uni-heidelberg.degranulomatosis, but also occurs in other forms of vasculi-

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