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A limiting-dilution analysis of activated circulating B cells in Crohn's disease

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A limiting-dilution analysis of activated circulating B cells in Crohn's disease
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  Journal of Clinical Immunology Vol. 10 No. 2 1990 A Limiting-Dilution Analysis of Activated Circulating B Cells in Crohn s Disease MONICA BOIRIVANT, 1 FRANCESCA QUINTIERI, 20RSOLA PUGLIESE, 2 GIUSEPPE FAMULARO, 3 STEFANO FAIS, 1 and FRANCESCO PALLONE 4 Accepted: November 2 1989 In the present study the spontaneous in vitro production of immunoglobulins G, A, and M by peripheral mononu- clear ceils was evaluated, in patients with Crohn's dis- ease, in relation to the state of B-cell activation and further characterized by limiting-dilution analysis. A total of 25 patients with Crohn's disease and 10 healthy con- trois was studied. The proportion of the transferrin re- ceptor-bearing cells in the B7+ subset was higher in active Crohn's disease patients than in either those with quiescent disease or controls. There was a significant rise in the in vitro IgG, IgM, and IgA production in patients with untreated active Crohn's disease compared to either those with untreated quiescent disease or controls. When patients were followed up from the active phase to clinical remission, a significant decrease in the production of IgG and IgM was observed. IgA levels also showed a decrease, although not reaching statistical significance. When the Ig production was analyzed by limiting dilution, no difference was observed between patients and controls in terms of either precursor frequency of Ig-producing cells or patterns of frequency distribution. In both pa- tients and controls a biphasic limiting-dilution profile was observed. This study shows that a significant B-cell activation is present in Crohn's disease patients, which is accompanied by an increase in immunoglobulin produc- tion. This study also indicates that in Crohn's disease the increased immunoglobulin production is related to an augmented B-cell clone size rather than to an increased precursor frequency. KEY WORDS: Peripheral activated B cells; immunoglobulin production; limiting-dilution analysis; Crohn's disease. ~Cattedra di Gastroenterologia I, Universit~i La Sapienza, Roma, Italy. 2Laboratorio di Immnnologia, Istituto Superiore di Sanitfi, Roma, Italy. 3Clinica Medica, Universit~i dell'Aquila, L'Aquila, Italy. 4Unith di Gastroenterologia, stituto di Oncologia Sperimentale e Clinica, Universitfi di R. Calabria, Catanzaro, Italy. INTRODUCTION In inflammatory bowel disease the frequent finding of circulating antibodies against a number of bacte- rial and food antigens, the presence of immunocom- plexes, and the detection of autoantibodies (1-5) appear to reflect a chronic activation of the immune system. In Crohn's disease evidence for an in vivo polyclonal B-cell activation has been provided in patients with active disease, apparently related to massive antigen stimulation secondary to the mu- cosal damage (6-8). In these patients, an increased proportion of peripheral blood lymphocytes dis- playing early activation antigens such as the 4F2 antigen and the transferrin receptor (TFR) is found (9-10). Several reports have shown that when cul- tured in vitro peripheral cells from these patients exhibit an increased spontaneous production of immunoglobulins of all three classes (6, 8). The mechanisms underlying this increased immunoglob- ulin production are not fully understood and it is unclear whether in Crohn's disease there is a dis- turbed cellular regulation of B-cell activation and function (6, 7, 11, 12). In the present study the spontaneous in vitro production of immunoglobulins G, A, and M by peripheral blood mononuclear cells (PBMC) was evaluated, in patients with Crohn's disease, in rela- tion to the proportion of circulating B cells express- ing activation antigens. The spontaneous in vitro immunoglobulin (Ig) production by PBMC was fur- ther characterized by performing limiting-dilution analysis (LDA). LDA is a quantitative approach to the study of immune phenomena allowing both the estimation of frequencies of cells involved in a given response and the partition analysis of these cells (13, 14). In the present study LDA was used to determine the precursor frequency of immunog]ob- 128 0271-9142/90/0300-0128506.00/0 © 1990 Plenum Publishing Corporation  ACTIVATED B CELLS IN CROHN'S DISEASE 129 ulin-producing PBMC and to investigate further the regulation of Ig production in Crohn's disease. MATERIALS AND METHODS Patients A total of 25 patients with Crohn's disease and I0 healthy controls was studied. There were 14 pa- tients with clinically active disease and I 1 in clinical remission. Disease activity was evaluated accord- ing to the simple index (15). At the time of the study 16 patients (9 active and 7 inactive) were not receiving any treatment, 5 with active disease were taking full doses of prednisone (25-30 mg/day), and 4 in clinical remission were still on prednisone (mean daily dosage, 5 mg). Six of the patients with active disease not taking any treatment at the first observation were followed-up until clinical remis- sion, achieved after 12 weeks of steroids. Four weeks from treatment withdrawal, a second blood collection from these six patients was obtained. Cell Preparation Peripheral blood mononuclear cells (PBMC) were isolated from heparinized venous blood by centrif- ugation on a Ficoll-Hypaque gradient. Cells were washed three times and suspended in complete culture medium consisting of RPMI 1640 supple- mented with 2 mM L-glutamine, 25 mM Hepes buffer, 100 U/ml penicillin, 100 txg/ml streptomycin, and 10% heat-inactivated fetal calf serum (FCS). Immunofluorescence Study Aliquots of freshly isolated PBMC were incu- bated for I hr with a monoclonal antibody specific for transferrin receptor (anti-TFR; Becton-Dickin- son, Inc., Mountain View, CA) or with a monoclo- nal antibody specific for interleukin-2 receptor (IL- 2R; CD 25 antigen) (anti-IL-2 R, Becton- Dickinson). A cytotoxic anti-B-cell monoclonal antibody (OKB7, Ortho-mune, Raritan, NJ) and 200 p~l of undiluted rabbit complement (Behring, Italy) were then added. After 30 min of incubation at 37°C, the dead B7+ cells were stained with ethid- ium bromide at a concentration of 50 txg/ml. The B cells appeared red in the nuclei and the TFR+ (or IL-2 R+) cells appeared green on the surface. The cells expressing the double phenotype B7+TFR+ (or B7+IL-2R+) showed a red nucleus and a green surface (16). Cell Cultures Cells (1 x 106) were cultured in 1.0 ml of com- plete culture medium in round-bottomed 12 x 75- mm tubes at 37°C in 5% COz. After 7 days of culture supernatants were removed after centrifugation and stored at -20°C for immunoglobulins assays. lmmunoglobulin Quantification An ELISA system was employed to quantitate IgG, IgA, IgM, or total Ig production, using a goat anti-human F(ab) 2 Ig antibody and a peroxidase- coupled goat anti-human IgG, IgA, IgM, or total Ig (Cappel-Cooper Biomedical, West Chester, PA). Serial dilutions of standard immunoglobulin G, M, and A preparations served as standards, and from these the sample IgG, IgM, and IgA values were calculated. This assay proved to detect Ig concen- trations as low as 4.6 -+ 2.4 ng/ml for total Ig, 7.2 - 3.7 ng/ml for IgG, 8.4 + 5 ng/ml for IgM, and 8 -+ 5 ng/ml for IgA. Limiting-Dilution Cultures PBMC were cultured in flat-bottomed 96-well microtiter plates at graded concentrations ranging from 20 to 6000 cells/well with adherent irradiated (4000 R) autologous cells as a feeder layer in 0.2 mt of complete medium. Autologous adherent cells were obtained by seeding PBMC at a concentra- tion of 500,000/ml in flat-bottomed 96 well micro- titer plates (100 I~l/well). After a 2-hr incubation at 37°C, nonadherent cells were removed and the adherent cells were irradiated (4000 rads). Prelim- inary experiments have shown that with this feeder layer, immunoglobulin production could be measured at concentrations as low as 6000 cells/ well or less, and that the immunoglobulin produc- tion at high cell concentrations was not modified. When tested with the ELISA system, superna- tants from adherent cells alone showed OD values comparable with those obtained with complete medium alone. After 7 days of culture the super- natants of each well were collected after plate centrifugation and stored at -20°C until tested for total immunoglobulin content. Routinely, 24 wells were set up for each cell concentration. Superna- tants were considered positive for Ig production Journal of Clinical Immunology Vol. 10 No. 2 1990  130 BOIRIVANT ET AL. percentoge of TFR cells within B7 cells 100 80 60 40 20 't',',, ~,,,z:I_z ACTIVE CO ~IIESCENT CD CONTROLS Fig, 1. Percentage of TFR cells in the subset of B7-positive cells. Open areas are the B7 subset and dashed areas are the percent- ages of B7+TFR+ cells. when OD values were greater than the mean + 3 SD of the OD values of supernatants of wells containing adherent cells alone. Frequency data were calculated from the number of negative wells at the different cell doses tested and plotted in a semilogarithmic way. The intercept of regression lines at 0.37 negative cultures extrapolates the cell dose that contains an average of one Ig-producing precursor cell per well (12). LDA was performed in 5 healthy controls and in 13 patients with Crohn's disease (7 active and 6 inactive). Seven patients (four active and three inactive) were not receiving any treatment, while six (three active and three inactive) were on prednisone. Statistical Analysis Data from immunofluorescence studies and immunoglobulin production assays were analyzed by nonparametric statistic tests: Mann-Whitney test or Wilcoxon rank sum test. LDA data were analyzed by a linear least-squares regression anal- ysis of the semilogarithmic plot of the number of cells per well vs the fraction of negative cultures. A correlation value of less than -0.98 suggests good- ness of fit with Poisson distribution. Single-hit ki- netics were tested for goodness of fit by the chi- square method. A chi-square-derived probability of >0.05 was accepted as single-hit kinetics. A com- puted y-intercept approximating 1.0 also supports the single-hit model. Each line describing frequency distribution was first drawn manually and then tested with the above-mentioned methods. Table I. Proportion of B Cells (B7+), T9+ Cells, and Activated B Cells (B7+/T9+) in PBMC B7+T9+ n B7+ (%) T9+ (%) (%) Controls 10 10.3 -+ 3.7 4.4 +- 3.4 0.8 -+ 1.7 Active Crohn's 14 13.0 + 3.0 11.5 + 5.5 4.5 +-- t.2 Quiescent Crohn's 11 9.5 -+ 5.3 8.7 -+ 6.6 1.1 +- 0.99 *P < 0.001 active vs quiescent Crohn's disease and vs controls. RESULTS Immunofluorescence Studies TFR-bearing circulating PBMC were higher in Crohn's disease patients than in controls. The pro- portion of the TFR-bearing cells in the B7+ subset was higher in active Crohn's disease patients than in either those with quiescent disease or controls (P < 0.001, Table I and Fig. 1). There was no differ- ence in TFR+, B7+, and B7+TFR+ cells between patients treated with prednisone and those without medical treatment (data not shown). A significant reduction in the proportion of B7+TFR+ cells was observed in the six patients prospectively followed-up from the active phase of the disease to clinical remission (4.3 -+ 1.3 vs 1 -+ 1%, P < 0.005) (Fig. 2). CD25+ PBMC were slightly higher in patients with active Crohn's disease (4 -+ 0.45%) than in either patients with quiescent disease (1.6 -+ 0.3%) or controls (1 -+ 0.5%). The proportion of CD25+ cells in the B7 subset was 0.66% + 0.3 in patients with active Crohn's disease and 0.3 -+ 0.3 in pa- tients with quiescent disease. No B7+ cells ap- 10Pe .rce n toge of B7+TFR+ ceils 4 2 0 ........... oct Ire remt ii |on Fig, 2, Percentage of B7+TFR+ PBMC in six patients during the active phase of the disease (left) and after remission was achieved (right). Journal of Clinical Immunology Vol. 10 No. 2 1990  ACTIVATED B CELLS IN CROHN'S DISEASE 131 Table II. Immunoglobulin t~oduction (X - SE) by Unstimulated PBMC in Healthy Controls and Crohn's Disease Patients Immunoglobulin concentration in supernatants (ng/ml) n IgG IgM IgA Controls Active Crohn' s Quiescent Crohn's 10 314 -+. 71 112 --_ 13 143 +- 52 9 4247 -+ 1039" t440 -+- 489* 2888 -+ 840* 7 1072 -+ 339 187 - 59 539 -+ 179 *P < 0.005 active vs quiescent Crohn's and controls. peared to react with the anti-CD25 moAb in con- trois. Immunoglobulin Production There was a significant rise in in vitro IgG, IgM, and IgA production in patients with untreated ac- tive Crohn's disease compared to either those with untreated quiescent disease or controls (Table II). In the five active patients receiving high-dose ste- roid treatment, the in vitro production of IgG, IgM, and IgA was within the control range (IgG, 539 --. 185 ng/ml; IgM, 131 -4-- 53 ng/ml; IgA, 339 -+ 151 ng/ml). In contrast, in the four patients requiring chronic continuous treatment with low-dose ste- roids to maintain clinical remission, the in vitro production of IgG, IgM, and IgA was significantly higher than in patients with stable clinical remission (IgG, 4069 -+ 1680 ng/ml; IgM, 645 -+ 436 ng/ml; IgA, 135 -+ 982 ng/ml; P < 0.05). In the six patients followed-up from the active phase to the clinical remission achieved with ste- roids, a significant decrease in the production of IgG and IgM was observed. IgA levels also showed a decrease, although not reaching statistical signif- icance (IgG, 3999 - 1600 vs 1035 - 493 ng/ml, P < 0.05; IgM, t0t5 + 377 vs 157 -+ 67 ng/ml, P < 0.05; IgA, 2613 -+ 1245 vs 583 - 264 ng/ml, P = 0.09) (Fig. 3). Serum levels of IgG, IgM, and IgA in the patient populations were 1228 _+ 298, 140 _+ 73, and 455 -+ 250 mg/dl in patients with active disease and 180 + 251,232 + 94, and 270 -+ 99 mg/dl in patients in remission, respectively. No correlation was found between the serum concentration of each class and the in vitro spontaneous production (IgG, r = 0.14; IgM, r = 0.12; IgA, r = 0. I0, P > 0.05). Limiting-Dilution Analysis To analyze Ig production further, experiments of limiting-dilution analysis were carried out. A bipha- sic LDA profile was observed both in controls and in patients with Crohn's disease. Figure 4 shows the plots of representative experiments with cells from controls (4a), active Crohn's disease (4b), and qui- escent Crohn's disease (4c) with no medical treat- ment. The biphasic profile showed an initial slope of increasing proportion of positive wells, followed by a decrease in positive wells at higher cell input and by a second slope of increasing responding wells. Thus, two straight lines appeared to cross the ordinate at 1.0 on the semilogarithmic plot. As shown in Fig. 4, the LDA profile of Ig-producing cell precursors of patients with Crohn's disease, either active or quiescent, did not differ from that of controls. In controls, the frequency of precursor cells of the first phase ranged from 1/30 to 1/60, while the frequency of precursor cells of the second phase ranged from 1/200 to 1/400. There was no significant difference between patients and controls in terms of overall precursor frequency either in the first slope (mean values were t/33 for active Crohn's disease, 1/56 for quiescent Crohn's dis- ease, and 1/40 for controls) or in the second slope (mean values were 1/202 for active Crohn's disease, act rem act rm~ oct rem Ig G Ig M lg A Fig. 3. IgG, IgM, and IgA concentrations (ng/ml) in the super- natants of cultured PBMC from six patients with Crohn's disease during the active phase (act) of the disease and after remission (rein) was achieved. Journal of Clinical Immunology Vol. 10 No. 2 1990  132 BOIRIVANT ET AL. cells / well 0 DO 200 300 400 500 600 ~ OA g 001 ~3 cells / wel 0 100 200 300 400 500 60O o o~ b cells well 0 ~00 ZOO 3OO 4O0 ~0 o, o ....................... i 0.01t C Fig. 4. Limiting-dilution assay of Ig-producing cell precursors of controls (a), untreated active Crohn's disease (b), and untreated quiescent Crohn's disease (c). Number of cells added per culture is plotted on the abscissa. Fraction of negative cultures is plotted on the ordinate. Frequency data were calculated from the number of negative wells at the different cell doses tested. The intercept of regression lines at 0.37 negative cultures extrapolates to the cell dose that contains an average of one Ig-producing cell precursor per well. 1/333 for quiescent Crohn's disease, and 1/280 for controls). There was a wide overlap between pa- tients and controls in the precursor-cell frequency in both the initial and the second slope of the LDA profile. In active patients on prednisone (Fig. 5) the frequency of precursor cells appeared to be reduced at low cell concentrations (range, 1/360-1/500). In these patients a deviation from linearity of the semilog plot was observed with high cell concentra- tions. In this condition it is not possible to estimate of number of precursor cells. In patients with clin- ical remission receiving low-dose steroids we ob- served a monophasic distribution of values, with a number of precursor cells ranging from 1/25 to 1/40. DISCUSSION We have shown here that in active Crohn's disease peripheral B cells are activated as indicated Journal of Clinical Immunology Vol. 10 No. 2 1990
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