Eur J Clin Chem Clin Biochem 1997; 35(3): 199-205 © 1997 by Walter de Gruyter · Berlin · New York

Distribution of Lymphocyte Subsets in Bone Marrow and PeripheralBlood Is Associated with Haptoglobin TypeBinding of Haptoglobin to the B-Cell Lectin CD221)

Michel Langlois1, Joris Delanghe1, Jan Philippe1, Jin Ouyang1, Dirk Bernard1, Marc De Buyzere1,Guido Van Nooten2 and Geert Leroux-Roels1

1 Central Laboratory,2 Department of Cardiac Surgery,University Hospital Gent, Gent, Belgium

Summary: Haptoglobin is an acute phase protein showing a genetic polymorphism with 3 major types: Hp 1 -1, Hp 2-1,and Hp 2-2. In this study, flow cytometric analysis demonstrated that all three haptoglobin types bind to CD22 on humanB-lymphocytes with equal affinity. Comparison of reference values for lymphocyte subsets in peripheral blood and bonemarrow showed significant differences between haptoglobin types. Haptoglobin 2-2 is associated with higher peripheralB-lymphocyte counts (P < 0.001) and CD4+ T-lymphocyte counts (P < 0.05). In bone marrow, CD4+ T-cell percent-ages were highest (P < 0.001) but B-cell percentages were lowest (P < 0.001) in haptoglobin 2-2 type. A negative cor-relation between serum haptoglobin 1-1 concentration and peripheral B-cell counts was observed (r = —0.663). Ourresults suggest that haptoglobin is involved in lymphocyte distribution. The present findings are a potential cause ofover- or underestimation of lymphocyte subset counts in the clinical staging of immunodeficiency diseases.

Introduction

Haptoglobin is an acute phase sialoglycoprotein withhaemoglobin-binding capacity (1). Haptoglobin is char-acterized by a molecular heterogeneity with three maingenetic types: Hp 1-1, Hp 2-1, and Hp 2-2 (2). Thispolymorphism arises from variant α-polypeptide chains(Q! and a2), since the -polypeptide chains of the threehaptoglobin types are identical (3). The α-chains are ge-netically determined by two autosomal alleles, Hpl andHp2 (3, 4). The homozygote Hp 1-1 is a small protein(Mr = 86000) with the formula (01 )2 (5). The hetero-zygote Hp 2-1 with the formula [(01 )2 + (α2β)η](η = 0, l, 2, ...) is characterized by polymerization (Mr

= 165 000) (5). In Hp 2-2 higher molecular mass formsare found (Mr =200000) with the formula (α2β)η

(η = 3, 4, 5, ...) (5). Many functional differences be-tween haptoglobin types have been described (6).

The protein plays an important role in the immunologi-cal and inflammatory response (5, 6). Haptoglobin in-hibits prostaglandin synthesis, but this anti-inflamma-tory effect is less pronounced for Hp 2-2 (6-8). The Hp2-2 type is over-represented among patients with auto-immune diseases (9, 10). Hp 2-2 individuals show a

') This study was supported by a grant of the Nationaal Fonds voorGeneeskundig Wetenschappelijk Onderzoek (NFGWO) (No.33003695).

stronger immune reactivity, as evidenced by higher anti-body titres following vaccination (11 — 13).

Interactions of haptoglobin with neutrophils and mono-cytes are well described (14-16), but haptoglobin bind-ing to lymphocytes has never been investigated. Hapto-globin is known to inhibit various forms of lectin-inducedlymphocyte transformations and there is some indicationthat haptoglobin modulates B-cell proliferation (17—19).More recently, it has been demonstrated that the carbohy-drate moiety of human haptoglobin binds to the B-cell ad-hesion glycoprotein CD22 (20), suggesting that haptoglo-bin is involved in trafficking of lymphocytes. On the otherhand, B- and T-lymphocyte counts are used as a diagnos-tic test in a number of diseases, particularly in the clinicalstaging of HIV-infection (21,22).

In this study, we performed a flow cytometric analysisof haptoglobin binding to human lymphocytes. In orderto study the physiological and clinical importance of thisbinding, we investigated the relationship between hapto-globin type and reference values for lymphocyte subsetsin peripheral blood and in bone marrow.

Materials and MethodsSubjects

A population of 107 healthy Caucasian volunteers of Flemish des-cent was studied. Blood was obtained by venipuncture, allowed toclot, and centrifuged (1000g, 10 minutes, room temperature). The

200 Langlois et al.: Haptoglobin polymorphism and lymphocyte distribution

supernatant serum was collected for analysis. Simultaneouslydrawn peripheral blood was collected in K2EDTA-coated tubes forcytological study. In order to reduce biological variations, bloodwas drawn between 09:00 h and 10:00 h. All subjects gave in-formed consent to participate in this study. The study group con-sisted of 44 males (age: 40 ± 11 years) and 63 females (age: 42±11 years). Subjects under corticoid therapy or with recent infec-tion, vaccination (within 12 months prior to sampling), and serumC-reactive protein concentrations > 10 mg/1 were excluded. Therewas no clinical evidence of any haematological or other disease.The haptoglobin type of the subjects was determined using starchgel electrophoresis of haemoglobin supplemented serum, followedby peroxid se staining (2).

Bone marrow aspiratesHuman bone marrow samples were aspirated by sternal puncturefrom 45 haematologically normal patients undergoing cardiac sur-gery (33 males aged 64 ± 7 years and 12 females aged 58 ± 8years). Informed consent was also obtained from these subjects.Bone marrow was collected in tubes containing 10 ml RPMI 1642medium (Gibco, Paisley, UK) and 50 μΐ sodium heparin (5 Χ 106

IU/1). The haptoglobin type was determined in serum collectedfrom these patients before surgery. This study was approved by theethical committee of the University Hospital of Gent.

Immunophenotyping of peripheral blood lymphocytesHaematological examinations were always performed on freshEDTA blood. Properties measured included the total counts of leu-kocytes and differential white blood cell count (Sysmex NE-8000,Toa Medical Electronics Co., Kobe, Japan) (23). Lymphocyte sub-sets were analysed by flow cytometry. For T-cell marker analysis,5 μΐ of TriTEST™ reagent (Becton Dickinson, Mountain View,CA, USA) were applied to 50 μΐ EDTA-blood. This reagent is acombination of CD4-phycoerythrin, CD8-fluorescein isothiocya-nate, and CD3-peridinin chlorophyll conjugated mouse monoclonalantibodies (isotypes: IgGiK). B-lymphocytes were analyzed usingphycoerythrin-labelled CD19-mouse antibodies (isotype: IgGiK)(Coulter, Hialeah, FL, USA) (5 μΙ/50 μΐ blood). Five microlitersof CD56-phycoerythrin and CD3-fluorescein isothiocyanate mouseantibodies (isotypes: IgGjK, both purchased from Coulter) wereapplied to 50 μΐ blood for the determination of Natural Killer (NK,CD3~CD56+) cells. After incubation (4 °C, 30 minutes), erythro-cytes were lysed by hypotonic shock and leukocytes were fixedusing a Q-Prep Workstation (Coulter, Hialeah, FL, USA). The per-centages of lymphocyte subsets in peripheral blood were deter-mined on a FACSort flow cytometer, equipped with theCellquest™ software (Becton Dickinson, Mountain View, CA,USA). The absolute numbers of lymphocyte subsets were calcu-lated by multiplying the percentages of CD3+-, CD3+CD4+-,CD3+CD8+-, CD19+-, and CD3-CD56+-cells by the totalnumber of peripheral blood lymphocytes.

Immunophenotyping of bone marrow lymphocytesImmediately after collection of heparinized bone marrow, mononu-clear cells were separated by density gradient centrifugation onLymphoprep® (Nycomed, Oslo, Norway), washed three times inHank's balanced salt solution (Gibco, Paisley, UK), and resus-pended in RPMI 1642 medium to a final cell count of 109/1. Lym-phocyte subsets were evaluated using Simultest™ reagents Leuco-GATE™ (CD45/CD14), Control γι/γ^ (IgGj/IgGO, CD3-fluores-cein isothiocyanate/CD4-phycoerythrin, CD3-fluorescein isothio-cyanate/CD8-phycoerythrin, CD3-fluorescein isothiocyanate/CD56+ CD16-phycoerythrin, and CD3-fluorescein isothiocyanate/CD19-phycoerythrin (Becton Dickinson). Twenty microliters ofSimultest™ reagents were applied to 100 μΐ of cell suspensions.Phenotyping was performed on a FACSort flow cytometer, makinguse of the SimulSET™ software (Becton Dickinson, MountainView, CA, USA).

Determination of haptoglobin and C-reactive proteinConcentrations of haptoglobin and C-reactive protein in serumwere measured by fixed-time immunonephelometry on a BN II

nephelometer (Behringwerke AG, Marburg, Germany) (24). Rabbitanti-human protein antisera, controls, and standards were obtainedfrom Behringwerke. For each assay, we used one single batch ofantiserum. Calibrations were done with the same standard batch.The serum protein concentrations were expressed according toIFCC-standards (25, 26).

Direct haptoglobin binding to lymphocytesPurified human haptoglobin type 1-1, 2-1, and 2-2 preparationswere a kind gift of Behringwerke AG (Marburg, Germany). Thepurity of the material was checked by SDS-PAGE. Haptoglobintypes were conjugated with fluorescein isothiocyanate (SigmaChemical Co., St. Louis, MO, USA), (0.05 mg fluorescein isothio-cyanate per mg haptoglobin) in a 0.25 mol/1 carbonate buffer, 0.15mol/1 NaCl, pH 9 at 4 °C overnight. The solution was dialyzed inorder to separate haptoglobin-fluorescein isothiocyanate from freefluorescein isothiocyanate.For haptoglobin binding analysis, 100 μΐ of peripheral blood mono-nuclear cell suspensions (109/1) were incubated (4 °C, 30 min) with100 μΐ of haptoglobin-fluorescein isothiocyanate (1 g/1). Haptoglo-bin was always applied to cells obtained from subjects of the samehaptoglobin type. In order to phenotype the haptoglobin bindingcells, 5 μΐ of phycoerythrin-labelled mouse antibodies against CD4(IgG!K, Coulter), CDS (IgGiK, Coulter), CD3 (IgG^, BectonDickinson), CD 19 (IgG^, Coulter), or CD56 (IgG^, Coulter)were applied as well. After incubation (4 °C, 30 min), the cellswere washed twice and resuspended to 0.5 ml with phosphate-buffered saline. Flow cytometric analysis was then performed on aFACSort flow cytometer. Lymphocytes were gated using a forwardscatter-side scatter plot.

Indirect haptoglobin detection on lymphocytes

Haptoglobin binding to lymphocytes was also investigated by indi-rect immunofluorescence. Therefore, 100 μΐ of mononuclear cellsuspensions (109/1) were incubated (4 °C, 30 min) with 1 ml ofpurified haptoglobin (1 g/1) and washed in phosphate-buffered sa-line. In a second step, 100 μΐ of cell suspensions were incubated(4 °C, 30 min) with 5 μΐ of sheep antihuman haptoglobin antiserum(The Binding Site, Birmingham, UK), diluted 1 :10 in phosphate-buffered saline, and were washed twice in phosphate-buffered sa-line. Five microliters of donkey anti-sheep IgG-fluorescein isothio-cyanate (The Binding Site), diluted 1 :20 in phosphate-bufferedsaline, and 5 μΐ of phycoerythrin-labelled antibodies (CD4, CDS,CD3, CD 19, CD56) were then applied to the cells. In order toeliminate (false-positive) Fc binding of the first antibody, cellswere preincubated (4 °C, 30 min) with normal sheep Ig (SigmaChemical Co.). As a negative control for the second antibody, cellswere incubated with donkey anti-sheep IgG-fluorescein isothiocya-nate and with phycoerythrin-labelled mouse antibodies only.Affinity of haptoglobin to the plasma membrane of lymphocyteswas studied using a similar procedure but with decreasing concen-trations of purified haptoglobin (1 g/1), diluted 1 : 2 to 1 : 1024 inphosphate-buffered saline. The affinity constant K was calculatedusing the equation

_ [occupied binding sites][free binding sites] [Hp]

where [Hp] represents the molar concentration of haptoglobin (27).

Interference of haemoglobin and CD22 antibodiesHuman haemoglobin was purchased from Sigma Chemical Co.(St. Louis, MO, USA). In order to study the effect of haemoglo-bin on haptoglobin binding to lymphocytes, 10 μΐ of the haemo-globin solution (40 g/1) were applied 15 minutes after applicationof purified haptoglobin to 100 μΐ of the mononuclear cell sus-pension (30 min, 4 °C). In order to study inhibition of haptoglo-bin binding by CD22 antibodies, the cells were preincubated for30 min at 4 °C with 10 μΐ of mouse anti-human CD22 antibodies(140 mg/1) (Dako A/S, Glostrup, Denmark). In both experiments,haptoglobin binding to lymphocytes was detected as previously

Langlois et al.: Haptoglobin polymorphism and lymphocyte distribution 201

Tab. 1 Age, gender, peripheral blood leukocyte counts, lympho-cyte subset counts, CD4+/CD8+ Τ cell ratio, and serum haptoglo-bin and C-reactive protein concentrations of the study populationaccording to haptoglobin (Hp) type.

Hp 1-1 Hp 2-1 Hp 2-2

nAge (years)Males/femalesLeukocytes (109/1)Neutrophils (109/1)Eosinophils (109/1)Basophils (109/1)Monocytes (109/1)Lymphocytes (109/1)

CD19+ (109/1)CD3+ (109/1)CD3+CD4+ (109/1)CD3+CD8+ (109/1)CD3~CD56+ (109/1)CD4+/CD8+

Haptoglobin (g/1)C-reactive protein

(mg/1)

2143 ± 13a

9/126.314.050.150.030.531.540.171.030.630.390.161.841.313.1

± 1.72± 1.31±0.08±0.02±0.16±0.47±0.06±0.29±0.13±0.17±0.06±0.64±0.53±2.8

5141 ±922/296.22 ±3.67 ±0.20 ±0.05 +0.56 ±1.73 ±0.21 ±1.21 ±0.76 ±0.42 ±0.20 +2.00 +1.00 ±2.9 ±

3540 ± 1213/22

1.561.160.140.030.190.490.060.390.220.210.110.730.403.5

6.623.990.190.040.571.830.261.240.790.430.212.030.773.0

-+·±·+·+++++++-H

+

+

+

1.961.640.150.030.200.47b

0.09C

0.380.28b

0.160.080.990.43C

2.9

a All values: Mean ± SD.b Ρ < 0.05 (ANOVA).c P < 0.001 (ANOVA).

described. The mean fluorescence and the number of positivecells obtained without haemoglobin or anti-CD22 incubation wasequalized to 100% (control). Mean fluorescence and the numberof positive cells after incubation with haemoglobin or anti-CD22were expressed as percentage of control. The difference betweenboth percentages was a measure for the degree of inhibition ofhaptoglobin binding.

StatisticsValues are expressed as means ± SD. Validity of Hardy-Weinbergequilibrium was tested using the chi-square test. Comparisons ofcell counts, cell percentages, and protein concentrations betweenhaptoglobin type groups were carried out using ANOVA. Corre-lations between data were examined using regression analysis. Sta-tistical significance was considered at the level P < 0.05. Adjust-ment for multiple testing was done when appropriate using theBonferroni correction.

Results

Haptoglobin gene frequency of thestudy population

In the study population, haptoglobin type frequencieswere 0.196 (Hp 1-1), 0.477 (Hp 2-1), and 0.327 (Hp 2-2). Allele frequencies of 0.40 (Hpl) and 0.60 (Hp2)were obtained. These results are in agreement withHardy-Weinberg'1 s equilibrium and with earlier studieson Flemish subjects (13, 28). Age and gender of thesubjects according to haptoglobin type are given intable 1.

Total and differential leukocyte counts

In peripheral blood, no differences between haptoglobintypes were found for total leukocyte counts (tab. 1). Dif-ferential counts of granulocytes and monocytes were

comparable but total lymphocyte count was significantlyhigher in haptoglobin 2-2 subjects (P < 0.05).

Peripheral blood lymphocyte subsets

The flow cytometric phenotype data presented in table1 correspond to normal reference values for peripheralblood lymphocyte subsets in Caucasians (29). No sig-nificant differences were found in percentages ofCD3+-, CD3+CD4+-, CD3+CD8+-, CD19+-, andCD3~CD56+-cells between haptoglobin types. Abso-lute peripheral CD3+, CD3+CD8+, and CD3~CD56+

counts (109/1) were comparable as well. However,CD3+CD4+-cell counts were significantly higher inHp 2-2 subjects (P < 0.05). The CD4+/CD8+ T-cellratio was comparable in the three groups. Furthermore,peripheral CD19+-cell counts showed marked differ-ences according to the haptoglobin type (P < 0.001).In the Hp 1-1 group, peripheral CD19+ counts arelow. Hp 2-2 is associated with the highest CD19+

counts. Neither age nor gender of the subjects wererelated to peripheral CD19+- and CD3+CD4+-cellcounts.

Serum haptoglobin and C-reactive proteinconcentrations

The concentrations of haptoglobin measured in serumcorrespond to earlier studies (tab. 1) (6, 13). In theoverall population, peripheral CD19+-cell counts werenot related to serum haptoglobin levels. However, inthe Hp 1-1 group, a negative correlation of CD19+

count with serum haptoglobin concentration was ob-tained: y (peripheral CD19+-cells, 109/1) = -67.73*(serum Hp 1-1 concentration, g/1) + 262.91;r = -0.663, Syx = 44.83 (P < 0.01) (fig. 1). Such cor-relations were not observed in Hp 2-1 and Hp 2-2subjects. Similarly, peripheral CD3+CD4+ counts werenot related to serum haptoglobin levels. Serum C-reactive protein concentrations were comparable be-tween all haptoglobin types (tab. 1).

„ 0.30 π==.o>£ °·25 -Ϊ2 0.20 ^

0.15 -

0.10 -

0.05 -

0.0I

0.5I

1.0 1.5I

2.0I

2.5Serum Hp 1-1 concentration [ g/l ]

Fig. 1 Relation between peripheral Β cell count (109/1) (y) andserum haptoglobin (Hp) concentration (g/l) (je) in Hp 1-1 subjects:y = -67.73* + 262.91, r = -0.663, P < 0.01, n = 21.

202 Langlois et al.: Haptoglobin polymorphism and lymphocyte distribution

Tab. 2 Lymphocyte subset percentages and CD4+/CD8+ T cellratio in human bone marrow according to haptoglobin (Hp) type.

nCD19+ (%)CD3+ (%)CD3+CD4+ (%)CD3+CD8+ (%)CD3-CD56+ (%CD4+/CD8+

Hp 1-1

838.4±11.0a

49.8 ± 16.5C

21.8 ± 9.1b

24.9 ± 14.2) 9.6 ± 5.4

1.21 ± 0.99

Hp 2-1

2627.9 ± 6.463.5 ± 5.239.0 ± 6.524.4 ± 5.98.5 ±2.8

1.75 ±0.72

Hp 2-2

1118.4± 7.97b

68.8 ± 9.444.5 ± 10.824.7 ± 10.814.5 ± 5.62.23 ± 1.35

a All values: Mean ± SD.b P < 0.001 (ANOVA).c P < 0.05 (ANOVA).

Bone marrow lymphocyte subsets

In human bone marrow aspirates, percentages ofCD3+-, CD3+CD4+-, CD3+CD8+-, CD19+-, andCD3~CD56+-cells were compared between haptoglo-bin types. As shown in table 2, total CD3+ bonemarrow cell percentages were lowest in Hp 1-1 type(P < 0.05), which was exclusively caused by lowerCD3+CD4+ percentages (P < 0.001). Significant dif-ferences for CD3+CD8+ and CD3~CD56+ bone mar-row cells were not observed. A nearly significant hap-toglobin type-dependence of CD4+/CD8+ T-cell ratiowas obtained in bone marrow (P = 0.079). In contrastto peripheral blood, Hp 2-2 is associated with thelowest CD19+ bone marrow cell percentages(P < 0.001).

Relations between bone marrow andperipheral blood lymphocytes

Peripheral blood lymphocyte subset counts were deter-mined in EDTA-blood obtained from 10 patients beforecardiac surgery and compared with the data obtained intheir bone marrow aspirates. Bone marrow CD3+CD4+

percentages (y) correlated positively with peripheralCD3+CD4+ counts (x, 109/1): y = 0.03* +11.38,r = 0.742, Syx = 7.53 (P < 0.05). In contrast, a negativecorrelation was found between CD19+ bone marrowpercentages (y) and peripheral CD19+ counts (jc, 109/1):y = -0.21* + 66.36, r = -0.779, S^ = 6.75(P < 0.01).

Relation between serum haptoglobinconcentrations and bone marrow lymphocytepercentages

In the overall group, no correlations were obtained be-tween serum haptoglobin concentrations and lymphocytesubset percentages in bone marrow. However, in Hp 1-1subjects, CD19+ percentages correlated positively withserum haptoglobin concentrations: y (% CD19+ bonemarrow cells) = 34.30* (serum Hp 1-1 concentration, g/1)-7.50, r = 0.770, Syx = 7.58 (P < 0.05). This is in con-trast with the negative correlation of Hp 1-1 concentration

with peripheral CD19+ count. In all haptoglobin types,CD3+CD4+ bone marrow percentages were not related toserum haptoglobin concentrations.

Binding of haptoglobin-fluoresceinisothiocyanate to lymphocytes

Figure 2 is a typical flow cytometric dot plot showinghaptoglobin-fluorescein isothiocyanate binding to thecell-surface of CD19+-lymphocytes. Such Hp+CD19+-cell populations were obtained with Hp 1-1 (n = 5), Hp2-1 (n = 5), and Hp 2-2 (n = 5). No haptoglobin-fluo-rescein isothiocyanate binding was detected on CD4+-,CD8+-, CD3+-, and CD56+-cells. As demonstrated inprevious studies, haptoglobin binding was also observedin the monocyte and granulocyte populations (14-16).

Indirect haptoglobin detection onlymphocytes

Identical results were obtained after indirect haptoglobinstaining using sheep anti-haptoglobin antiserum anddonkey anti-sheep IgG-fluorescein isothiocyanate. In allhaptoglobin types, the flow cytometric dot plots ofHp+CD19+-lymphocytes were comparable with the di-rect haptoglobin staining (fig. 3). Control experimentswere performed in order to exclude Fc binding of thetwo antibodies. Hp"l"CD19+-lymphocytes were also ob-tained after preincubation of the cells with normal sheepimmunoglobulin. On the other hand, no Hp+CD19+

double-positive staining was observed after incubationof the cells with donkey anti-sheep IgG-fluoresceinisothiocyanate and phycoerythrin-labelled CD 19 anti-bodies only.

Effect of haemoglobin and CD22-antibodieson haptoglobin binding

Flow cytometric analysis showed that the addition ofhaemoglobin to the cell suspensions did not cause a sig-

10"

LU 8Q. ~2§ io2-Ο

* 10°10"10" 10' 10* 10"

Direct Hp staining (FITC)[relative fluorescence intensity]

Fig. 2 Illustration of haptoglobin (Hp) binding to Β cells. Shownis a flow cytometric dot plot of log fluorescein isothiocyanate(FITC) fluorescence (Hp) vs log phycoerythrin (PE) fluorescence(CD 19) of lymphocytes. The plot was obtained after incubation ofperipheral blood mononuclear cells with human Hp-FITC andmouse anti-CD 19 PE.

Langlois et al.: Haptoglobin polymorphism and lymphocyte distribution 203

nificant reduction of Hp 1-1 (n = 3), Hp 2-1 (n = 3),or Hp 2-2 (n = 3) binding to CD19+-lymphocytes. Incontrast, binding of haptoglobin to CD 19+-lymphocytescould be inhibited by preincubation of the cells withCD22 antibodies. The CD22 antibodies reduced themean fluorescein isothiocyanate fluorescence by 78± 5% and the number of positive cells by 96 ± 2%(n = 9). Comparable results were obtained for all threehaptoglobin types.

Affinity of haptoglobin towards CD22

The affinity constants Κ observed for Hp-CD22 bindingwere 2.62 ± 0.53 ΙΟ7 ηιοΓ1 X 1 (Hp 1-1; n = 5), 2.71± 1.52 107 mol~1 X 1 (Hp 2-1; n = 5), and 3.20 ± 1.47ΙΟ7 ηιοΓ1 X 1 (Hp 2-2; n = 5). Figure 4 shows thebound/free ratios (occupied CD22 binding sites/freeCD22 binding sites) at increasing molar concentrationsof haptoglobin. Approximately 104 CD22 molecules are

1U "

Ι ίο3·§8 102-Φο<is ;s.

10°-

•*

• "

• ·'.. .ι

••^y.^»

·* · * " "λτ^Α,ΐτ ' .• ^"tjjf' ^-i* " ** ""*% yV · ··* "t.'.. ·

'

10° 101 10^ 10J 10^Indirect Hp staining (FITC)

[relative fluorescence intensity]

Fig. 3 Indirect detection of haptoglobin (Hp) binding to B cells.The flow cytometric dot plot showing log fluorescein isothiocya-nate (FITC) fluorescence (Hp) vs log phycoerythrin (PE) fluores-cence (CD 19) was obtained after incubation of lymphocytes withhuman Hp, sheep anti-Hp, donkey anti-sheep FITC, and mouseanti-CD 19 PE.

2.0-

1.5-

u_ω

1.0-

0.5-

0.01 4 6

Hp concentration [10"8mol/l ]

Fig. 4 Binding of Hp types 1-1, 2-1, and 2-2 to Β lymphocytes.Shown are the B/F ratios (bound/free) of CD22 binding sites atincreasing molar Hp concentrations. The percentages of Hp-bind-ing CD19+ lymphocytes were multiplied by the mean FITC fluo-rescence of the positive CD19+ cell populations. The B/F ratioswere calculated from the maximum Hp-CD22 saturation: B/F =[observed binding]/[maximal binding — observed binding].1 · Hp 1-1, 2 A Hp 2-1, 3 · Hp 2-2

present on B-lymphocytes (30). Taking into account thecalculated mean B-cell counts (tab. 1), this correspondsto CD22 molecule number concentrations of approxi-mately 1.7 X 1012/1 (Hp 1-1), 2.1 X 1012/1 (Hp 2-1), and2.6 X 1012/1 (Hp 2-2) in peripheral blood. From themean molar concentrations of haptoglobin types 1-1 (14X 10~6 mol/1), 2-1 (6 X 10~6 mol/1), and 2-2 (2.85X 10~6 mol/1), we can estimate that a negligible fraction(< 0.0001%) of plasma haptoglobin is present on B-lymphocytes. However, the occupation of CD22 mole-cules by haptoglobin is type-dependent: the percentagesof free CD22 molecules are approximately 0.25% (Hp1-1), 0.70% (Hp 2-1), and 1.20% (Hp 2-2).

Discussion

Flow cytometric analysis demonstrated that haptoglobinbinds to human B-lymphocytes in Hp 1-1, Hp 2-1, andHp 2-2 individuals. In contrast, no significant bindingwas detected for T-cells and NK-cells.

The values for the affinity constant observed for hapto-globin binding (2.80 ± 0.53 107 mol"1 X 1) are inagreement with those found by Hanasaki et al. who de-monstrated that the carbohydrate moiety of the hapto-globin -chain binds to human recombinant CD22 (20).The affinity constants were comparable for the threehaptoglobin types. This is not surprising, since the hap-toglobin -chain is identical in all haptoglobin types.However, the number of free CD22 binding sites wereestimated to be higher in Hp 2-2 blood.

Antibodies to CD22 block haptoglobin binding to fi-celle, indicating that CD22 is indeed the binding site forhaptoglobin. In contrast, addition of haemoglobin hadno effect on haptoglobin binding to B-cells. Althoughthe binding of haemoglobin to the -chain of haptoglo-bin is characterised by a very high affinity (> 1010

mol"1 X 1) (31), it does not interfere with the haptoglo-bin-CD22 binding. This suggests that the haptoglobin -binding region for CD22 is different from the haemoglo-bin-binding site.

Reference values for peripheral blood lymphocytes dif-fer significantly depending on the haptoglobin type. To-tal peripheral blood lymphocyte counts are highest inHp 2-2 individuals, which is exclusively caused byhigher B-cell and helper T-cell counts. This effect waseven more pronounced in bone marrow helper T-cellpercentages. In contrast, the lowest B-cell percentageswere observed in bone marrow aspirates from Hp 2-2subjects. Haptoglobin type dependence was not ob-served for cytotoxic/suppressor T-cells and NK-cells inboth peripheral blood and bone marrow.

The association of Hp 2-2 type with a higher numberof peripheral circulating B-lymphocytes and helper T-lymphocytes partly explains the strong immunological

204 Langlois et al.: Haptoglobin polymorphism and lymphocyte distribution

reactivity in Hp 2-2 individuals (5, 6). The present find-ings can improve the interpretation of lymphocyte subsetanalysis in the practice of laboratory medicine, since B-and T-cell counts are used as a diagnostic test in anumber of diseases, such as primary or acquired immu-nodeficiencies and sarcoidosis (21).

Particularly, the knowledge of the patients' haptoglobintype is of interest for the interpretation of CD4+ T-cellcounts in the clinical staging of HIV-infected patients(22). Clinicians using the fixed classification criteria ofHIV-disease may overestimate CD4+ T-cell counts inHp 2-2 patients (22). Therefore, absolute CD4+ T-cellcounts should be interpreted with caution.

Measurements of serum haptoglobin concentration cor-respond to earlier studies and showed lowest haptoglo-bin levels in Hp 2-2 individuals (6, 13). The distributionvolume of haptoglobin is about 1.5 times the plasmavolume, so the measured serum concentration is in equi-librium with the concentration in bone marrow (4). Theassociation of the haptoglobin type with reference val-ues for B-cells and CD4+ T-cells cannot be explainedby an effect of haptoglobin concentration. However, se-rum Hp 1-1 concentration showed a negative correlationwith peripheral B-lymphocyte count. In bone marrow,serum Hp 1-1 concentration correlated positively withB-cell percentage. These findings suggest a negative ef-fect of Hp 1-1 on the number of peripheral circulatingB-lymphocytes. Such correlations were not observed inthe Hp 2-1 and Hp 2-2 groups.

Peripheral helper T-lymphocyte counts correlated posi-tively with bone marrow helper T-cell percentages.However, for the B-cell markers, an inverse relationwas observed between peripheral counts and bonemarrow percentages. These observations support theview of Hanasaki et al. who postulated a role forhaptoglobin in homing and trafficking of B-lympho-cytes (20).

A lectin-like binding of haptoglobin to lymphocyteshas been suggested by several authors (5, 6). Using aflow cytometric approach, we confirmed here the re-cent finding that haptoglobin binds to CD22 (20).CD22 is a cell-surface phosphoglycoprotein found onthe majority of B-lymphocytes (32). The glycoproteinis a member of the immunoglobulin superfamily andis involved in antigen-induced B-cell activation andsignal transduction (32, 33). CD22 is also an adhesionreceptor and mediates interactions of B-cells witherythrocytes, T-lymphocytes, monocytes, neutrophils,and endothelial cells by specific binding to glycopro-teins containing N-linked oligosaccharides with termi-nal a2-6-linked sialic acid residues (34, 35). Addition-

ally, CD22 has a function in T-cell activation viabinding to CD45RO, a T-cell surface glycoproteinwhich modulates the signal transduced by the T-cellreceptor (CD3/TCR complex) (36, 37).

The haptoglobin ß-chain contains terminal a2-6 linkedsialic acid residues (38), and is capable to inhibit theCD22 binding to TNF-a-activated human umbilical veinendothelial cells (20). Our data demonstrate an impor-tant biological effect of haptoglobin on B-lymphocytedistribution as well, and further support earlier indic-ations that haptoglobin modulates lymphocyte prolifera-tion (28). Antibody production, however, is not influ-enced by the haptoglobin type: reference values for se-rum immunoglobulins are not type-dependent, exceptfor IgA (39).

Many questions remain to be answered. These include:how can the haptoglobin type-dependence of B-cell ref-erence values be explained? Hp 1-1, Hp 2-1, and Hp 2-2 differ in a-polypeptide chain composition and in thepresence of high molecular mass forms (4). Therefore, itis possible that the biological effect of Hp-CD22 bindingdepends upon the presence or absence of multiple ar ora2-polypeptide chains in a specific orientation or confor-mation. Furthermore, the molar concentrations of Hp 2-2 in serum are low due to the presence of higher poly-mers, and the number of CD22 molecules occupied byhaptoglobin is lowest in Hp 2-2.

It also remains to be explained why reference values forCD4+ T-cells are influenced by the haptoglobin type.Since CD22-CD45 binding regulates TCR-mediated sig-nal transduction (36, 37), haptoglobin may also partici-pate in T-B-cell interaction. This could result in a nega-tive or positive regulation of T-cell proliferation (36,37).

Conclusion

We have demonstrated that Hp 1-1, Hp 2-1, and Hp 2-2bind to CD22 on the cell-surface of human B-lympho-cytes. Haptoglobin type is associated with the numbersof B-lymphocytes and helper T-lymphocytes present inperipheral blood and in bone marrow. This view pro-vides new insights in the immunoregulatory mechanismof haptoglobin and improves the interpretation of lym-phocyte subset analysis, particularly in the clinical stag-ing of HIV-infection.

AcknowledgementsThe authors wish to thank Mrs. L. Claeys and /. Verhelle for theirskillful assistance. We are grateful to Prof. Dr. S. Baudner f (Beh-ringwerke AG, Marburg, Germany) for kindly providing the puri-fied haptoglobin preparations.

Langlois et al.: Haptoglobin polymorphism and lymphocyte distribution 205

References1. Bowman BH. Haptoglobin. In: Bowman BH, editor. Hepatic

plasma proteins. San Diego: Academic Press, 1993:159—67.2. Smithies O. Zone electrophoresis in starch gels: group varia-

tions in the serum proteins of normal human adults. BiochemJ 1955; 61:629-41.

3. Bowman BH, Yang F. DNA sequencing and chromosomal lo-cations of human plasma protein genes. In: Putnam FW, editor.The plasma proteins. Structure, function and genetic control.2nd ed. London: Academic Press, 1987:12-48.

4. Schultze HE, Heremans JF, editors. Molecular biology of hu-man proteins. Volume 1: Nature and metabolism of extracellu-lar proteins. Amsterdam: Elsevier, 1966.

5. Lange V. Der Haptoglobin Polymorphismus — nicht nur einegenetische Markierungshilfe. Anthrop Anzeiger 1992;50:281-302.

6. Langlois M, Delanghe J. Biological and clinical significanceof haptoglobin polymorphism in humans. Clin Chem 1996;42:1589-600.

7. Kendall PA, Saeed SA, Collier HOJ. Identification of an en-dogenous inhibitor of prostaglandin synthetase with haptoglo-bin and albumin. Biochem Soc Transact 1979; 7:543—5.

8. Jue D-M, Shim B-S, Kang Y-S. Inhibition of prostaglandinsynthase activity of sheep seminal vesicular gland by humanserum haptoglobin. Molec Cell Biochem 1983; 51:141-7.

9. Rantapää Dahlqvist S, Frohlander N. Haptoglobin groups andrheumatoid arthritis. Hum Hered 1985; 35:207-11.

10. Rantapää Dahlqvist S, Beckman G, Beckman L. Serum proteinmarkers in systemic lupus erythematosus. Hum Hered 1988;38:44-7.

11. Nevo S, Sutton H. Association between response to typhoidvaccination and known genetic markers. Am J Hum Genet1979; 20:461-9.

12. Friedel E, Schmidt M, Apstoloff E, Geserick G, Montag T,Kuhlmay J, Bode F. Ein wahrscheinlicher Zusammenhang zwi-schen Haptoglobintyp und immunogener Reaktivität nach Te-tanusimmunierung. Dt Gesundh-wesen 1979; 34:376—7.

13. Louagie H, Delanghe J, De Sombere I, De Buyzere M, HauserP, Leroux-Roels G. Haptoglobin polymorphism and immuneresponse after hepatitis B vaccination. Vaccine 1993;11:1188-90.

14. Oh SK, Pavlotski N, Tauber AI. Specific binding of haptoglo-bin to human neutrophils and its functional consequences. JLeukoc Biol 1990; 47:142-8.

15. Wagner L, Gessl A, Baumgartner Parzer S, Base W, WaldhäuslW, Pasternack MS. Haptoglobin phenotyping by newly devel-oped monoclonal antibodies. Demonstration of haptoglobinuptake into peripheral blood neutrophils and monocytes. J Im-munol 1996; 156:1989-96.

16. El Ghmati SM, Van Hoeyveld EM, Van Strijp JAG, CeuppensJL, Stevens EAM. Identification of haptoglobin as an alterna-tive ligand for CD lib/CD 18. J Immunol 1996; 156:2542-52.

17. Baskies AM, Chretien PB, Weiss JF, Makuch RW, BeveridgeRA, Catalona WJ, et al. Serum glycoproteins in cancer pa-tients: first reports of correlations with in vitro and in vivoparameters of cellular immunity. Cancer 1980; 45:3050—60.

18. Kudo J, Okubo H, Ikuta T, Hirata Y, Ishibashi H. Interactionof acute phase reactive (APR) proteins with lectins: its rela-tionship to lymphocyte transformation. Biomed Res 1982;3:417-21.

19. Basler MW, Burrel R. Purification of haptoglobin and its ef-fects on lymphocytes and alveolar lymphocyte responses. In-flammation 1983; 7:387-400.

20. Hanasaki K, Powell LD, Varki A. Binding of human plasmasialoglycoproteins by the B cell specific lectin CD22. Selectiverecognition of immunoglobulin M and haptoglobin. J BiolChem 1995; 270:7543-50.

21. Friedman RB, Young DS, editors. Effects of disease on clinicallaboratory tests. 2nd ed. Washington: AACC Press, 1989.

22. Saag MS. Natural history of HIV-1 disease. In: Broder S, Meri-gan TC, Bolognesi D, editors. Textbook of AIDS medicine.Baltimore: Williams & Wilkins, 1994:45-53.

23. Devreese K, De Logi E, Francart C, Heyndrickx B, PhilippeJ, Leroux-Roels G. Evaluation of the automated haematologyanalyser Sysmex NE-8000. Eur J Clin Chem Clin Biochem1991; 29:339-45.

24. Fink PC, Roemer M, Haeckel R, Fateh-Moghadam A, Delan-ghe J, Gressner AM, et al. Measurement of proteins with theBehring Nephelometer: a multicentre evaluation. J Clin ChemClin Biochem 1989; 27:261-76.

25. Whicher JT, Ritchie RF, Johnson AM, Baudner S, Bienvenu J,Blirup-Jensen S, et al. A new international reference prepara-tion for proteins in human serum (RPPHS 5). Clin Chem1994; 40:934-8.

26. Dati F, Schumann G, Thomas L, Aguzzi F, Baudner S, Bien-venu J, et al. Consensus of a group of professional societiesand diagnostic companies on guidelines for interim referenceranges for 14 proteins in serum based on standardizationagainst the IFCC/BCR/CAP reference material (CRM 470).Eur J Clin Chem Clin Biochem 1996; 34:517-20.

27. Barrow GM. Equilibria in chemical and biological systems. In:Barrow GM, editor. Physical chemistry for the life sciences.2nd ed. New York: McGraw-Hill, 1981:172-213.

28. Maes M, Cooreman W, Delanghe J, Schärpe S, Wauters A,Neels H, et al. Components of biological variation in plasmahaptoglobin: relationships to plasma fibrinogen and immunevariables, including interleukin-6 and its receptor. Clin ChimActa 1995; 239:23-35.

29. Macey MG. Immunophenotypic analysis of lymphocytes andleukemias. In: Macey MG, editor. Flow cytometry: clinical ap-plications. Oxford: Blackwell Scientific Publications,1994:67-100.

30. Shaw S. Leucocyte differentiation antigen database. In:Schlossmann SF, Boumsell L, Gilks W, Harlan JM, KishimotoT, Morimoto C, Ritz J, Shaw S, Silverstein R, Springer T, Ted-der TF, Todd RF, editors. Leucocyte typing V. Proceedings ofthe fifth International Workshop on Human Leucocyte Differ-entiation Antigens; 1993 Nov 3—7; Oxford: University Press,1995:74.

31. McCormick DJ, Atassi MZ. Hemoglobin binding with hapto-globin: delineation of the haptoglobin binding site on thealpha-chain of human hemoglobin. J Prot Chem 1990;9:735-42.

32. Barclay AN, Birkeland ML, Brown MH, Beyers AD, DavisSJ, Somoza C, Williams AF, editors. The leucocyte antigenFactsBook. London: Academic Press, 1993:148-9.

33. Law C-L, Sidorenko SP, Chandran KA, Zhao Z, Shen S-H,Fischer EH, et al. CD22 associates with protein tyrosine phos-phatase 1C, Syk, and phospholipase C- upon B cell activa-tion. J Exp Med 1996; 183:547-60.

34. Stamenkovic I, Seed B. The B cell antigen CD22 mediatesmonocyte and erythrocyte adhesion. Nature 1990; 345:74—7.

35. Engel P, Nojima , Rothstein D, Zhou L-J, Wilson GL, KehrlJH, et al. The same epitope on CD22 of B lymphocytes medi-ates the adhesion of erythrocytes, T and B lymphocytes, neu-trophils and monocytes. J Immunol 1993; 150:4719-32.

36. Law C-L, Sidorenko SP, Clark EA. Regulation of lymphocyteactivation by the cell-surface molecule CD22. Immunol Today1994; 15:442-9.

37. Clark EA, Ledbetter JA. How B and T cells talk to each other.Nature 1994; 367:425-8.

38. Turner GA. N-glycosylation of serum proteins in disease andits investigation using lectins. Clin Chim Acta 1992;208:149-71.

39. Langlois M, Delanghe J, De Buyzere M. Relation between se-rum immunoglobulin A concentration and haptoglobin type.Clin Chem 1996; 42:1722-3.

Received September 6/December 19, 1996Corresponding author: Prof. Dr. J. Delanghe, Central Laboratory1B2, University Hospital Gent, De Pintelaan 185, B-9000 Gent,Belgium