immunological abnormalities in family membersof patients

Original Article

Immunological Abnormalities in Family Members of Patientswith IgA Nephropathy

Masanobu MlYAZAKl

Weexamined several immunological parameters such as the numberof T cells with CD4antigen andthe receptor for the Fc portion of IgA (Ta cells), in vitro immunoglobulin production by peripheral bloodlymphocytes with or without pokeweedmitogen and serum levels of immunecomplexes in 19 healthy familymembers of patients with IgA nephropathy (IgAN). It was shown that the levels of serum IgA, T4 cells,CD4/CD8ratio of T cell subsets, T a cells, IgA circulating immune-complexes and spontaneous synthesisof immunoglobulins were significantly increased in family members of patients with IgAN. No family membershad proteinuria or hematuria. It was concluded that immunological abnormalities, including abnormalitiesof T cells and B cells, were found not only in patients with IgAN but also in their healthy family members.It was suggested that some factors in addition to these immunological abnormalities may be involved in thedevelopment of IgAN.

Key words: T a cells, Family study, Cellular immunity

IgA nephropathy (IgAN) is the most commonprimary glomerulonephritis in Japan as well as inseveral other countries (1 , 2). A marked geographicalheterogeneity has been observed in the incidence ofthis disease (2). Cases of familial IgAN (3), an

occasional association of IgAN with certain HLAantigens (4, 5) and polymorphism in the immuno-globulin heavy chain switch region (6) have beenreported. These results suggested that somegeneticfactors might be involved in the development ofIgAN.

IgAN is considered to be an immune-mediateddisorder (7) and many immunological abnormalitieshave been observed, including elevated serum IgAlevels (8), enhanced production of IgA by peripheralblood lymphocytes (9, 10), increases in IgA-bearinglymphocytes (1 1), elevated CD4/CD8ratio in T cellsubsets (12) and various types of autoantibodies ofthe IgA class in the sera (13, 14). Some of theseimmunological abnormalities have also been ob-

served in healthy relatives of patients with IgAN.

IgA-bearing lymphocytes were increased in some ofthe family members of patients with IgAN (15). Bcells of healthy relatives of patients with IgANshowed an enhanced production of immunoglobulin(10, 16, 17).

IgA synthesis is thought to be highly T celldependent. IgA-specific switching T cells were foundin murine Peyer's patches (18). It was shown thatT cells with the receptor for the Fc portion of IgA(Ta cell) had IgA-specific helper activity (19, 20)and that these cells might play some role in IgA-specific switching (21).

In the present study, T cell subsets including T a 4cells, and in vitro immunoglobulin production inhealthy relatives of patients with IgANwere in-vestigated to elucidate immunological abnormalitiesin family members of patients with IgAN.

MATERIALS AND METHODS

Su bjectsNineteen first-degree relatives (1 1 parents and

From Department of Internal Medicine, School of Medicine, Tokai University, IseharaReceived for publication January 29, 1990; Accepted for publication June 28, 1990Reprint requests should be addressed to MasanobuMiyazaki, MD,Department of Internal Medicine,

School of Medicine, Tokai University, Bouseidai, Isehara 259-ll, Japan

Jpn J Med Vol 29, No 5 (September, October 1990) 469

Miyazaki

eight siblings) in eight families of patients with IgANwere studied. Fifteen healthy adults served as con-trols. The diagnosis of IgAN was confirmed by openrenal biopsy, followed by light microscopic andimmunofluorescence studies. Systemic diseases suchas lupus erythematosus, rheumatoid arthritis,diabetes mellitus and liver diseases were excluded.Noneof the patients took any medications prior tothis study. All patients had proteinuria and/ormicroscopic hematuria, but none of them hadelevated levels of serum creatinine or blood ureanitrogen. None of the family members or healthycontrols had proteinuria, microhematuria or anyother abnormalities in urinary sediments. Bloodsamples were collected early in the morning beforebreakfast.Purification of human IgA myeloma proteinHuman IgA protein was purified from IgAl(lambda) myeloma serum by jacalin affinity columnchromatography (22). Briefly, the IgA-rich fractionwas precipitated by 50% ammoniumsulfate satura-tion, and was applied to a jacalin column (Sepharose4B coupled with protein extracted from the seeds ofthe jack fruit). After washing with borate-bufferedsaline, IgA was eluted with 0.8 Mgalactose solu-tion. Purified IgA showed a single precipitating lineagainst anti-whole humanserum as examinedbyimmunoelectrophoresis; it was not contaminatedwith IgG or IgM as proven by enzyme-linkedimmunosorbent assay (ELISA). Polyacrylamidegradient gel electrophoresis (PAA2/ 16, Pharmacia,Piscataway, NJ) showed that this fraction containedpolymeric IgAl. This purified human IgA myelomaprotein was conjugated with FITC using the methodof Kawamura (23). The F/P ratio of this FITC-labeled IgA was 1.8.T cell subsets

Peripheral blood mononuclear cells (PBMC)wereseparated by Ficoll-Paque (Pharmacia LKB

Biotechnology Inc.) density gradient centrifugationafter collection of heparinized blood. Monocytes andmacrophages were removed by an adherent pro-cedure. Phycoerythrin-labeled anti-CD3, CD4 andCD8 (OKT3, OKT4 and OKT8, Ortho DiagnosticSystems Inc., Raritan, NJ) were added to onemillion mononuclear cells and incubated for 30min on ice. After washing three times with 1%

bovine serum albumin (BSA) in phosphate-buffered

saline (PBS), the receptors for the Fc portion of IgA(Fc a R) were stained by FITC-labeled human IgAfor 30 min on ice. To examine the specific bindingof IgA to Fc receptor of IgA on T cells, human IgG,IgM or IgA was added to FITC-labeled IgA. Two-color fluorescent flow cytometry (FACStar, BectonDickinson, Mountain View, CA) was employed toenumerate Ta cells which had CD3, CD4or CD8antigen and Fc a R. A pure lymphocyte populationwas obtained by two-dimensional gating (forwardlight scattering vs. side light scattering). Ten

thousand lymphocytes were enumerated each timeto study T cell subsets.Ba cells

When measuring Ta3 cells (T a cells with a CD3surface marker), the numberof non-T cells withreceptors for the Fc portion of IgA was enumerated;these cells were defined as Ba cells. The subset ofnon-T3 cells included less than 0.5% monocytes, asconfirmed by flow cytometry.Cell culture conditions

After separating mononuclear cells fromperipheral blood, washed cells were cultured at aconcentration of 1 x 106 cells/ml with or withoutthe additon of 10 //g/ml of pokeweed mitogen(PWM, Sigma, St. Louis, MO) in RPMI 1640 con-taining 100 U/ml of penicillin and 100 /zg/ml ofstreptomycin supplemented with 10% heat-inactivated fetal calf serum (GIBCO, Grand Island,NY) at 37°C in 5% CO2 in air at 100% humidity.The culture supernatants were collected on day 7 andstored at -20°C until assay.Enzyme-linked im munosorbent assayThe concentrations of IgG, IgA and IgM in the

supernatant of lymphocytecultures were measuredby ELISA as follows. A 96-well polystyrene

microplate (Linbro/Titertek, Flow Laboratories,Netherlands) was coated with F(ab')2 fragments ofgoat anti-human IgG, IgA or IgM antisera (CappelLaboratories, Malvern, PA), and then blocked with1% BSA in PBS. Appropriately diluted sampleswere added to the wells, incubated at 4°C overnight,and then washed three times with 0.1% Tween inPBS. After incubation with alkaline phosphatase-conjugated affinity-purified goat anti-human IgG,IgA and IgM antisera (Sigma), the wells were washedand p-nitrophenyl phosphate disodium solution(Sigma) was added. The optical absorption was

470 Jpn J Med Vol 29, No 5 (September, October 1990)

Family Study of IgA Nephropathy

measured by a microplate photometer (ModelMTP-22, Corona Electric, Japan) at a wave lengthof 405 nm. Log linear curves were obtained fromstandard sera and were used for quantitation ofimmunoglobulins.

Concentration of serum immunoglobulinsThe levels of serum IgG, IgA and IgM weremeasured by laser nephelometry (Behring Institute,West Germany).Levels of circulating immune-complexesThe levels of IgG and IgA immune-complexeswere measured by solid phase anti-C3 Facb ELISAmethods (Teijin Lab, Tokyo, Japan) (24).Statistical analysisThe Mann-Whitney U test and the Spearman-Kendall test were employed for statistical analysis.

RESULTS

Levels of serum IgAAs shown in Fig. 1, the serum levels of IgA weresignificantly increased not only in patients withIgAN but also in their healthy relatives (IgANpatients: 429±138 mg/dl; family members:332± 111 mg/dl; controls: 269± 82 mg/dl, p<0.01).

There were no differences in the levels of serum IgGand IgM among these three groups.

Ta cellsFigure 2 shows the amount of Ta cells. No

significant differences in the number of T a 3 cellswere observed among IgAN patients, their familymembers and controls (patients: 5.8 ± 2.6%; family

- P < 0 . 0 1 -

6 0 0

p 5 0 0e nE

- N . S .- ,r P < 0 .0 1~ 1

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t I IP a tie n t F a m ily C o n tro l( n = 8 ) ( n = 19 ) ( n = 15 )

Fig. 1. Levels of serum IgAin patientswith IgAN, their family members and con-trols. The patients and family membersshowed higher levels of serum IgA(p <0.01).

-N.S.-I P<0.005 1

|-p<o.o5-|rp<o.oi-|

1010

3

Patient Family Control(n=8) (n=!9) (n=15)

-!--P<0.01-

-N.S. n N.

Patient(n=8)

10

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Family Control(n=!9) (n=15) Patient

(n=8)Family

(n=!9)Control

(n=15)

Fig. 2. Amount of Ta3, Ta4 and Ta8 cells in peripheral blood. No differences in Ta 3 cells were observed amongthe three groups. To: 4 cells in the patients (p <0.005) and relatives (p <0.01) were significantly increased as compared tothe controls. Patients had higher levels of T a 4 cells than their family members (p <0.05). Significant increases in T a 8 cellsin patients were found compared to controls (p <0.01). There was no difference in T a 8 cells between family members andcontrols.


Miyazaki

members: 4.2± 1.6%; controls: 4.3± 1.5%). Thenumber of T a 4 cells was significantly increased inpatients with IgAN (5.5±3.0%; controls: 2.3±1.0%, p <0.005). Their relatives also had signifi-cantly elevated levels of T«4 cells (3.9±2.1%,

p <0.01). Patients with IgAN had higher levels ofT a 4 cells than their family members (p <0.05). Thenumber of T a8 cells was significantly higher inpatients with IgAN (6.0±2.5%; controls: 3.9±

1.2%, p<0.01). Some of the family members hadmore T a 8 cells in their peripheral blood than con-trols although statistical analysis showedno signifi-cant differences (family members: 4.3 ± 2.1%). Nocorrelations were observed between increased T acells and histological severity, the level of proteinuriaor other clinical data in patients with IgAN. Thebinding of IgA to Fc receptor of IgA on T cells wasnot inhibited by IgG or IgM; it was only inhibitedby IgA. Therefore, IgA binding to T cells was con-sidered as a immunoglobulin class-specificphenomenon.

Ba cellsThe percentages of Ba cells in patients with

IgAN, family members and controls were 5.9±3.4%, 6.4±4.3% and4.3±3.1% (Fig. 3). Three out

of eight patients and eight of 19 family membersshowed an increase in B a cells comparedto the con-trol group although there was no statisticalsignificance.

CD4- and CD8-positive T cells and CD4/CD8 ratiosCD4-positive T cells was increased in the

peripheral blood from patients compared with con-trols (patients: 45.0±5.8%; controls: 39.3 ±5.1%,p<0.05). A significant increase in CD4 cells in

family members was also observed (family members:48.7 ± 8.4%, p <0.005). There were no significantdifferences in the amount of CD8-positive T cellsamongthese three groups. The ratio of CD4/CD8was significantly increased in family members com-

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Patient Family Control(n=8) (n=l9) (n=l5)

Fig. 3. AmountofBa cellsin

peripheral blood. Some patients andtheir relatives showed higher levelsof these cells.

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P a tie n t F a m ily C o n t ro l P a tie n t F a m ily C o n tr o(n = 8 ) (n = 19 ) (n = !2 ) (n = 8 ) (n = !9 ) (n = 12 )

Fig. 4. Circulating IgG and IgA immune complexes in sera. Althoughsome patients showed an increase in the levels of IgG-CIC, no significantdifferences were observed among the three groups. The patients and relativeshad significantly higher levels of IgA-CIC than controls (p <0.05).



pared with controls (family members: 1.86 ±0.59;controls: 1.31 ±0.17, p<0.005). Only one patientand about one half of the family members had aCD4/CD8 ratio of more than 1.65 (mean±2SD)compared with the control group.Circulating immune-complexes(CIC)As shown in Fig. 4, the levels of IgA class CICwere significantly increased in the patients withIgANand their family memberscompared with con-trols (patients: 4.8 ±2.8 /ig/ml; family members:2.8 ± 1.2^g/ml; controls: 2,1 ±0.9^g/ml, p <0.05).There was no significant difference in the levels ofIgA-CIC between the patients and their familymembers. No significant increase in IgG-CIC wasobserved among these three groups (patients:3.6 ±2.3 /*g/ml; family members: 2.5 ±0.8 /ig/ml;controls: 2.2± 0.7 //g/ml).

In vitro immunoglobulin production by peripheralblood mononuclear cells (PBMC)

Significant increases were observed inspontaneous IgA synthesis by PBMCfrom patientswith IgAN (p<0.05) and their family members(p <0.005) compared with the controls (Fig. 5). Thedegree of the increases of IgA production did notdiffer in patients and family members. Marked in-creases in IgA synthesis (p <0.05) were observed inPWM-stimulated PBMCcultures only from familymembers (Fig. 6).

Results of IgG, IgA and IgM production withand without PWMare summarized in Table 1.Spontaneous IgG, IgA and IgM synthesis was in-creased in patients and their family memberscom-pared with the controls (p<0.05). In PWM-stimulated cultures, significant increases in IgG, IgAand IgM production were observed only in thefamily members (p <0.05). Immunoglobulin pro-duction with PWMin the culture supernatant wasnot increased in patients with IgANwhen comparedto the controls. The increased IgA synthesis withPWMwas observed in ten of eleven controls, butit was not a statistically increase.The altered IgA synthesis due to the addition ofPWMis shown in Table 2. IgA synthesis in cultureswith PWMdecreased in all patients compared tothose without PWM.Only two persons in the con-trol group showed a decrease in IgA synthesis in the

PWM(-)

, P<0.05 ,, N.S.-,rP<0.005-,

J= 5000

i }< » _L

i ~ i à"jS 1000

I à" : -^ 500

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Patient Family Control(n=8) (n=J9) (n=ll)

Fig. 5. In vitro IgA production byperipheral lymphocytes without pokeweedmitogen (PWM). Patients (p<0.05) and

their relatives (p <0.005) produced muchmore IgA in PBMCculture than controls.There was no difference between patientsand their relatives.

PWM(+)

-N.S.-rP<0.05-]rP<0.05-1

Patient Family Control(n=8) (n=19) (n=11)

Fig. 6. In vitro IgA production with the

addition of pokeweed mitogen (PWM). Lympho-cytes from family membersshowed enhanced IgAproduction with PWMcompared those frompatients and controls (p <0.05). No significantincrease in IgA production was observed in thepatients.


Miyaz aki

Table 1. Immunoglobulin production by PBMC.PWM(-)

PWM(+)IgG

IgANPatient 1749 ± 1365

(n=8)

Family 1331±923 -,* 1709±1093(n=19)

Control 383 ± 353(n=ll)

*IgA

1534±1091 n

689±964*

IgM

902±905

1489±1387IgG

IgAIgM

330±335Jj

1670±1335 -i 835±955 -i 1420±1469i* *

4214±3232:j 2892±3443^ 3231±2228*1767±1108 J 758±668 J 4567±3672J

*p<0.05

Without pokeweed mitogen (PWM), patients and family members showed a significantly enhanced production of IgG,IgA and IgM as compared to controls (p <0.05). Whencultured with PWM,only the family members produced a significantamount of IgG, IgA and IgM in comparison with patients and controls (p <0.05). No increase in immunoglobulin productionwas observed in the patient.

Table 2. Altered IgA synthesis with PWMcompared to thatwithout PWM.

Increase

Patient 0(n=8)

Family 1 1 *(n=19)

Control 9

(n=ll)

*p<0.05

All patients showed decreased IgA synthesis when culturedwith pokeweed mitogen (PWM)compared to that withoutPWM.An increase in IgA production with PWMwas foundin two of the control group and in ll of the family members.About one half of the family membersmanifested an increasein IgA synthesis, while others showed a decrease in IgAsynthesis.

cultures with PWMcompared to those withoutPWM.Eight of 19 family members showed adecrease in IgA synthesis in cultures with PWM.However, other family members showed an increasein IgA synthesis in PBMCcultures with PWM.

DISCUSSION

It has been reported that an elevated serum level

of IgA was observed in approximately 50% ofpatients (8). One-third of the family members ofpatients with IgAN had higher serum levels of IgAthan those of controls (16). In this study, we showedthat healthy relatives also had significantly elevatedserum IgA levels. Although the elevated serum IgAin patients with IgAN might be due to the hyper-production of IgA, an impaired clearance of IgAremains a possible cause. IgA synthesis is known tobe highly T cell dependent. Activated T cells whichhave IL-2 (interleukin-2) receptors are reported tobe increased in patients with IgAN (25). The helperactivity of T cells in patients was found to be parallelwith the elevated CD4/CD8ratio (12). In the currentstudy, CD4-positive T cells were increased in bothpatients and family members, and the CD4/CD8ratio was also elevated in family members. It isinteresting to note that all patients had at least onerelative with an elevated CD4/CD8ratio (greaterthan the mean of the controls) in this study. Anincreased CD4/CD8ratio might be due to hyper-activity of CD4-positive T cells because the numbersof CD8-positive T cells did not differ among thesethree groups. T cells with receptors for the Fcportion of IgA (Ta cells) were considered asprospective IgA-specific helper T cells (19, 20).

Furthermore, the majority of Ta cells have CD4antigen on their surface (T a 4 cells) (26) and these



cells have been shown to switch from IgM-bearingB cells to IgA-bearing B cells in vitro (21). T a4 cellshave been reported to be significantly increased inpatients (27) and the number of Ta4 cells is re-ported to correlate with the amount of spontaneousIgA synthesis (28). The number of Ta4 cells wasalso increased in family members in the present studywithout any significant increase in the number ofT a 3 or T a 8 cells. It is suggested that the increasein T#4 cells might play an important role in theelevated serum IgA in both patients and their familymembers.

There have been several studies on in vitro

immunoglobulin production by lymphocytes frompatients with or without the addition of mitogens.Several authors have reported that in vitro IgAsynthesis is increased with or without the presenceof mitogens (9, 10), but others have indicatedno increase (29). In this study, not only thespontaneous production of IgA, but also thatof IgG and IgM was significantly increased in bothpatients and their family members. Enhanced T cellhelper activity might have effects on not only IgAbut also on IgG and IgM via a non-specificmechanism. However, only family members showedenhanced immunoglobulin production in PWM-

stimulated cultures compared with patients and con-trols. There was no difference in immunoglobulinsynthesis by PBMCwith PWMbetween patients andthe control group. When IgA synthesis by PBMCwith PWMwas compared to that without PWM,IgA production with PWMwas decreased in allpatients. This decrease of IgA synthesis with PWMwas observed in two of eleven controls and eight ofnineteen family members. The results of the presentstudy suggested that about one half of the healthyrelatives had almost the same T cell function as thepatients.

Impaired immunoglobulin synthesis by peri-pheral blood lymphocytes in systemic lupuserythematosus (SLE) is well known (30). Onepossible explanation for this depressed PWM-stimulated immunoglobulin response is that lym-

phocytes from patients with IgANwere alreadyactivated in vivo and that these preactivated cells didnot respond to a second stimulation such as PWM,as observed in SLE. It might also be speculated thathelper T cell activity is inhibited in in vitro cultures

with PWMbecause CD4T cells are known tobecome refractory to IL-2 growth promotion earlierthan CD8-positive T cells (3 1). This enhanced helperactivity of T cells in peripheral blood might beassociated with a hyperresponse to exogenousantigens in patients and family members with IgAN.It has been shown that the administration of in-fluenza vaccines to patients with IgANinduces notonly an increase in serum levels of IgA-class anti-influenza antibodies but also a simultaneous increasein IgA-class rheumatoid factor (32); this pheno-menonwas also observed in somehealthy relativesof patients with IgAN (33).The level of IgA-class CIC was shown to beelevated in patients and their relatives. Although thephysiochemical properties of these immunecom-plexes were not examinedin this study, a mechanismfor the enhancement of IgA-CIC might be presentin family members of patients with IgAN, probablydue to the hyperactivation of lymphocytes.B cells with receptors for the Fc portion of IgA(B a cells) have been observed in human peripheralblood (34) and are reported to be increased in theperipheral blood of patients with IgAN (35).Although the role of these cells remains obscure,they are reported to be activated B cells (36).Furthermore, activated B cells with the Fc receptorfor IgE, known as CD23, were also increased in

patients with IgAN(unpublished data). In this study,more Ba cells were seen in somefamily membersof IgAN patients than in the controls. It is postulatedthat some of the healthy family members of patientswith IgAN might have activated B cells in theirperipheral blood.

These observations suggest that several immuno-logical abnormalities might exist even in healthyfamily members who have no clinical signs ofglomerulonephritis. Someunknown factors in thedevelopment of IgAN might exist in addition to thedysregulation of IgA synthesis.In conclusion, healthy relatives of patients withIgAN were "high responders", especially in IgAsynthesis. Several immunological abnormalities,

including an increase in the concentration of serumIgA, elevated IgA-CIC and enhanced in vitro IgAsynthesis with and without PWMwere observed inthe healthy relatives of patients with IgAN. Thesephenomena might be associated with an increase in


Miyazaki

T a 4 cells observed not only in the patients but alsoin the healthy family members. Factors other thanthe high IgA response might also be involved in thedevelopment of IgAN.

ACKNOWLEDGEMENT:The author is grateful to Dr.Masayuki Endoh and Dr. Hideto Sakai for their very helpfultechnical advice and discussions.

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immunological abnormalities in family membersof patients

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