ADONIS 000991049100031 S
Clin. exp. Immunol. (1991) 83, 163-168
Human rib bone marrow mononuclear cells spontaneously synthesize and secrete IgE in vitro R. P. MACDERMOTT*, G. A. S. JENDRISAKt, G. S. NASHt, S. SCHREIBER*, M. J. BERTOVICH*, M. NAHMt, M. NONAKAJ, P. FITZGERALDI, D. H. KATZ: & J. F. MARCELLETTIJ *Gastroenterology Section, University of Pennsylvania School of Medicine, Philadelphia, PA, tDivision of Gastroenterology, Washington University School of Medicine, St Louis, MO, and tDivision of Immunology, Medical Biology Institute, La Jolla, CA, USA
(Acceptedfor publication 23 July 1990)
SUMMARY We have examined spontaneous secretion of IgE by human rib bone marrow mononuclear cells (MNC). Bone marrow MNC from nine out of 12 rib specimens synthesized and secreted substantial amounts of IgE during 14 days of in vitro culture. The 14-day supernatants from these bone marrow MNC contained a mean of 2589 pg/ml of IgE (n = 12) with a maximum production of 15 408 pg/ml of IgE compared with small amounts of IgE (80-200 pg/ml) produced by similarly cultured normal and inflammatory bowel disease intestinal lamina propria MNC. Using two rib specimens, timecourse studies revealed spontaneous secretion of IgE to be minimal during the first 2 days of culture (152 pg/ml), followed by a steady increase between days 4 (517 pg/ml) and 14 (3588 pg/ml). The addition of pokeweed mitogen resulted in 72% suppression of spontaneous IgE production by bone marrow MNC. The bone marrow MNC isolated from the ribs consisted of 22% Leul2+ (B) cells of which 3-2% were surface IgE positive. Staining for cytoplasmic immunoglobulin revealed I % of the bone marrow MNC to be cytoplasmic IgE+. The presence of IgE-bearing and IgE-secreting MNC in human bone marrow is consistent with the observation that allergen-specific IgE-mediated hypersensitivity is adoptively transferred by human bone marrow transplantation and demonstrates the usefulness of human bone marrow MNC for examination of IgE secretory and regulatory events. Keywords bone marrow IgE antibody secretion
INTRODUCTION Increased understanding of the production and regulation of IgE in humans is of particular importance, because of the role of IgE in allergic diseases. Thus, it is obviously important to develop a good model of in vitro human IgE synthesis. In vitro studies of human IgE synthesis have been hampered in the past by the lack of a reliable and sensitive assay to quantify IgE. Even after the development of sensitive assays, in vitro production of IgE was focused on spontaneous production by peripheral blood mononuclear cells (MNC) from atopic individuals (Fiser & Buckley, 1979; Saxon, Morrow & Stevens, 1980; Romagnani et al., 1980a, 1980b; Nonaka et al., 1981), and there is still controversy regarding the demonstration of in vitro IgE production with normal lymphocytes from a variety of tissues, including peripheral blood, spleen, or tonsils (Romagnani et al., 1980a, 1980b; Matsumoto et al., 1985; Ricci et al., 1985).
Differences in the findings have been attributed to the assays for IgE, selection of donors, in vitro culture conditions, and the use of polyclonal activators (Fiser & Buckley, 1979; Tijo, Hall & Gleica, 1979; Romagnani et al., 1980a, 1980b; Saxon et al., 1980b; Zuraw et al., 1981). Thus, there is a need for developing a good in vitro model. In order to develop an in vitro system for IgE production using MNC from healthy individuals, we selected bone marrow MNC. We chose bone marrow as the source of MNC because there have been reports of increased levels of serum IgE and the presence of IgE-bearing cells in patients with acute graft-versushost disease (GVHD) following bone marrow transplant (Saryan et al., 1983; Sehuurman et al., 1986), and also because we have previously seen heightened spontaneous production of IgG and IgA by 14-day cultured human rib bone marrow MNC from non-atopic donors (Alley, Nash & MacDermott, 1982; Alley et al., 1983; MacDermott et al., 1983). Our present study demonstrates that bone marrow MNC produce significant amounts of IgE spontaneously in the absence of any in vitro mitogenic or antigenic stimulation. The ability to study spontaneous IgE production by human bone marrow MNC will enable
Correspondence: Richard P. MacDermott, M.D., T. Grier Miller Professor of Medicine, Chief, Gastrointestinal Section, University of Pennsylvania School of Medicine, Clinical Sciences Research Building, Room 600, 422 Curie Blvd, Philadelphia, PA 19104-6144, USA.
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a better understanding of the regulatory control mechanisms involved in human IgE synthesis and secretion.
MATERIALS AND METHODS Isolation of MNCfrom human rib bone marrow, peripheral blood and intestine Ribs were obtained at the time of surgery from patients (age range 35-75 years) undergoing thoracotomy for lung cancer. None of the patients had metastatic disease, and none was malnourished. None had received immunosuppressives, chemotherapeutic agents, or radiation therapy. The ribs were immediately transported from the operating room to the laboratory. As previously described (Alley & MacDermott, 1980), utilizing sterile techniques, the ribs were split open and the bone marrow removed by scraping into 50 ml of HBSS without Ca2+ or Mg'+, and with 2-5 mm EDTA plus 500 U of sodium heparin. The EDTA-heparinized bone marrow from the human rib specimens was then vortexed, and bone spicules allowed to settle for 10 min at room temperature. Fat globules were aspirated off the top and the cell-rich supernatant harvested. The bone spicules and debris in the bottom of the tube were discarded. The cells in the supernatants were then centrifuged at 400 g for 40 min over Ficoll-Hypaque (SG 1-077). The cells were harvested from the interface, washed once in HBSS/EDTA, and three times in HBSS without EDTA. Cells were then washed four times through 5 ml of fetal bovine serum (FBS) gradients, washed and resuspended in culture medium. MNC from human intestinal mucosa were isolated from seven control subjects; five patients with Crohn's disease and five with ulcerative colitis, as previously described (MacDermott et al., 1980). The mucosa was dissected free, minced into 2-5mm pieces and washed repeatedly in calcium-magnesium-free HBSS containing antibiotics (medium I). The mucosal pieces were washed in multiple changes of medium I containing 0.75 mM EDTA and 5% heat-inactivated pooled human serum (PHS) in order to remove the epithelial cells. The tissue was washed with HBSS (with Ca2+ and Mg2+), then incubated overnight in HBSS (with Ca2+ and Mg2+)/collagenase medium containing 60 U/ml of chromatographically purified collagenase, antibiotics, and 20% heat-inactivated PHS. Following digestion, the cells were centrifuged at 400 g for 40 min over a Ficoll-Hypaque gradient, the interface harvested, diluted with HBSS, washed, resuspended in 10 ml Percoll solution (SG 1-040), and centrifuged at 500 g for 15 min to remove debris from the collagenase digestion procedure. The resultant isolated intestinal MNC were then centrifuged through four FBS gradients, washed, and resuspended in RPMI 1640 medium. In vitro antibody synthesis and secretion Human rib bone marrow MNC and intestinal MNC were cultured in vitro in RPMI 1640 medium (with 10% FBS and antibiotics as described above) with or without a 1/100 dilution of pokeweed mitogen (PWM) (Alley et al., 1982, 1983; MacDermott et al., 1983); 0-2 ml MNC at a concentration of 2 x 106 cells/ml in RPMI 1640 assay medium were incubated at 37°C, in 5% C02, in flat-bottomed microtitre plates. Each FBS lot used was previously tested, found not to be mitogenic, but to be capable of supporting PWM-driven peripheral blood MNC antibody synthesis. Furthermore, mesenteric lymph node MNC and peripheral blood MNC processed through the same
isolation steps as bone marrow or intestine did not exhibit high spontaneous secretion of immunoglobulins (data not shown). Two rib specimens were cultured in time-course assays, and after 1, 2, 4, 7, 10, or 14 days the supernatants were harvested. Supernatants from cultured MNC were assayed for the levels of secreted IgE (performed at the Molecular Biology Institute, La Jolla, CA), or IgA, IgM, or IgG (performed at Washington University School of Medicine, St Louis, MO). Radioimmunoassay (RIA) for IgE A solid-phase RIA system was employed to measure human IgE in culture supernatants (Chen et al., 1984). The quantitative RIAs were performed at the Medical Biology Institute, La Jolla, CA, in 96-well flexible polyvinyl plates coated with 100 p1 of borate-buffered saline (BBS) containing 1 ,ug/ml extensively absorbed, affinity-purified goat anti-human IgE. After washing the plates extensively with BBS, the wells were blocked by addition of 250 p1/well of BBS containing 5% normal fetal calf serum (FCS). After incubation for I h at 23°C, 100 p1 of test sample were added to each well. After a 3-h incubation at room temperature, the plates were washed and the amount of IgE protein bound to each well was detected by the addition of 100 P1 of affinity-purified '25l-labelled goat anti-human IgE at a concentration of 200 ng/ml. After a final 2-h incubation at room temperature, the plates were washed 10 times, the wells were cut out, and the radioactivity in the individual wells was determined using an automatic gamma counter. The levels of detectability, defined as three times background binding in the absence of IgE, was on the order of 50 pg/ml. We have confirmed that this human IgE-specific RIA detects only the epsilon heavy chain, and does not cross-react with determinants present on other heavy chain classes, or with idiotypic determinants (Chen et al., 1984).
RIAs for IgA, IgG, IgG subclasses and IgM The amount of IgA, IgG, and IgM in cultured supernatants was determined at Washington University School of Medicine by separate RIAs for each isotype, using a solid-phase system (Alley et al., 1982, 1983; MacDermott et al., 1983) with commercially obtained rabbit anti-IgA, anti-IgM, and antiIgG, covalently bound to cross-linked polyacrylamide beads (BioRad, Richmond, CA). The system employed 96-well roundbottomed microtitre plates; 10 p1 of culture supernatants were added to 50 p1 of radioiodinated IgA, IgG, or IgM (25 000 ct/min) and 50 ul of the appropriate solid-phase antiserum. The plates were incubated overnight at 25°C followed by harvesting on glass fibre filter strips with a Skatron microharvester (Skatron, Norway) and the individual discs counted, using an automated gamma counter (Beckman, Palo Alto, CA). The results were expressed as ng of antibody per ml of supernatant and were determined by linear regression analysis of the points on the standard curve. The amounts of IgG1, IgG2 and IgG3 were assayed at Washington University School of Medicine as previously described (Scott et al., 1986).
Surface immunoglobulin Cells (I x 106 were washed in phosphate-buffered saline (PBS) with 0-2% bovine serum albumin (BSA) and 0-2% NaN3, labelled with FITC-conjugated F(ab')2 fragments of goat antihuman IgM, IgG, IgA and IgE (Cappel Laboratories, Cochranville, PA) and incubated for 30 min at 4°C. The cells were then
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Spontaneous IgE secretion by bone marrow MNC Table 1. IgE levels in supernatants of 14-day cultured bone marrow mononuclear cells (MNC)
30r E
25F
CP
C
IgE (pg/ml)
M
784
F M F F F M M M M F M
12 1450 633 30 1117 4171 3850 269 30 15 408 3325
None None None None None None None None Morphine None None
Arithmetic mean+(s.e.m.) Geometric mean (s.e.m.)
2589 594
(1245)
Age (years)
212 214 216 227 228 229 230 238 239 240 241 242
50 60 75 48 72 56 71 35 61 66 60 61
20K -o(n 0 0 c 0
15 p
0 c
E 01
1o
0.
c
To
co
I 5
[T
en
n,
IgM
IgG
I. -
IgGI
IgG2
IgG3
IgA
Fig. 1. Spontaneous immunoglobulin secretion by human bone marrow mononuclear cells. Bars represent the arithmetic mean+ s.e.m. of the concentration of IgM, IgG, IgGl, IgG2, IgG3, and IgA present in 14-day culture supernatants without pokeweed mitogen (n = 8).
Allergy history
Sex
Rib sample number
Two-million MNC/well
were
None
(1-92)
cultured and super-
natants collected after 14 days of culture. The amount
of IgE was determined by radioimmunoassay.
washed twice and fixed with 1% paraformaldehyde. The percentage of surface-immunoglobulin-positive cells was determined using a Becton Dickinson FACS 440 after gating for the lymphocyte population using forward angle and 900 light scatter
(a) 4000 r
signals. 3000K
Cytoplasmic staining Cytopreps of bone marrow MNC were prepared and fixed with 2% paraformaldehyde for 20 min on ice. The slides were washed three times with PBS+ 1% FCS. FITC-conjugated F(ab')2 fragments of goat anti-human IgM, IgG, IgA or IgE (ICN Immunobiological) were added and the slides incubated for 30 min in humidified chambers. The slides were washed three times with PBS + 1% FBS, a drop of 1: 1 glycol/PBS was added and the slides viewed using a Zeiss photomicroscope-11. RESULTS As shown in Fig. 1, high levels of IgA, substantial amounts of IgG but minimal amounts of IgM are spontaneously secreted by pure human bone marrow MNC cultured in media alone for 14 days. Further analysis revealed that the secreted IgG was primarily IgGI, with some IgG2, and very little IgG3 (Fig. 1). Bone marrow MNC from nine out of 12 rib specimens secreted substantial amounts of IgE during 14 days of in vitro culture, as shown in Table 1. The 14-day supernatants from all 12 bone marrow cultures contained an arithmetic mean of 2589 pg/ml (geometric mean 594 pg/ml) of IgE (Table 1 and Fig. 2a) with a maximum production of 15 408 pg/ml of IgE (Table 1). As with bone marrow MNC, isolated normal intestinal MNC also exhibit high spontaneous synthesis and secretion of IgA (MacDermott et al., 1980). High spontaneous synthesis and secretion of IgG is observed by intestinal MNC from inflammatory bowel disease specimens (Scott et al., 1986). As shown in Fig. 2a and Table 2, intestinal MNC from normal Crohn's
E
2000K Ca.
1000o
ol
an
(b)
E N.1
0'1
Zi'
Donors
Fig. 2. Spontaneous IgE (a) and IgA (b) secretion by normal human bone marrow mononuclear cells (MNC) (0) (N= 12) and intestinal MNC: seven normal (U); five Crohn's disease (-); and five ulcerative colitis (U). Bars represent the arithmetic mean + s.e.m. of the concentration of immunoglobulin present in 14-day culture supernatants without pokeweed mitogen.
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R. P. MacDermott et al. Table 2. IgE secretion by human intestinal mononuclear cells (MNC) during a 14-day culture period
Immunoglobulin concentration (pg/ml) Source of intestinal MNC
Control Crohn's disease Ulcerative colitis
PWM
Medium
200 + 133 (7) 271±+ 80 (3) 82 + 39 (5) 235 + 73 (4) 111 + 79 (5) 30 +0 (3)
Mean+ s.e.m. for 14 day supernatants from the number of specimens shown in parentheses for each group. PWM, pokeweed mitogen. *
a=
In
-
IgM
cL
IgA
IgE
Fig. 4. The expression of surface IgM, IgG, IgA and IgE by human rib bone marrow mononuclear cells on day 0. Mean per cent + s.e.m. of cells positive for surface immunoglobulin (slg+). The cells were obtained from three separate human rib specimens.
0
a)
0 a 0
uL-
IgG
I
0)1
6 8 Day of harvest
10
12
Fig. 3. Time-course studies of spontaneous secretion of IgE by human bone marrow mononuclear cells. Cells from two human rib thoracotomy specimens were cultured in vitro in the absence (spontaneous secretion) (0) or presence (0) of pokeweed mitogen, and the supernatants from separate wells harvested after 1, 2, 4, 7, 10, or 14 days of culture. The amount of IgE synthesized and secreted into the supernatants was determined using a solid-phase radioimmunoassay, and the results (arithmetic mean + s.e.m.) presented in pg/ml (n = 2). 0'
disease and ulcerative colitis intestinal specimens spontaneously secreted minimal amounts of IgE, compared with cultured bone marrow MNC. Although these intestinal MNC specimens produced very little IgE (normal intestinal MNC secreted 200 pg/ml), they were able to secrete significant amounts of IgA during the same culture period (Fig. 2b). This observation demonstrates the unique aspect of the spontaneous secretion of IgE by human bone marrow MNC, and also serves as an important control since intestinal MNC spontaneously secrete large amounts of immunoglobulins (IgA, IgG and IgM) which were not non-specifically detected in the IgE RIA. To investigate the time course of IgE secretion by bone marrow cells, we examined the synthesis and secretion of IgE by two bone marrow specimens after 1, 2, 4, 7, 10 or 14 days of in vitro culture. As shown in Fig. 3, minimal IgE secretion was seen during the first 2 days (152 pg/ml) followed by steadily
0)
+ H
Fig. 5. Cytoplasmic IgM, IgG, IgA and IgE expression by human rib bone marrow mononuclear cells. The cells were obtained from three separate human rib specimens. Mean per cent + s.e.m. of cells positive for cytoplasmic immunoglobulin (cIg+).
Spontaneous IgE secretion by bone marrow MNC increasing IgE secretion after 4 days (517 pg/ml), 7 days (910 pg/ml) and 14 days (3588 pg/ml) ofculture. The addition of PWM at culture initiation resulted in 72% suppression of the spontaneous IgE production, as previously observed by us for IgA and IgG production (Alley et al., 1982, 1983; MacDermott et
al., 1983).
The expression of cell surface and cytoplasmic IgE by human bone marrow MNC was also examined. Bone marrow MNC were isolated from ribs and stained with F(ab')2 fragments of FITC-labelled goat anti-human IgM, IgG, IgA and IgE antibodies. As shown in Fig. 4, freshly isolated bone marrow MNC predominantly expressed surface (s) IgG (8%), sIgM (5%), followed by sIgE (3-2%) and sIgA (2%). Analysis of cytoplasmic (c) immunoglobulin expression on day 0 revealed the predominant expression of cIgG (11%) and cIgA (6%) followed by lower numbers of cIgM (15%) and cIgE (0 6%) bone marrow MNC (Fig. 5). No expression of sIgE or cIgE on isolated intestinal MNC was detectable. DISCUSSION We have demonstrated that human bone marrow MNC
spontaneously secrete IgE during a 14-day culture period, and contain significant number of cells with surface and cytoplasmic IgE when freshly isolated. This extends previous studies, where we observed the spontaneous production of IgG and IgA by human bone marrow MNC isolated from ribs (Alley et al., 1982). Time-course studies show predominant secretion during the latter part of the culture period, and suggest that IgEsecreting cells in human bone marrow exist as precursor or preactivated B cells which differentiate and mature during the 14 days of culture, and that IgE secreted from these cells is synthesized de novo and is not cytoplasmic carry-over from plasma cells. In addition, some immunoglobulin-secreting cells have been pre-activated and differentiated in vivo, as confirmed by the presence of cIgA, cIgG and cIgE in freshly isolated bone marrow MNC. The suppression of spontaneous IgE production by PWM is consistent with previous observations of PWM suppression of IgA (Alley et al., 1983) and suggests that additional stimulatory signals result in the down-regulation of IgE production in human bone marrow. Moreover, PWMmediated inhibition suggests that the majority of IgE secreted is newly synthesized in vitro because suppression is observed in the presence of the mitogen. There was large individual variation (up to 1000-fold) in the amount of IgE secreted by different human bone marrow MNC samples, with three out of 12 specimens secreting only minimal amounts of IgE. This could in part reflect individual variability in the propensity to secrete IgE. None of the rib donors, however, had any history of atopic disorders. The heterogeneity of IgE secretion is also similar to our previous results with regard to IgA, IgG and IgM in 14-day cultures. However, while IgA and IgG levels closely correlated with each other (specimens with high IgG had increased levels of IgA (Alley et al., 1982)), there was no correlation between IgA or IgG and IgE secretion. Although high serum levels of IgE are commonly associated with atopic conditions and parasitic infections, elevation in serum IgE is also seen in patients who develop acute GVHD following allogeneic bone marrow transplantation (Saryan et al., 1983; Sehuurman et al., 1986). Increased numbers of IgE-
167
containing plasma cells in these patients, which were suggested to be of donor origin (Sehuurman et al., 1986). related to increased serum IgE levels and in parallel to an increase in activated IgE-specific helper T cells with diminished IgE-specific suppressor T cells (Saryan et al., 1983). The present study shows that human bone marrow contains cells capable of IgE production which could be transferred during allogeneic bone marrow transplantation, ultimately leading to the observed elevated levels of IgE in patients with acute GVHD. Consistent with this idea, recent studies (Agosti et al., 1988) demonstrated bone marrow transplantation transfers IgE-mediated hypersensitivity in eight out of 11 cases, including allergic rhinitis and asthma. The observations in our present study support and extend the clinical findings of Agosti et al. (1988) in that we have observed that IgE-bearing cells and IgE precursor cells are present within the human bone marrow MNC population. Furthermore, because we used human ribs, rather than aspirates, the results are not due to contamination with peripheral blood MNC (MacDermott et al., 1983). The pattern of expression at day 0 of surface and cytoplasmic immunoglobulin differs from the 14-day culture secreted immunoglobulin isotype profile. In freshly isolated bone marrow MNC, there is predominant expression of sIgG with lower levels of sIgA, sIgM and sIgE expression, while the predominant cytoplasmic immunoglobulin is IgG, followed by IgA. In contrast, the spontaneous immunoglobulin-secretion pattern is different, with little secretion of IgM and predominant secretion of IgA followed by IgG. These results suggest that factors which regulate development and maturation of immunoglobulin-secreting cells may be important in controlling the in vitro production of antibodies by human bone marrow MNC. IgE responses are controlled by several factors, including specific lymphokines, such as interleukin-4 (IL-4), and interleukin-5 (IL-5) produced by activated T cells, IgE-binding factors, and IgE Fc-receptor-positive cells (Katz, 1982, 1984; Marcelletti & Katz, 1984, 1986; Snapper & Paul, 1987 (murine); Murray et al., 1987 (murine); Snapper, Finkelman & Paul, 1988 (murine); Miyajima et al., 1988). IL-4 is capable of stimulating both IgG 1, and IgE synthesis by murine B cells (Snapper & Paul, 1987; Snapper et al., 1988). IL-5 stimulates the production of IgA in the murine system (Murray et al., 1987; Miyajima et al., 1988). Since bone marrow MNC spontaneously produce immunoglobulin with the predominant isotypes being IgA, IgG 1, and IgE, it is possible that IL-4 and IL-5 are important contributors to normal human bone marrow function. The role of lymphokines in the regulation of human bone marrow B cell function, particularly with regard to those cytokines derived from bone marrow T cells, will be an important area for future
investigation. Mononuclear cells from the intestine also secrete large quantities of immunoglobulins spontaneously, in particular IgA, and in inflammatory bowel disease IgG. It was of interest to determine whether spontaneous synthesis and secretion of IgE by intestinal MNC may be of importance in the pathophysiology of inflammatory bowel disease (MacDermott & Stenson, 1988a & b). The acute and chronic cell mediated processes which induce intestinal damage in ulcerative colitis and Crohn's disease have been demonstrated to result in the production of inflammatory mediators (MacDermott & Stenson, 1988a, 1988b). However, in this study little or no secretion of IgE was observed from control, ulcerative colitis, or Crohn's disease
R. P. MacDermott et al.
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intestinal MNC. These results argue against IgE-mediated immediate hypersensitivity events contributing significantly to intestinal inflammation in either ulcerative colitis or Crohn's disease. This observation also serves as an important negative control for the present human bone marrow experiments. IgE is an important molecule for host defence mechanisms as well as being the initiating molecule responsible for human atopic diseases. The observation that IgE-secreting cells are present in human bone marrow will now provide the opportunity to examine aspects of IgE B cell development into mature plasma cells, within human bone marrow. Human bone marrow MNC will provide a valuable source for the study of the mechanisms involved in spontaneous IgE production and regulation of the IgE response in humans. This capacity will be of particular interest with regard to disorders in which elevated IgE levels are observed. ACKNOWLEDGMENTS This reasearch was supported in part by U.S. Public Health Service grants DK21474, DK33487, Al19476, and A124526, and by a grant of the Deutsche Gesellschaft fur Verdauungs- und Stoffwechselst6rungen. This work was presented in part at the 1988 Annual Meeting of the American Association of Immunologists and was published in part in abstract form in Fed. Proc. (1988) 2, 1250 (Abstract no. 5549).
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MAcDERMOTT, R.P., BEALE, M.G., ALLEY, C.D., NASH, G.S., BERTOVICH, M.J. & BRAGDON, M.J. (1983) Synthesis and secretion of IgA, IgM, and IgG by peripheral blood mononuclear cells in human disease states, by isolated human intestinal mononuclear cells, and by human bone marrow mononuclear cells from ribs. Ann. NYAcad. Sci. 409, 498. MARCELLETTI, J.F. & KATZ, D.H. (1984) FcRe+ lymphocytes and regulation of the IgE antibody system. IV. Delineation of target cells and mechanisms of action of SFA and EFA in inhibiting in vitro induction of FcRe expression. J. Immunol. 133, 2845. MARCELLETTI, J.F. & KATZ D.H. (1986) FcRe+ lymphocytes and regulation of the IgE antibody system. V. Preliminary physicochemical characterization of the T cell-selective IgE-induced regulant EIR. J. Immunol. 137, 2599. MATSUMOTO, T., YAMAGUCHI, K., SHIMAMURA, M. & HARADA, Y. (1985) In vitro IgE secreting cells in man. I. Mitogen-independent anddependent subpopulations. Clin. Allergy, 15, 61. MIYAJIMA, A., MIYATAKE, S., SCHREURS, J., DEVRIES, J., ARAI, N., YOKOTA, T. & ARAI, K.I. (1988) Coordinate regulations of immune and inflammatory responses by T cell-derived lymphokines. FASEB J. 2, 2462. MURRAY, P.D., MCKENZIE, P.T., SWAIN, S.L. & KAGNOFF, M.F. (1987) Interleukin 5 and interleukin 4 produced by Peyers patch T cells selectively enhance immunoglobulin A expression. J. Immunol. 139, 2669. NONAKA, M., ZURACO, B.L., O'HAIR, C.H. & KATZ, D.H. (1981) Stimulation of primary in vitro IgE antibody response in cultures of human peripheral mononuclear cells. J. exp. Med. 153, 1574. Ricci, M., PRETE, G.F., MAGGI, E., LANZAVECCHIA, A., SALA, P.G. & ROMAGNANI, S. (1985) In vitro synthesis of human IgE: reappraisal of a 5-year-study. Int. Arch. Allergy appl. Immunol. 77, 32. ROMAGNANI, S., MAGGI, E., PRETE, G., TRONCONE, R. & Ricci, M. (1980a) In vitro production of IgE by human peripheral blood mononuclear cells. I. Rate of IgE biosynthesis. Clin. exp. Immunol. 42, 167. ROMAGNANI, S., PRETE, G.F., MAGGI, E., TRONCONE, R., GIUDIZI, G.M., ALMERIGOGNA, F. & Ricci, M. (1980b) In vitro production of IgE by human peripheral blood mononuclear cell. II. Cells involved in the spontaneous IgE production in atopic patients. Clin. exp. Immunol. 42, 579. SARYAN, J.A., RAPPEPORT, J., LEURG, D.V., PARKMAN, R. & GEHA, R.S. (1983). Regulation of human immunoglobulin and synthesis in acute graft versus host disease. J clin Invest. 71, 556. SAXON, A., MORROW, A.L. & STEVENS, R.H. (1980) Subpopulations of circulating B cells and regulatory T cells involved in in vitro immunoglobulin E synthesis. J. clin. Invest. 65, 1457. SCOTT, M.G., NAHM, M.H., MACKE, K., NASH, G.S., BERTOVICH, M.J. & MAcDERMOTT, R.P. (1986) Spontaneous secretion of IgG subclasses by intestinal mononuclear cells: differences between ulcerative colitis, Crohn's disease, and controls. Clin. exp. Immunol. 66, 209. SEHUURMAN, H.J., VERDONCK, L.F., GEERTZEMA, J.G., LINDEN, J.A. & DEGAST, G.C. (1986) Monotypc immunoglobulin E plasma cells in an allogeneic bone marrow transplant recipient. Histopathology, 10, 963. SNAPPER, C.M. & PAUL, W.E. (1987) Interferon-gamma and B cell stimulatory factor-i reciprocally regulate Ig isotype production. Science, 236, 944. SNAPPER, C.M., FINKELMAN, F.D. & PAUL, W.E. (1988) Differential regulation of IgG, and IgE synthesis by interleukin 4. J. exp. Med. 167, 183. TIJO, A.H., HALL, W.H. & GLEICA, G.J. (1979) Production of human immunoglobulin E antibody in vitro. J. Immunol. 122, 2131. ZURAW, B.L., NONAKA, M., O'HAIR, C. & KATZ, D.H. (1981) Human IgE antibody synthesis in vitro: stimulation of IgE responses by pokeweed mitogen and selective inhibition of such responses by human suppressive factor of allergy (SFA)I. J. Immunol. 127, 1169.
Clin. exp. Immunol. (1991) 83, 163-168
Human rib bone marrow mononuclear cells spontaneously synthesize and secrete IgE in vitro R. P. MACDERMOTT*, G. A. S. JENDRISAKt, G. S. NASHt, S. SCHREIBER*, M. J. BERTOVICH*, M. NAHMt, M. NONAKAJ, P. FITZGERALDI, D. H. KATZ: & J. F. MARCELLETTIJ *Gastroenterology Section, University of Pennsylvania School of Medicine, Philadelphia, PA, tDivision of Gastroenterology, Washington University School of Medicine, St Louis, MO, and tDivision of Immunology, Medical Biology Institute, La Jolla, CA, USA
(Acceptedfor publication 23 July 1990)
SUMMARY We have examined spontaneous secretion of IgE by human rib bone marrow mononuclear cells (MNC). Bone marrow MNC from nine out of 12 rib specimens synthesized and secreted substantial amounts of IgE during 14 days of in vitro culture. The 14-day supernatants from these bone marrow MNC contained a mean of 2589 pg/ml of IgE (n = 12) with a maximum production of 15 408 pg/ml of IgE compared with small amounts of IgE (80-200 pg/ml) produced by similarly cultured normal and inflammatory bowel disease intestinal lamina propria MNC. Using two rib specimens, timecourse studies revealed spontaneous secretion of IgE to be minimal during the first 2 days of culture (152 pg/ml), followed by a steady increase between days 4 (517 pg/ml) and 14 (3588 pg/ml). The addition of pokeweed mitogen resulted in 72% suppression of spontaneous IgE production by bone marrow MNC. The bone marrow MNC isolated from the ribs consisted of 22% Leul2+ (B) cells of which 3-2% were surface IgE positive. Staining for cytoplasmic immunoglobulin revealed I % of the bone marrow MNC to be cytoplasmic IgE+. The presence of IgE-bearing and IgE-secreting MNC in human bone marrow is consistent with the observation that allergen-specific IgE-mediated hypersensitivity is adoptively transferred by human bone marrow transplantation and demonstrates the usefulness of human bone marrow MNC for examination of IgE secretory and regulatory events. Keywords bone marrow IgE antibody secretion
INTRODUCTION Increased understanding of the production and regulation of IgE in humans is of particular importance, because of the role of IgE in allergic diseases. Thus, it is obviously important to develop a good model of in vitro human IgE synthesis. In vitro studies of human IgE synthesis have been hampered in the past by the lack of a reliable and sensitive assay to quantify IgE. Even after the development of sensitive assays, in vitro production of IgE was focused on spontaneous production by peripheral blood mononuclear cells (MNC) from atopic individuals (Fiser & Buckley, 1979; Saxon, Morrow & Stevens, 1980; Romagnani et al., 1980a, 1980b; Nonaka et al., 1981), and there is still controversy regarding the demonstration of in vitro IgE production with normal lymphocytes from a variety of tissues, including peripheral blood, spleen, or tonsils (Romagnani et al., 1980a, 1980b; Matsumoto et al., 1985; Ricci et al., 1985).
Differences in the findings have been attributed to the assays for IgE, selection of donors, in vitro culture conditions, and the use of polyclonal activators (Fiser & Buckley, 1979; Tijo, Hall & Gleica, 1979; Romagnani et al., 1980a, 1980b; Saxon et al., 1980b; Zuraw et al., 1981). Thus, there is a need for developing a good in vitro model. In order to develop an in vitro system for IgE production using MNC from healthy individuals, we selected bone marrow MNC. We chose bone marrow as the source of MNC because there have been reports of increased levels of serum IgE and the presence of IgE-bearing cells in patients with acute graft-versushost disease (GVHD) following bone marrow transplant (Saryan et al., 1983; Sehuurman et al., 1986), and also because we have previously seen heightened spontaneous production of IgG and IgA by 14-day cultured human rib bone marrow MNC from non-atopic donors (Alley, Nash & MacDermott, 1982; Alley et al., 1983; MacDermott et al., 1983). Our present study demonstrates that bone marrow MNC produce significant amounts of IgE spontaneously in the absence of any in vitro mitogenic or antigenic stimulation. The ability to study spontaneous IgE production by human bone marrow MNC will enable
Correspondence: Richard P. MacDermott, M.D., T. Grier Miller Professor of Medicine, Chief, Gastrointestinal Section, University of Pennsylvania School of Medicine, Clinical Sciences Research Building, Room 600, 422 Curie Blvd, Philadelphia, PA 19104-6144, USA.
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a better understanding of the regulatory control mechanisms involved in human IgE synthesis and secretion.
MATERIALS AND METHODS Isolation of MNCfrom human rib bone marrow, peripheral blood and intestine Ribs were obtained at the time of surgery from patients (age range 35-75 years) undergoing thoracotomy for lung cancer. None of the patients had metastatic disease, and none was malnourished. None had received immunosuppressives, chemotherapeutic agents, or radiation therapy. The ribs were immediately transported from the operating room to the laboratory. As previously described (Alley & MacDermott, 1980), utilizing sterile techniques, the ribs were split open and the bone marrow removed by scraping into 50 ml of HBSS without Ca2+ or Mg'+, and with 2-5 mm EDTA plus 500 U of sodium heparin. The EDTA-heparinized bone marrow from the human rib specimens was then vortexed, and bone spicules allowed to settle for 10 min at room temperature. Fat globules were aspirated off the top and the cell-rich supernatant harvested. The bone spicules and debris in the bottom of the tube were discarded. The cells in the supernatants were then centrifuged at 400 g for 40 min over Ficoll-Hypaque (SG 1-077). The cells were harvested from the interface, washed once in HBSS/EDTA, and three times in HBSS without EDTA. Cells were then washed four times through 5 ml of fetal bovine serum (FBS) gradients, washed and resuspended in culture medium. MNC from human intestinal mucosa were isolated from seven control subjects; five patients with Crohn's disease and five with ulcerative colitis, as previously described (MacDermott et al., 1980). The mucosa was dissected free, minced into 2-5mm pieces and washed repeatedly in calcium-magnesium-free HBSS containing antibiotics (medium I). The mucosal pieces were washed in multiple changes of medium I containing 0.75 mM EDTA and 5% heat-inactivated pooled human serum (PHS) in order to remove the epithelial cells. The tissue was washed with HBSS (with Ca2+ and Mg2+), then incubated overnight in HBSS (with Ca2+ and Mg2+)/collagenase medium containing 60 U/ml of chromatographically purified collagenase, antibiotics, and 20% heat-inactivated PHS. Following digestion, the cells were centrifuged at 400 g for 40 min over a Ficoll-Hypaque gradient, the interface harvested, diluted with HBSS, washed, resuspended in 10 ml Percoll solution (SG 1-040), and centrifuged at 500 g for 15 min to remove debris from the collagenase digestion procedure. The resultant isolated intestinal MNC were then centrifuged through four FBS gradients, washed, and resuspended in RPMI 1640 medium. In vitro antibody synthesis and secretion Human rib bone marrow MNC and intestinal MNC were cultured in vitro in RPMI 1640 medium (with 10% FBS and antibiotics as described above) with or without a 1/100 dilution of pokeweed mitogen (PWM) (Alley et al., 1982, 1983; MacDermott et al., 1983); 0-2 ml MNC at a concentration of 2 x 106 cells/ml in RPMI 1640 assay medium were incubated at 37°C, in 5% C02, in flat-bottomed microtitre plates. Each FBS lot used was previously tested, found not to be mitogenic, but to be capable of supporting PWM-driven peripheral blood MNC antibody synthesis. Furthermore, mesenteric lymph node MNC and peripheral blood MNC processed through the same
isolation steps as bone marrow or intestine did not exhibit high spontaneous secretion of immunoglobulins (data not shown). Two rib specimens were cultured in time-course assays, and after 1, 2, 4, 7, 10, or 14 days the supernatants were harvested. Supernatants from cultured MNC were assayed for the levels of secreted IgE (performed at the Molecular Biology Institute, La Jolla, CA), or IgA, IgM, or IgG (performed at Washington University School of Medicine, St Louis, MO). Radioimmunoassay (RIA) for IgE A solid-phase RIA system was employed to measure human IgE in culture supernatants (Chen et al., 1984). The quantitative RIAs were performed at the Medical Biology Institute, La Jolla, CA, in 96-well flexible polyvinyl plates coated with 100 p1 of borate-buffered saline (BBS) containing 1 ,ug/ml extensively absorbed, affinity-purified goat anti-human IgE. After washing the plates extensively with BBS, the wells were blocked by addition of 250 p1/well of BBS containing 5% normal fetal calf serum (FCS). After incubation for I h at 23°C, 100 p1 of test sample were added to each well. After a 3-h incubation at room temperature, the plates were washed and the amount of IgE protein bound to each well was detected by the addition of 100 P1 of affinity-purified '25l-labelled goat anti-human IgE at a concentration of 200 ng/ml. After a final 2-h incubation at room temperature, the plates were washed 10 times, the wells were cut out, and the radioactivity in the individual wells was determined using an automatic gamma counter. The levels of detectability, defined as three times background binding in the absence of IgE, was on the order of 50 pg/ml. We have confirmed that this human IgE-specific RIA detects only the epsilon heavy chain, and does not cross-react with determinants present on other heavy chain classes, or with idiotypic determinants (Chen et al., 1984).
RIAs for IgA, IgG, IgG subclasses and IgM The amount of IgA, IgG, and IgM in cultured supernatants was determined at Washington University School of Medicine by separate RIAs for each isotype, using a solid-phase system (Alley et al., 1982, 1983; MacDermott et al., 1983) with commercially obtained rabbit anti-IgA, anti-IgM, and antiIgG, covalently bound to cross-linked polyacrylamide beads (BioRad, Richmond, CA). The system employed 96-well roundbottomed microtitre plates; 10 p1 of culture supernatants were added to 50 p1 of radioiodinated IgA, IgG, or IgM (25 000 ct/min) and 50 ul of the appropriate solid-phase antiserum. The plates were incubated overnight at 25°C followed by harvesting on glass fibre filter strips with a Skatron microharvester (Skatron, Norway) and the individual discs counted, using an automated gamma counter (Beckman, Palo Alto, CA). The results were expressed as ng of antibody per ml of supernatant and were determined by linear regression analysis of the points on the standard curve. The amounts of IgG1, IgG2 and IgG3 were assayed at Washington University School of Medicine as previously described (Scott et al., 1986).
Surface immunoglobulin Cells (I x 106 were washed in phosphate-buffered saline (PBS) with 0-2% bovine serum albumin (BSA) and 0-2% NaN3, labelled with FITC-conjugated F(ab')2 fragments of goat antihuman IgM, IgG, IgA and IgE (Cappel Laboratories, Cochranville, PA) and incubated for 30 min at 4°C. The cells were then
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Spontaneous IgE secretion by bone marrow MNC Table 1. IgE levels in supernatants of 14-day cultured bone marrow mononuclear cells (MNC)
30r E
25F
CP
C
IgE (pg/ml)
M
784
F M F F F M M M M F M
12 1450 633 30 1117 4171 3850 269 30 15 408 3325
None None None None None None None None Morphine None None
Arithmetic mean+(s.e.m.) Geometric mean (s.e.m.)
2589 594
(1245)
Age (years)
212 214 216 227 228 229 230 238 239 240 241 242
50 60 75 48 72 56 71 35 61 66 60 61
20K -o(n 0 0 c 0
15 p
0 c
E 01
1o
0.
c
To
co
I 5
[T
en
n,
IgM
IgG
I. -
IgGI
IgG2
IgG3
IgA
Fig. 1. Spontaneous immunoglobulin secretion by human bone marrow mononuclear cells. Bars represent the arithmetic mean+ s.e.m. of the concentration of IgM, IgG, IgGl, IgG2, IgG3, and IgA present in 14-day culture supernatants without pokeweed mitogen (n = 8).
Allergy history
Sex
Rib sample number
Two-million MNC/well
were
None
(1-92)
cultured and super-
natants collected after 14 days of culture. The amount
of IgE was determined by radioimmunoassay.
washed twice and fixed with 1% paraformaldehyde. The percentage of surface-immunoglobulin-positive cells was determined using a Becton Dickinson FACS 440 after gating for the lymphocyte population using forward angle and 900 light scatter
(a) 4000 r
signals. 3000K
Cytoplasmic staining Cytopreps of bone marrow MNC were prepared and fixed with 2% paraformaldehyde for 20 min on ice. The slides were washed three times with PBS+ 1% FCS. FITC-conjugated F(ab')2 fragments of goat anti-human IgM, IgG, IgA or IgE (ICN Immunobiological) were added and the slides incubated for 30 min in humidified chambers. The slides were washed three times with PBS + 1% FBS, a drop of 1: 1 glycol/PBS was added and the slides viewed using a Zeiss photomicroscope-11. RESULTS As shown in Fig. 1, high levels of IgA, substantial amounts of IgG but minimal amounts of IgM are spontaneously secreted by pure human bone marrow MNC cultured in media alone for 14 days. Further analysis revealed that the secreted IgG was primarily IgGI, with some IgG2, and very little IgG3 (Fig. 1). Bone marrow MNC from nine out of 12 rib specimens secreted substantial amounts of IgE during 14 days of in vitro culture, as shown in Table 1. The 14-day supernatants from all 12 bone marrow cultures contained an arithmetic mean of 2589 pg/ml (geometric mean 594 pg/ml) of IgE (Table 1 and Fig. 2a) with a maximum production of 15 408 pg/ml of IgE (Table 1). As with bone marrow MNC, isolated normal intestinal MNC also exhibit high spontaneous synthesis and secretion of IgA (MacDermott et al., 1980). High spontaneous synthesis and secretion of IgG is observed by intestinal MNC from inflammatory bowel disease specimens (Scott et al., 1986). As shown in Fig. 2a and Table 2, intestinal MNC from normal Crohn's
E
2000K Ca.
1000o
ol
an
(b)
E N.1
0'1
Zi'
Donors
Fig. 2. Spontaneous IgE (a) and IgA (b) secretion by normal human bone marrow mononuclear cells (MNC) (0) (N= 12) and intestinal MNC: seven normal (U); five Crohn's disease (-); and five ulcerative colitis (U). Bars represent the arithmetic mean + s.e.m. of the concentration of immunoglobulin present in 14-day culture supernatants without pokeweed mitogen.
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R. P. MacDermott et al. Table 2. IgE secretion by human intestinal mononuclear cells (MNC) during a 14-day culture period
Immunoglobulin concentration (pg/ml) Source of intestinal MNC
Control Crohn's disease Ulcerative colitis
PWM
Medium
200 + 133 (7) 271±+ 80 (3) 82 + 39 (5) 235 + 73 (4) 111 + 79 (5) 30 +0 (3)
Mean+ s.e.m. for 14 day supernatants from the number of specimens shown in parentheses for each group. PWM, pokeweed mitogen. *
a=
In
-
IgM
cL
IgA
IgE
Fig. 4. The expression of surface IgM, IgG, IgA and IgE by human rib bone marrow mononuclear cells on day 0. Mean per cent + s.e.m. of cells positive for surface immunoglobulin (slg+). The cells were obtained from three separate human rib specimens.
0
a)
0 a 0
uL-
IgG
I
0)1
6 8 Day of harvest
10
12
Fig. 3. Time-course studies of spontaneous secretion of IgE by human bone marrow mononuclear cells. Cells from two human rib thoracotomy specimens were cultured in vitro in the absence (spontaneous secretion) (0) or presence (0) of pokeweed mitogen, and the supernatants from separate wells harvested after 1, 2, 4, 7, 10, or 14 days of culture. The amount of IgE synthesized and secreted into the supernatants was determined using a solid-phase radioimmunoassay, and the results (arithmetic mean + s.e.m.) presented in pg/ml (n = 2). 0'
disease and ulcerative colitis intestinal specimens spontaneously secreted minimal amounts of IgE, compared with cultured bone marrow MNC. Although these intestinal MNC specimens produced very little IgE (normal intestinal MNC secreted 200 pg/ml), they were able to secrete significant amounts of IgA during the same culture period (Fig. 2b). This observation demonstrates the unique aspect of the spontaneous secretion of IgE by human bone marrow MNC, and also serves as an important control since intestinal MNC spontaneously secrete large amounts of immunoglobulins (IgA, IgG and IgM) which were not non-specifically detected in the IgE RIA. To investigate the time course of IgE secretion by bone marrow cells, we examined the synthesis and secretion of IgE by two bone marrow specimens after 1, 2, 4, 7, 10 or 14 days of in vitro culture. As shown in Fig. 3, minimal IgE secretion was seen during the first 2 days (152 pg/ml) followed by steadily
0)
+ H
Fig. 5. Cytoplasmic IgM, IgG, IgA and IgE expression by human rib bone marrow mononuclear cells. The cells were obtained from three separate human rib specimens. Mean per cent + s.e.m. of cells positive for cytoplasmic immunoglobulin (cIg+).
Spontaneous IgE secretion by bone marrow MNC increasing IgE secretion after 4 days (517 pg/ml), 7 days (910 pg/ml) and 14 days (3588 pg/ml) ofculture. The addition of PWM at culture initiation resulted in 72% suppression of the spontaneous IgE production, as previously observed by us for IgA and IgG production (Alley et al., 1982, 1983; MacDermott et
al., 1983).
The expression of cell surface and cytoplasmic IgE by human bone marrow MNC was also examined. Bone marrow MNC were isolated from ribs and stained with F(ab')2 fragments of FITC-labelled goat anti-human IgM, IgG, IgA and IgE antibodies. As shown in Fig. 4, freshly isolated bone marrow MNC predominantly expressed surface (s) IgG (8%), sIgM (5%), followed by sIgE (3-2%) and sIgA (2%). Analysis of cytoplasmic (c) immunoglobulin expression on day 0 revealed the predominant expression of cIgG (11%) and cIgA (6%) followed by lower numbers of cIgM (15%) and cIgE (0 6%) bone marrow MNC (Fig. 5). No expression of sIgE or cIgE on isolated intestinal MNC was detectable. DISCUSSION We have demonstrated that human bone marrow MNC
spontaneously secrete IgE during a 14-day culture period, and contain significant number of cells with surface and cytoplasmic IgE when freshly isolated. This extends previous studies, where we observed the spontaneous production of IgG and IgA by human bone marrow MNC isolated from ribs (Alley et al., 1982). Time-course studies show predominant secretion during the latter part of the culture period, and suggest that IgEsecreting cells in human bone marrow exist as precursor or preactivated B cells which differentiate and mature during the 14 days of culture, and that IgE secreted from these cells is synthesized de novo and is not cytoplasmic carry-over from plasma cells. In addition, some immunoglobulin-secreting cells have been pre-activated and differentiated in vivo, as confirmed by the presence of cIgA, cIgG and cIgE in freshly isolated bone marrow MNC. The suppression of spontaneous IgE production by PWM is consistent with previous observations of PWM suppression of IgA (Alley et al., 1983) and suggests that additional stimulatory signals result in the down-regulation of IgE production in human bone marrow. Moreover, PWMmediated inhibition suggests that the majority of IgE secreted is newly synthesized in vitro because suppression is observed in the presence of the mitogen. There was large individual variation (up to 1000-fold) in the amount of IgE secreted by different human bone marrow MNC samples, with three out of 12 specimens secreting only minimal amounts of IgE. This could in part reflect individual variability in the propensity to secrete IgE. None of the rib donors, however, had any history of atopic disorders. The heterogeneity of IgE secretion is also similar to our previous results with regard to IgA, IgG and IgM in 14-day cultures. However, while IgA and IgG levels closely correlated with each other (specimens with high IgG had increased levels of IgA (Alley et al., 1982)), there was no correlation between IgA or IgG and IgE secretion. Although high serum levels of IgE are commonly associated with atopic conditions and parasitic infections, elevation in serum IgE is also seen in patients who develop acute GVHD following allogeneic bone marrow transplantation (Saryan et al., 1983; Sehuurman et al., 1986). Increased numbers of IgE-
167
containing plasma cells in these patients, which were suggested to be of donor origin (Sehuurman et al., 1986). related to increased serum IgE levels and in parallel to an increase in activated IgE-specific helper T cells with diminished IgE-specific suppressor T cells (Saryan et al., 1983). The present study shows that human bone marrow contains cells capable of IgE production which could be transferred during allogeneic bone marrow transplantation, ultimately leading to the observed elevated levels of IgE in patients with acute GVHD. Consistent with this idea, recent studies (Agosti et al., 1988) demonstrated bone marrow transplantation transfers IgE-mediated hypersensitivity in eight out of 11 cases, including allergic rhinitis and asthma. The observations in our present study support and extend the clinical findings of Agosti et al. (1988) in that we have observed that IgE-bearing cells and IgE precursor cells are present within the human bone marrow MNC population. Furthermore, because we used human ribs, rather than aspirates, the results are not due to contamination with peripheral blood MNC (MacDermott et al., 1983). The pattern of expression at day 0 of surface and cytoplasmic immunoglobulin differs from the 14-day culture secreted immunoglobulin isotype profile. In freshly isolated bone marrow MNC, there is predominant expression of sIgG with lower levels of sIgA, sIgM and sIgE expression, while the predominant cytoplasmic immunoglobulin is IgG, followed by IgA. In contrast, the spontaneous immunoglobulin-secretion pattern is different, with little secretion of IgM and predominant secretion of IgA followed by IgG. These results suggest that factors which regulate development and maturation of immunoglobulin-secreting cells may be important in controlling the in vitro production of antibodies by human bone marrow MNC. IgE responses are controlled by several factors, including specific lymphokines, such as interleukin-4 (IL-4), and interleukin-5 (IL-5) produced by activated T cells, IgE-binding factors, and IgE Fc-receptor-positive cells (Katz, 1982, 1984; Marcelletti & Katz, 1984, 1986; Snapper & Paul, 1987 (murine); Murray et al., 1987 (murine); Snapper, Finkelman & Paul, 1988 (murine); Miyajima et al., 1988). IL-4 is capable of stimulating both IgG 1, and IgE synthesis by murine B cells (Snapper & Paul, 1987; Snapper et al., 1988). IL-5 stimulates the production of IgA in the murine system (Murray et al., 1987; Miyajima et al., 1988). Since bone marrow MNC spontaneously produce immunoglobulin with the predominant isotypes being IgA, IgG 1, and IgE, it is possible that IL-4 and IL-5 are important contributors to normal human bone marrow function. The role of lymphokines in the regulation of human bone marrow B cell function, particularly with regard to those cytokines derived from bone marrow T cells, will be an important area for future
investigation. Mononuclear cells from the intestine also secrete large quantities of immunoglobulins spontaneously, in particular IgA, and in inflammatory bowel disease IgG. It was of interest to determine whether spontaneous synthesis and secretion of IgE by intestinal MNC may be of importance in the pathophysiology of inflammatory bowel disease (MacDermott & Stenson, 1988a & b). The acute and chronic cell mediated processes which induce intestinal damage in ulcerative colitis and Crohn's disease have been demonstrated to result in the production of inflammatory mediators (MacDermott & Stenson, 1988a, 1988b). However, in this study little or no secretion of IgE was observed from control, ulcerative colitis, or Crohn's disease
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intestinal MNC. These results argue against IgE-mediated immediate hypersensitivity events contributing significantly to intestinal inflammation in either ulcerative colitis or Crohn's disease. This observation also serves as an important negative control for the present human bone marrow experiments. IgE is an important molecule for host defence mechanisms as well as being the initiating molecule responsible for human atopic diseases. The observation that IgE-secreting cells are present in human bone marrow will now provide the opportunity to examine aspects of IgE B cell development into mature plasma cells, within human bone marrow. Human bone marrow MNC will provide a valuable source for the study of the mechanisms involved in spontaneous IgE production and regulation of the IgE response in humans. This capacity will be of particular interest with regard to disorders in which elevated IgE levels are observed. ACKNOWLEDGMENTS This reasearch was supported in part by U.S. Public Health Service grants DK21474, DK33487, Al19476, and A124526, and by a grant of the Deutsche Gesellschaft fur Verdauungs- und Stoffwechselst6rungen. This work was presented in part at the 1988 Annual Meeting of the American Association of Immunologists and was published in part in abstract form in Fed. Proc. (1988) 2, 1250 (Abstract no. 5549).
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MAcDERMOTT, R.P., BEALE, M.G., ALLEY, C.D., NASH, G.S., BERTOVICH, M.J. & BRAGDON, M.J. (1983) Synthesis and secretion of IgA, IgM, and IgG by peripheral blood mononuclear cells in human disease states, by isolated human intestinal mononuclear cells, and by human bone marrow mononuclear cells from ribs. Ann. NYAcad. Sci. 409, 498. MARCELLETTI, J.F. & KATZ, D.H. (1984) FcRe+ lymphocytes and regulation of the IgE antibody system. IV. Delineation of target cells and mechanisms of action of SFA and EFA in inhibiting in vitro induction of FcRe expression. J. Immunol. 133, 2845. MARCELLETTI, J.F. & KATZ D.H. (1986) FcRe+ lymphocytes and regulation of the IgE antibody system. V. Preliminary physicochemical characterization of the T cell-selective IgE-induced regulant EIR. J. Immunol. 137, 2599. MATSUMOTO, T., YAMAGUCHI, K., SHIMAMURA, M. & HARADA, Y. (1985) In vitro IgE secreting cells in man. I. Mitogen-independent anddependent subpopulations. Clin. Allergy, 15, 61. MIYAJIMA, A., MIYATAKE, S., SCHREURS, J., DEVRIES, J., ARAI, N., YOKOTA, T. & ARAI, K.I. (1988) Coordinate regulations of immune and inflammatory responses by T cell-derived lymphokines. FASEB J. 2, 2462. MURRAY, P.D., MCKENZIE, P.T., SWAIN, S.L. & KAGNOFF, M.F. (1987) Interleukin 5 and interleukin 4 produced by Peyers patch T cells selectively enhance immunoglobulin A expression. J. Immunol. 139, 2669. NONAKA, M., ZURACO, B.L., O'HAIR, C.H. & KATZ, D.H. (1981) Stimulation of primary in vitro IgE antibody response in cultures of human peripheral mononuclear cells. J. exp. Med. 153, 1574. Ricci, M., PRETE, G.F., MAGGI, E., LANZAVECCHIA, A., SALA, P.G. & ROMAGNANI, S. (1985) In vitro synthesis of human IgE: reappraisal of a 5-year-study. Int. Arch. Allergy appl. Immunol. 77, 32. ROMAGNANI, S., MAGGI, E., PRETE, G., TRONCONE, R. & Ricci, M. (1980a) In vitro production of IgE by human peripheral blood mononuclear cells. I. Rate of IgE biosynthesis. Clin. exp. Immunol. 42, 167. ROMAGNANI, S., PRETE, G.F., MAGGI, E., TRONCONE, R., GIUDIZI, G.M., ALMERIGOGNA, F. & Ricci, M. (1980b) In vitro production of IgE by human peripheral blood mononuclear cell. II. Cells involved in the spontaneous IgE production in atopic patients. Clin. exp. Immunol. 42, 579. SARYAN, J.A., RAPPEPORT, J., LEURG, D.V., PARKMAN, R. & GEHA, R.S. (1983). Regulation of human immunoglobulin and synthesis in acute graft versus host disease. J clin Invest. 71, 556. SAXON, A., MORROW, A.L. & STEVENS, R.H. (1980) Subpopulations of circulating B cells and regulatory T cells involved in in vitro immunoglobulin E synthesis. J. clin. Invest. 65, 1457. SCOTT, M.G., NAHM, M.H., MACKE, K., NASH, G.S., BERTOVICH, M.J. & MAcDERMOTT, R.P. (1986) Spontaneous secretion of IgG subclasses by intestinal mononuclear cells: differences between ulcerative colitis, Crohn's disease, and controls. Clin. exp. Immunol. 66, 209. SEHUURMAN, H.J., VERDONCK, L.F., GEERTZEMA, J.G., LINDEN, J.A. & DEGAST, G.C. (1986) Monotypc immunoglobulin E plasma cells in an allogeneic bone marrow transplant recipient. Histopathology, 10, 963. SNAPPER, C.M. & PAUL, W.E. (1987) Interferon-gamma and B cell stimulatory factor-i reciprocally regulate Ig isotype production. Science, 236, 944. SNAPPER, C.M., FINKELMAN, F.D. & PAUL, W.E. (1988) Differential regulation of IgG, and IgE synthesis by interleukin 4. J. exp. Med. 167, 183. TIJO, A.H., HALL, W.H. & GLEICA, G.J. (1979) Production of human immunoglobulin E antibody in vitro. J. Immunol. 122, 2131. ZURAW, B.L., NONAKA, M., O'HAIR, C. & KATZ, D.H. (1981) Human IgE antibody synthesis in vitro: stimulation of IgE responses by pokeweed mitogen and selective inhibition of such responses by human suppressive factor of allergy (SFA)I. J. Immunol. 127, 1169.
