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(Hypertension. 1995;26:432-435.)
© 1995 American Heart Association, Inc.

Articles

Enhanced Immunoglobulin Formation of Immortalized B Cells From Hypertensive Patients

Dieter Rosskopf; Kathrin Hartung; Jörg Hense; Winfried Siffert

From the Institut für Pharmakologie and Abteilung für Hämatologie (J.H.), Universitätsklinikum Essen (Germany).

	Abstract

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Abstract Increased immunoglobulin levels and leukocyte counts have frequently been reported in essential hypertension. The underlying mechanisms, however, have remained obscure. Enhanced Na⁺-H⁺ exchanger activity is another frequently observed abnormality in essential hypertension that persists in immortalized B lymphoblasts and coincides with enhanced proliferation. We investigated the capacity of B lymphoblasts from essential hypertensive patients to synthesize and secrete immunoglobulins. Six B cell lines from essential hypertensive patients with enhanced Na⁺-H⁺ exchanger phenotype and six cell lines from normotensive subjects were studied. Lymphocyte markers were visualized by immunostaining. Immunoglobulin secretion was analyzed by enzyme-linked immunosorbent assay. These cell lines did not differ with respect to B cell markers. In response to 100 nmol/L platelet-activating factor, cells from hypertensive patients proliferated distinctly more quickly and their cell number increased by 3.9±0.4-fold (mean±SD) within 4 days, whereas the number of cells from normotensive subjects increased by only 2.6±0.1-fold. Furthermore, platelet-activating factor induced average increases in IgM and IgG formation of 13.3- and 5.4-fold, respectively, in lymphoblasts from hypertensive patients, which was significantly higher than increases in cells from normotensive subjects (1.4- and 1.2-fold, respectively). Thus, lymphoblasts from hypertensive patients proliferate more quickly and secrete more immunoglobulins in response to a physiological stimulus in vitro. This may contribute to the raised immunoglobulin levels and leukocyte counts reported in vivo.

Key Words: sodium-hydrogen antiporter • B lymphocyte • immunoglobulins • platelet activation • signal transduction

	Introduction

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Several epidemiological studies have demonstrated increased serum IgG and IgA levels in patients with essential hypertension (for review see Reference 1¹ ). The elevation of IgG levels has been reported to be independent of age, sex, treatment, duration of hypertension, and the severity of the disorder.^{2 3 4 5} Furthermore, several epidemiological studies have reported an association between an increased number of white blood cells and the incidence of coronary heart disease⁶ and essential hypertension.⁷ The mechanisms responsible for these observations are unclear. Ebringer and Doyle² suggested that the elevated blood pressure chronically damages the vessel wall, which in turn exposes otherwise hidden components of the arterial texture and ultimately induces the production of autoantibodies.^{3 5} However, no such antigenic epitopes have yet been discovered. Other possibilities, such as hemoconcentration caused by an increased filtration pressure in the hypertensive circulation, have been ruled out.⁴ A family history of hypertension has been reported to be predisposing to the elevation in serum immunoglobulin concentration.⁴ Hence, it has been suggested that leukocytes from hypertensive patients exhibit an inherited yet not understood ability "to react more intensely in response to physiologic stimuli."⁴

Increased NHE activity represents another frequently observed abnormality in essential hypertension (for review see Reference 8⁸ ) that is present in approximately 30% to 50% of all hypertensive patients. We have previously demonstrated that the enhanced NHE phenotype persists in immortalized B lymphoblasts from patients with essential hypertension,⁹ which strongly suggests that this abnormality is genetically fixed. Furthermore, this phenomenon coincides with increased cell proliferation.⁹ Since formation and secretion of immunoglobulins are the natural functions of B lymphoblasts, we examined whether B cells from hypertensive patients with enhanced NHE phenotype (HT cells) would differ from cells of normotensive origin and low NHE phenotype (NT cells) with respect to immunoglobulin secretion. Our results strongly suggest that B lymphoblasts from hypertensive patients synthesize more immunoglobulins and grow more quickly in response to PAF. This may provide an alternative explanation for the increase in serum immunoglobulin levels in essential hypertension.

	Methods

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Reagents
RPMI 1640 medium, fetal calf serum, penicillin, and streptomycin were supplied by Life Technologies. Antibodies (all immunopurified, goat)—anti-human IgM, anti-human IgA, and alkaline phosphatase–conjugated anti-human immunoglobulin (all subclasses)—were purchased from Tago Immunologicals. Anti-human IgE and anti-human IgG antibodies as well as alkaline phosphatase–conjugated anti-human IgG were from Vector Laboratories. Antibodies for immunostaining were purchased either from Becton Dickinson (anti-IgM, -IgD, -IgA_1/2, -IgG, -CD23, -CD10) or from DAKO (anti-Ki67, anti-CD19, -CD2, -CD34, -CD25, -CD30, -CD38, and polyclonal rabbit anti-mouse serum). Control serum for clinical chemistry (Kontrollogen L) was supplied by Behringwerke AG, Mayer's Hämalaun Solution from E. Merck Diagnostika, and p-nitrophenyl phosphate from Serva. Naphthol AS-BI phosphate, fast red TR salt (4-chloro-2-methylbenzenediazonium salt), BSA, and all other chemicals were supplied by Sigma Chemical Co. PAF was purchased from Calbiochem and dissolved in RPMI 1640 medium containing 0.1% (wt/vol) BSA (vehicle).

Subjects and Establishment of Cell Lines
The establishment of NT and HT cell lines has been detailed previously.⁹ In brief, lymphocytes were obtained from nine healthy male normotensive subjects (age, 40.3±3.9 years; blood pressure, 122.1/77.4±7.6/5.8 mm Hg; mean±SD) with normal NHE activity and 10 male hypertensive patients (46.1±9.9 years; blood pressure, 150.0/104.0±15.1/10.2 mm Hg) with increased NHE activity.^{9 10} The normotensive individuals were free of a family history of hypertension, whereas all hypertensive patients confirmed a family history of essential hypertension.⁹ Cell lines were routinely recultivated from frozen stocks every 3 to 4 months. For reasons of feasibility, the current study was performed on six different NT and HT cell lines with low and high NHE activities, respectively.

Determination of Cell Proliferation
B lymphoblasts were subcultured in serum-free RPMI 1640 medium modified according to Kovar and Franek¹¹ and stimulated by addition of PAF or vehicle, as indicated. Cells were counted daily with a CASY cell analyzer system (Schärfe).

Measurement of Immunoglobulin Secretion
Supernatants of PAF-stimulated and control cells were harvested, centrifuged at 12 000g for 10 minutes in a microcentrifuge, and stored at -20°C. Immunoglobulin concentrations were measured by ELISA according to Mazer et al.¹² The 96-well ELISA flat-bottomed plates were coated with 100 µL goat anti-human IgM (3 µg/mL), goat anti-human IgE (3 µg/mL), or goat anti-human IgA (0.2 µg/mL) antibody for 16 hours at 4°C in coating buffer (0.2 mol/L Na₂CO₃ and 0.2 mol/L NaHCO₃, pH 9.5). ELISA plates were washed three times with washing solution consisting of 135 mmol/L NaCl, 0.1% (wt/vol) Tween 20, and 25 mmol/L Tris-HCl, pH 7.4. Supernatants (150 µL, appropriately diluted in 100 mmol/L Tris-HCl, pH 7.5, and 1% [wt/vol] BSA) were added and incubated for 2 hours at room temperature. The ELISA plates were washed again, and alkaline phosphatase–conjugated anti-human Ig antibody (1:1000 dilution in 100 mmol/L Tris-HCl, pH 7.5, and 1% [wt/vol] BSA) was added. After a 2-hour incubation followed by three washes, the ELISA plates were developed with substrate solution (2 mmol/L p-nitrophenyl phosphate, 0.5 mmol/L MgCl₂, 10 mmol/L diethanolamine, pH 9.6). The reaction was stopped by addition of 100 µL of 0.1 mol/L EDTA, and the optical density was recorded at =405 nm with an automated ELISA reader. Optical densities were calibrated in terms of immunoglobulin concentrations with the use of serial dilutions of a standard serum. For determination of IgG concentrations the method was slightly modified. The plates were coated with goat anti-human IgG (0.4 µg/mL), subsequently postcoated for 2 hours at room temperature in 3% (wt/vol) skim milk powder in washing solution, and further processed as described above. Instead of the alkaline phosphatase–conjugated antibody directed against all subclasses of human immunoglobulins, the alkaline phosphatase–conjugated goat anti-human IgG antibody was used.

Immunocharacterization of B Cell Markers
B lymphoblasts were grown in RPMI 1640 medium in the presence of 10% fetal calf serum for 2 days. Cells were harvested, washed twice in phosphate-buffered saline, transferred to glass slides, fixed with acetone, and air-dried at room temperature. Surface and intracellular antigens of the cells were visualized by the alkaline-phosphatase anti–alkaline phosphatase staining technique.¹³ Slides were incubated for 30 minutes with one of the following monoclonal antibodies: CD23, CD3, CD10, IgM, IgD, IgA_1,2, IgG, CD19, CD22, CD34, CD25, CD30, CD38, or Ki67. After thorough washing with Tris-NaCl buffer, polyclonal rabbit anti-mouse serum was added. A further washing with Tris-NaCl buffer was followed by incubation with a conjugate of alkaline phosphatase and mouse anti–alkaline phosphatase monoclonal antibody (DAKO) for 30 minutes. These last two steps were repeated twice each. The enzymatic reaction was developed by addition of naphthol AS-BI phosphate as a substrate and fast red TR salt as an indicator. After contrast staining with hemalum the slides were reviewed by light microscopy.

Statistical Analysis and Presentation of Data
Data represent means and SEM. Comparisons were performed using paired and unpaired two-tailed Mann-Whitney U tests as indicated. Differences were regarded as significant at a value of P<.05.

	Results

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Expression of B Cell Markers and Immunoglobulins in NT and HT Cell Lines
In the first set of experiments we characterized the various cell lines in terms of typical lymphocyte lineage and proliferation markers. The results for each of four representative NT and HT cell lines are summarized in Table 1. The expression of typical B cell markers was identical in all cell lines regardless of their normotensive or hypertensive origin. As expected, none of the cell lines displayed T cell markers or the CD10 and CD34 differentiation antigens. Furthermore, all cell lines expressed the proliferation markers CD24, CD30, and Ki67, with the exception of one NT cell line that was Ki67 negative. Hence, the previously observed differences in proliferation and NHE activity of HT cells do not result from a fortuitous selection process occurring during long-term cell culture or from differences in the developmental stage of the individual B cell lines. Subsequently, we determined the classes of immunoglobulins expressed on the cell surface and secreted into the culture medium using each of six defined NT and HT cell lines. As shown in Table 2, we found no significant differences between the various cell lines irrespective of their normotensive or hypertensive origin.

View this table: [in this window] [in a new window]   Table 1. Lymphocyte Markers of NT and HT Cell Lines

View this table: [in this window] [in a new window]   Table 2. Immunoglobulin Formation of NT and HT Cells

Effect of PAF on NT and HT Cell Line Proliferation
Next we analyzed the effect of PAF on the proliferation of six NT and six HT cell lines. Cells were seeded at an initial density of 2x10⁵ cells per milliliter in serum-free medium and stimulated with 100 nmol/L PAF. The results are summarized in Fig 1. PAF stimulated proliferation in both NT and HT cells. This growth-promoting effect of PAF was statistically significant already on day 3 in HT cells (P=.03; paired Mann-Whitney U test) and reached statistical significance on day 4 for both NT and HT cells (P=.03 for both NT and HT cells; paired Mann-Whitney U test). Although all cell lines responded to stimulation with PAF, HT cells proliferated distinctly more quickly in the presence of PAF, and their cell number increased from 2x10⁵ to 7.8±0.8x10⁵cells per milliliter (mean±SD) within 4 days, whereas the NT cells proliferated to only 5.2±0.2x10⁵ cells per milliliter (mean±SD).

View larger version (12K): [in this window] [in a new window]   Figure 1. Line graph shows effect of PAF on the proliferation of NT and HT cell lines. Cells were seeded at a density of approximately 2x105 cells per milliliter in serum-free medium containing PAF (100 nmol/L; squares) or vehicle (circles). B lymphoblasts were counted immediately after seeding and on days 3 and 4. Shown are mean±SEM for each of six NT (open symbols) and HT (filled symbols) cell lines. Duplicate experiments were performed on each cell line. In the absence of PAF, HT cell lines exhibited a trend toward faster proliferation than NT cells that did not reach statistical significance. PAF stimulated the proliferation of both NT and HT cells. This effect was statistically significant on day 4 (P<.03 for both NT and HT cells; paired Mann-Whitney U test for unstimulated vs PAF-stimulated cell lines). In the presence of 100 nmol/L PAF the average increase in cell counts of HT cell lines was significantly enhanced compared with that of NT cell lines (indicated by asterisks: P<.05 and P<.03 on days 3 and 4, respectively).

Basal and Stimulated Immunoglobulin Secretion From NT and HT Cell Lines
Subsequently, we determined the effect of 100 nmol/L PAF on immunoglobulin secretion in these cell lines. Since the various immunoglobulin classes were not expressed homogeneously by all cell lines (see Table 2), we analyzed PAF-stimulated IgM formation of four NT and three HT cell lines as well as PAF-stimulated IgG formation of four NT and six HT cell lines. PAF induced a significant increase in IgM and IgG concentrations in the supernatants of HT and NT cells on day 4 after stimulation (P=.04 and P=.03 for NT and HT cells, respectively; paired Mann-Whitney U test; PAF-stimulated versus unstimulated cells). Absolute values of both basal and PAF-stimulated immunoglobulin secretion displayed a wide variation and were not significantly different between NT and HT cell lines. However, the PAF-stimulated increase in immunoglobulin formation was significantly enhanced in HT cells (Fig 2). The relative increase in immunoglobulin formation (PAF-stimulated over unstimulated control cells) amounted to 1.4±0.3-fold and 13.3±5.9-fold for IgM in NT and HT cells, respectively (P=.05). For IgG formation the respective values were 1.2±0.1-fold and 5.4±2.4-fold for NT and HT cells (P=.03). It is evident that the increase in immunoglobulin concentration does not only reflect the changes in cell number induced by PAF. Since there is only a 3- to 4-fold rise in cell number compared with a 5- to 10-fold increase of immunoglobulin formation in PAF-stimulated HT cells within the same time period, PAF promotes both proliferation and immunoglobulin secretion of B lymphoblasts.

View larger version (10K): [in this window] [in a new window]   Figure 2. Bar graph shows PAF-stimulated immunoglobulin secretion from NT (open bars) and HT (filled bars) cells. Cell culture supernatants from the experiment depicted in Fig 1 were harvested on day 4. IgM and IgG concentrations were determined by ELISA. Shown is the increase in IgM and IgG formation (mean±SEM) evoked by 100 nmol/L PAF over unstimulated cells. HT cells exhibited a significantly enhanced PAF-stimulated formation of both IgM and IgG. *P=.05, **P=.03.

	Discussion

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We have recently reported that enhanced NHE activity, which frequently occurs in essential hypertension, remains conserved in immortalized B lymphoblasts from hypertensive patients.⁹ Interestingly, these cells proliferate distinctly more quickly than those from normotensive control subjects with normal NHE activity.^{9 14}

The first important aspect of the present findings is the detailed immunocharacterization of the established cells. Our data clearly indicate that NT and HT cells do not differ with respect to typical lymphocyte markers. Furthermore, the types of immunoglobulins expressed on the cell surface and secreted into the supernatants are not significantly different in NT and HT cell lines. From these findings we conclude that the previously reported differences in NHE activity and proliferation between NT and HT cells are not the reflection of a fortuitous selection process in vitro, which could have led to the establishment of, for example, incomparable oligoclonal cell lines with completely different immunological phenotypes.

The second major finding is the enhanced proliferation of PAF-stimulated HT cells compared with NT cells. Our previous studies were conducted on cells stimulated with serum, ie, an undefined mixture of various growth factors.^{9 14} The results reported here clearly demonstrate that an enhanced proliferation of HT cells can also be observed on stimulation with a single, well-defined agonist.

Finally, the data presented here clearly show an enhanced functional response of PAF-stimulated HT cells, ie, an increased formation of IgG and IgM.

These findings raise two questions: (1) what are the potential mechanisms underlying the enhanced responsiveness of HT cells? and (2) how do these findings relate to in vivo abnormalities observed in patients with essential hypertension? We will consider both questions briefly.

Available evidence suggests that the enhanced NHE activity of HT cells results neither from an overexpression of the NHE nor from a mutation in its coding gene.⁹ Furthermore, the enhanced NHE activity of HT cells is not the main determinant of their enhanced proliferation.¹⁰ Therefore, we have compared in more detail PAF-evoked intracellular signal transduction in NT and HT cells. Preliminary results suggest that PAF-stimulated HT cells also display increased Ca²⁺ signals and formation of inositol 1,4,5-trisphosphate compared with NT cells, despite similar PAF receptor densities.¹⁵ This appears to result from an enhanced activation of G_i-type G proteins.¹⁵ The underlying molecular mechanisms are currently being investigated in our laboratory.

The second question relates to the potential pathophysiological significance of our findings. There exists general agreement that an enhanced proliferation tendency is frequently observed in animal models of hypertension. For example, spontaneously hypertensive rat vascular smooth muscle cells, which also exhibit an increased NHE activity, show an enhanced proliferation tendency compared with normotensive Wistar-Kyoto rat cells.¹⁶ Whether a similar enhanced proliferation tendency is a genetically fixed abnormality also in patients with primary hypertension is much more difficult to answer. Given the enhanced proliferation of HT cells in our model system, the most immediate question to ask is whether patients with essential hypertension display enhanced white blood cell counts. In fact, some studies have demonstrated significantly increased leukocyte counts in essential hypertension and cardiovascular disease.^{6 7} It is attractive to speculate that this is a reflection of an enhanced proliferative tendency of white blood cells in vivo that could be similar to our in vitro observations. However, it is clear that the number of circulating leukocytes in humans depends on many other variables, eg, immune status and hormonal influences, and that such a simple analogy is appealing but remains unproved. Similar considerations come into play on evaluation of the significantly increased immunoglobulin formation by agonist-stimulated HT cell lines. Independent studies have confirmed enhanced immunoglobulin levels in essential hypertension that appear to correlate with a genetic predisposition to hypertension,^{2 3 4 5} although the pathophysiological significance of these findings has remained obscure. It appears at least unlikely that this phenomenon reflects a specific, hypertension-related immune response. Our data could be taken to argue that the elevated immunoglobulin levels in vivo are just another result of an enhanced cellular signal transduction as mirrored in HT cells in vitro by an enhanced proliferation and immunoglobulin synthesis. Again, this appealing concept has to await further justification.

In summary, the present report extends our previous findings of a genetically fixed enhanced proliferation of immortalized HT cells and underscores the potential use of this system to unravel the molecular mechanisms that ultimately underlie the differences in signal transduction in NT and HT cells.

	Selected Abbreviations and Acronyms

 

BSA = bovine serum albumin ELISA = enzyme-linked immunosorbent assay HT cells = B cells from hypertensive patients with enhanced NHE phenotype NHE = Na+-H+ exchanger NT cells = B cells from normotensive subjects with low NHE phenotype PAF = platelet-activating factor

	Acknowledgments

 
This study was financially supported by the Deutsche Forschungsgemeinschaft and by the Ministerium für Wissenschaft und Forschung NRW. We wish to acknowledge the expert technical assistance of G. Siffert, U. Schmücker, and D. Vidoz.

	Footnotes

 
Reprint requests to Dr Dieter Rosskopf, Institut für Pharmakologie, Universitätsklinikum Essen, Hufelandstr 55, D-45122 Essen, FRG.

Received January 17, 1995; first decision February 27, 1995; accepted May 19, 1995.

	References

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This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rosskopf, D. Articles by Siffert, W. Search for Related Content PubMed PubMed Citation Articles by Rosskopf, D. Articles by Siffert, W.