Extramedullar B lymphopoiesis in liver schistosomal granulomas ...

International Immunology, Vol. 11, No. 4, pp. 509–518

© 1999 The Japanese Society for Immunology

Extramedullar B lymphopoiesis in liver schistosomal granulomas: presence of the early stages and inhibition of the full B cell differentiation ´ ´ Maria Isabel D. Rossi1, Helio S. Dutra2, Marcia C. El-Cheikh3, Adriana Bonomo4 2,3 and Radovan Borojevic ´ ´ de Patologia, Hospital Universitario Antonio Pedro, Universidade Federal Fluminense, ´ Niteroi, Brazil ` ´ 2Programa Avancado de Biologia Celular Aplicada a Medicina, Hospital Universitario Clementino Fraga ¸ Filho, Universidade Federal do Rio de Janeiro, Brazil ˆ ´ 3Departamento de Histologia e Embriologia, Instituto de Cie ncias Biomedicas, and 4Departamento de Imunologia, Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Brazil 1Departamento

Keywords: B220, B cell precursors, B lymphocytes, c-kit, CD43, IL-7, inflammation, hematopoietic stroma, λ5, RAG-1, schistosomiasis

Abstract Inflammatory granulomatous reactions in liver elicited by schistosomal infection have been shown to function as active extramedullar myelopoietic sites, producing potentially all the myeloid lineages. We have now addressed the question of the extramedullar B lymphopoiesis in these sites. We have shown the presence of early B cell precursors (pro-B cells) in the granulomas by immunophenotyping. Their total number in the liver was equivalent to the pro-B cells in the bone marrow of one femur. In agreement with their phenotype, the RT-PCR analysis showed that these cells expressed RAG-1 and λ5 genes. However, the conversion of the pro-B to pre-B cells was not observed and no clonogenic B cell precursors could be detected in semi-solid cultures stimulated by IL-7. The granulomatous stroma was shown to produce IL-7 and express c-kit, and was able to sustain the full B lymphopoiesis in vitro. Conversely, the granuloma supernatant was shown to inhibit actively the development of B lymphocytes. We conclude that the granuloma environment elicits homing and proliferation of totipotent hematopoietic precursors, and that it is permissive for early commitment to the B cell lineage, but the full extramedullar production of B cell is abrogated by soluble factors produced inside the granulomas. Introduction In embryos, the major site of hematopoiesis is the fetal liver. At the perinatal period, liver tissue looses the ability to sustain hematopoiesis, and both lymphopoiesis and myelopoiesis are progressively transferred into the bone marrow, where they are controlled by and depend upon the medullar hematopoietic environment. In contrast to this concept stands the fact that bone marrow hematopoiesis can be complemented or substituted by extramedullar production of blood cells. This occurs in pathologies that modify the bone marrow environment, such as severe myelofibrosis, or in those in which other cells occupy the hematopoietic space, such as leukemias (1). We understand

this extramedullar hematopoiesis as a passive phenomenon, in which hematopoietic cells are dislodged from their normal place. Conversely, extramedullar production of one or several hematopoietic lineages can be actively transferred into the peripheral tissues committed by pathologic processes or submitted to experimental manipulations. In this case, the extramedullar environment that mimics the hematopoietic stroma is formed, becoming able to induce homing, proliferation and differentiation of only one or a selected set of hematopoietic lineages. Peripheral production of myeloid cells that participate in inflammatory infiltrates has been described in several experimental models (2,3), including schistosomal

Correspondence to: R. Borojevic, Caixa Postal 68021, 21941-970 Rio de Janeiro, Brazil Transmitting editor: R. L. Coffman

Received 12 May 1998, accepted 9 December 1998

510 B lymphopoiesis in liver granulomas infection in mice, in which it is mostly associated with periovular granulomas (4–6). Hepatic granulomas can be isolated from the surrounding tissue and their cell production can be monitored ex vivo. We have shown that, similar to fetal liver tissue, liver granulomas harbor pluripotent progenitor cells, and the local stroma can sustain the production of the myeloid, erythroid and megakaryocytic cell lineages (7). Inflammatory granulomatous reactions are a hallmark of schistosomiasis. Schistosome worms live in the mesenteric venous system, depositing eggs in small veins of the intestinal wall. A part of eggs leads to the formation of microabscesses from which they are released into the intestinal lumen, but approximately one-third is washed by the venous blood flow, brought into the liver and embolized in the intrahepatic portal venous radiculi. They elicit inflammatory granulomatous reactions, composed essentially of macrophages. Since schistosome eggs are a potent source of antigens, lymphocytes and eosinophils become subsequently the major component of granulomas (8). A fibrotic reaction is progressively formed by mobilization of activated hepatic stellate cells from the adjacent parenchyma, which deposit a complex extracellular matrix (9). The classical concept of granuloma being a cicatricial reaction encapsulating a foreign body has now to be complemented by the notion that granulomas also produce inflammatory cells in situ. This activity is particularly evident in the chronic phase of schistosomiasis, when a peripheral production of inflammatory cells, mainly macrophages and eosinophils, can supplement in large part the systemic bone marrow production of these lineages (7,10). Conversely, an extensive and sustained extramedullar production of the B cell lineage has not been reported, with a possible exception of peritoneal B1 cells (11). In view of the complex kinetics of granuloma cell populations including B cells, and the presence of an internal rich source of schistosome antigens, on one side, and complex controls of B lymphopoiesis, on the other, we have addressed now the question of a potential production and selection of the B cell lineage in this extramedullar site. Methods

Schistosomal infection C3H/HeN mice of both sexes were obtained from the colony bred at the Federal University of Rio de Janeiro. At 1 month of age, they were infected by a transcutaneous penetration of 30 Schistosoma mansoni cercariae (BH strain, Instituto Oswaldo Cruz). Mice were sacrificed after 45–55 or 90–100 days of infection, corresponding to the beginning of the acute and chronic phases of the disease respectively (12).

Cell lines Primary cultures of normal C3H/HeN mouse skin fibroblasts, L929 cells that secrete macrophage colony stimulating factor (M-CSF), and NIH Swiss Albino 3T3 cells transfected with the murine IL-7 gene that constitutively secrete IL-7 were obtained from the Rio de Janeiro Cell Bank (Programa Avanc¸ado de ` Biologia Celular Aplicado a Medicina, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil).

Granulomas and granuloma cell cultures Hepatic granulomas were obtained as previously described under sterile conditions, by homogenization of liver tissue followed by repeated sedimentation (13). The granulomas were washed and incubated in RPMI 1640 medium (Sigma, St Louis, MO), supplemented with 5% FBS (batch 1137; Cultilab, Campinas, Brazil or batch 40K0242; Gibco/BRL, Gaithersburg, MD), 2 g/l HEPES buffer, 100 U/ml penicillin, 100 µg/ml streptomycin, 5310–5 M β-mercaptoethanol and 2 mM L-glutamine. Granulomas were pooled, and 0.5 ml of wet granulomas was plated into a 25 cm2 tissue culture flask (Nunc, Roskilde, Denmark) and covered with 10 ml culture medium. Cells produced into the culture medium were harvested, quantified and either identified morphologically after staining of cytocentrifuge-prepared smears by standard ¨ May–Grunwald/Giemsa stains or monitored for expression of cell surface antigens. Alternatively the cells and granulomas were discarded after 24 h of culture and the conditioned medium was collected. In order to obtain cells present inside the granulomas, freshly isolated granulomas were submitted to digestion with collagenase IA (Sigma) (1 mg/ml in DMEM), for two periods of 1 h. Cells were harvested after each digestion period, layered over Percoll gradient of 25–70% (1030–1080 density) and centrifuged. The cells retained over the 70% Percoll layer were harvested and left to adhere in plastic tissue culture plates for 2–3 h or passed through a nylon wool column. Adherent cells were discarded and non-adherent cells were further analyzed or used for cell cultures. Alternatively, cells harvested after collagenase digestion were plated into 25 cm2 tissue culture flasks, in DMEM containing 10% FBS for 3 h. Non-adherent cells were washed and the adherent cells were harvested by trypsinization. They contained connective tissue cells and macrophages. Cells were maintained in the same medium and trypsinized when coming to early confluence. Trypsin-resistant macrophages were hence lost and from the third passage on these cultures contained only connective tissue cells that had been involved in liver granulomatous reactions, designated ‘GR cells’.

Bone marrow cells and cultures Procedures for initiation and maintenance of long-term B cell-producing bone marrow cultures, known as Witlock–Witte culture conditions, have been described in detail (14). Briefly, bone marrow cells of 3- to 4-week-old BALB/c mice were seeded (3–53105 cells/ml) into 25 cm2 flasks (6.5 ml/flask). Cultures were fed weekly with RPMI 1640 medium supplemented with 5% FBS, 5310–5 M β-mercaptoethanol, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin, and maintained at 37°C under 5% CO2. To initiate pre-B and B cell cultures, we seeded 53105 fresh bone marrow cells onto the established (3- to 4-week-old) stromal feeder layer. The cells were harvested weekly, counted in a hemocytometer ¨ and stained with May–Grunwald/Giemsa stains or reacted with appropriate antibodies for the flow cytometry.

Clonogenic assays Soft agar cultures for pre-B cells were performed as previously described (15). Briefly, 53104 bone marrow

B lymphopoiesis in liver granulomas 511 cells or 53105 granuloma-derived cells were plated in 1 ml ˜ of 0.3% agar (Bacto-agar; Difco, Sao Paulo, Brazil) in McCoy’s 5A medium supplemented with 15% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 5310–5 M β-mercaptoethanol, 100 U penicillin, 100 µg streptomycin, 0.4% vitamin mixture, 0.8% essential amino acids, 0.4% non-essential amino acids (all from Gibco/BRL), 0.6% of a 7.5% NaHCO3 solution and 1% sheep red blood cells (SRBC), in 35 mm2 culture plates. The recommended concentration of IL-7 (supernatant of IL-7 transfected cells) was added prior to the addition of the cells in agar medium. The methylcellulose cultures were performed as described elsewhere (16–18). Briefly, 53104 bone marrow cells or 53105 granuloma cells were plated in 35 mm2 culture plates, in 0.8% methylcellulose (4000 cps; Sigma) in Iscove’s medium containing 30% FBS, 1% deionized fraction V BSA (Sigma) and the additives used for agar cultures, with the exception of SRBC. The pre-B colony formation was stimulated with 10 ng/ml of murine recombinant (mr) IL-7 (Genzyme, Cambridge, MA). The cultures were incubated at 37°C in a fully humidified atmosphere with 5% CO2 for 7 days. Colony formation was scored using an inverted microscope, after hemolysis with 0.3% acetic acid, when needed. For morphological characterization, individual colonies were picked up from methylcellulose cultures, cytocentrifuged, fixed with ¨ methanol and stained with May–Grunwald/Giemsa stains. Alternatively, the intact agar cultures were floated onto slides and, after drying, stained with Harris’ hematoxylin. For the soft-agar cultures for macrophage c.f.u., cells were plated in 35 mm tissue culture Petri dishes, in McCoy’s 5A medium containing 0.37% agar and 20% FBS. Proliferation of macrophage c.f.u. was stimulated with 10% supernatant of L929 cells that secrete constitutively M-CSF. Formation of colonies (.50 cells) was monitored after 12 days.

FACS analysis of cell markers Single-cell suspensions of non-adherent granuloma-derived cells or of cultured cells were washed 3 times with buffered saline solution, pH 7.2, containing 0.2% BSA. Then 106 cells were incubated with mouse serum in order to saturate the Fc receptors, followed by the appropriate dilution of the antibodies, for 30 min, on ice. Cells were washed 3 times with the staining medium, and incubated for 30 min with Quantum Red–streptavidin to reveal the biotinylated reagents and finally washed twice with the staining medium. Flow cytometry analysis was performed using an argon laser tuned to a 488 nm flow cytometer (Coulter Epics 751 flow cytometer, Beckman-Coulter, Fullerton, CA or FACScalibur, Becton´ Dickinson, San Jose, CA at the Instituto Oswaldo Cruz, Rio de Janeiro, Brazil).

Antibodies and cytokines We used FITC-conjugated rat anti-mouse λ 1 1 2 chain mAb (R26-46), FITC-conjugated anti-mouse κ chain mAb (R5-240), FITC-conjugated rat anti-mouse µ chain (R6-60.2), FITC- or biotin-conjugated rat anti-mouse c-kit (CD117; 2B8), phycoerythrin (PE)-labeled rat anti-mouse Mac-1 (CD11b; M1/70), PE- or CyChrome-labeled rat anti-mouse CD5 (53-7.3), PElabeled rat anti-mouse CD43 (S7) (all from PharMingen, San Diego, CA), PE- or FITC-conjugated rat anti-mouse B220 mAb (RA3-6B2) (Gibco/BRL or PharMingen), and FITC-conjugated

rat anti-mouse CD3 (145.2C11) (Sigma). Quantum Red-conjugated streptavidin (Sigma) was used as a second-step reagent for biotinylated antibodies.

RT-PCR The total RNA was isolated from stromal cell lines or from non-adherent granuloma cells. Cells were lysed with 4 M guanidine isothiocyanate containing 0.1 M β-mercaptoethanol. The lysates were layered onto 3 ml of 5.7 M cesium chloride and ultracentrifuged overnight at 35,000 r.p.m. Alternatively, cells were lysed with TRIZOL Reagent (Gibco/ BRL) and the RNA was purified following the manufacturer’s protocol. cDNA was prepared with total RNA using reverse transcriptase (Gibco/BRL) following the standard protocol, and amplified by PCR with IL-7, stem cell factor and β-actin primers, in 30–40 cycles. The PCR products were run in 1.5% agarose gels and stained by ethidium bromide. We used a mock cDNA as negative controls, prepared with the total RNA without reverse transcriptase. DNA mol. wt markers were 1 kb DNA ladders from Gibco/BRL. Primer oligonucleotides were chosen from published cDNA sequences for murine IL-7: sense 59-AGA TGA TGG ACA CAG AAA TGC AGC-39 and antisense 59-CAA GAC CAT TAT CCA TCA CCG TGA G-39 (19); murine β-actin: sense 59-GTG GGC CGC TCT AGG CAC CA-39 and antisense 59-CTC TTT GAT GTC ACG CAC GAT TTC-39 (20); murine RAG-1: sense 59-CCA AGC TGC AGA CAT TCT TAG CAC TC-39 and antisense 59-CAA CAT CTG CCT TCA CGT CGA TCC-39 (21); murine λ5: sense 59-ATG AAG CTC AGA GTA GGA CAG AC-39 and antisense 59-CTA AGA ACA CTC AGC AGG TGA C-39 (22). All the RT-PCR reactions yielded a single band of the expected size. Results During the first days of culture, schistosomal granulomas released non-adherent cells into the culture medium (Fig. 1A). Subsequently, their number decreased and granulomas released essentially macrophages, which readily adhered to the substrate. The release of eosinophils was higher than of lymphocytes, but in granulomas obtained from mice with the chronic disease the eosinophil release was exhausted prior to the cessation of the lymphocyte production (data not shown). Roughly, half of the released lymphocytes were B cells, as identified by the B220 marker (data not shown). The analysis of surface Ig (sIg) light chain (κ 1 λ) expression showed that the produced B cells contained similar quantities of positive and negative cells (Fig. 1B). The identification of sIg– cells suggested either the presence of immature B cells or the activated B cells in the terminal stages of differentiation. Standard morphological analysis of cytosmears indicated that plasma cells were not frequent, suggesting the search for potential B cell precursors. Since pro-B cells and early pre-B cells maintain a close contact with the stroma, and are expected to be largely retained inside the granulomas, further studies were done on the total cell population harvested from collagenase-digested granulomas. The obtained cells were let to adhere on plastic surface, in order to eliminate macrophages and connective tissue cells. Non-adherent granuloma-derived cells contained 60–80% eosinophils, as shown by cytosmear analysis. In the FACS

512 B lymphopoiesis in liver granulomas

Fig. 1. (A) Cells released in vitro by schistosomal granulomas obtained from liver of one mouse. Freshly isolated granulomas were placed in culture and the release of non-adherent cells was monitored daily. Cells were identified on cytocentrifuge slides stained by standard ¨ May–Grunwald/Giemsa stains. (B) B cells released by schistosomal granulomas in vitro, during the first 72 h of culture. Expression of surface B cell-related antigens was analyzed by FACS, on cell population gated on lymphocytes. Cells labeled as sIg1 correspond to the sum of cells labeled with antibodies to either κ or λ chain. Results represent mean values of three independent experiments, in which the non-adherent cells of three to four culture flasks were pooled and reaction with antibodies was done in duplicates.

analysis, the lymphocyte gate contained 30 and 50% T cells (CD51 B220–) in the cell populations harvested from the acute and the chronic phase granulomas respectively. The acute phase granulomas contained 25–30% B cells (B2201) and the chronic ones 35%. The large majority of granulomaderived cells in the lymphocyte gate were CD431 (data not shown), expressing the surface leukosialin, in agreement with their location inside the granuloma and their tight association with the granulomatous stroma. The majority of B cells were surface IgM1. The CD5/B220 expression (Fig. 2) shows that a fraction of lymphocytes was CD51 B2201 (B1 cells). They were Mac-1- and surface IgM1, like B1a cells of the spleen. Their relative numbers increased from 15% of B lymphocytes in the acute phase to 30% in the chronic one. The presence of the putative B cell precursors was analyzed comparing the bone marrow and granuloma-derived mononuclear cells (Fig. 3A–C). A small fraction of B2201 c-kit1 cells (upper right quadrant in the Fig. 3C) was further analyzed for the CD43 expression (Fig. 3D). All the B2201 c-kit1 cells were CD431 (Fig. 3E) and a majority of B2201 CD431 cells were c-kit1 (Fig. 3F). Both in the bone marrow and in the chronic phase granulomas, the B2201 CD431 c-kit1 cells reached 10% of B cells. This population corresponds to the pro-B phenotype (23). Albeit the cells with the pro-B phenotype represented only ~1% of all the cells obtained from granulomas, their total quantity per liver was equivalent to the pro-B cell population found in one femur (Table 1). At the pro-B stage of lymphopoiesis, B cells initiate the rearrangement of Ig genes and express the λ5 light chain. In

Fig. 2. The phenotype of B2201 CD51 cells obtained from granulomas of chronically infected mice; the same phenotype pattern was observed on cells obtained from mice with acute schistosomal infection. (A) Expression of the two markers on cells in the lymphocyte gate. Expression of the surface IgM (B) and Mac-1 (C) on the B2201 CD51 cells (full line) and the corresponding negative controls (dotted line). The same pattern was observed on three independent experiments.

order to confirm the presence of early B cells in granulomas, the cDNA was prepared from granuloma cells and assayed by RT-PCR for expression of the of the λ5 gene. The assay was positive for liver granuloma-derived cells and negative for the normal liver tissue (Fig. 4), indicating the presence of early B cell precursors. The Ig genes rearrangement requires also the product of the RAG genes, which are also expressed in early T cells and in the central nervous system (21). As predicted, the cDNA from granulomas was also positive for the RAG-1 gene (Fig. 4). The conversion of pro-B to pre-B lymphocytes is marked by the progressive appearance of differentiation markers, such as the µ chain, coincident with the down-regulation of CD43 and of c-kit, and the ability to grow in the semi-solid media in the presence of IL-7 (24). Granuloma-derived nonadherent cells were studied in clonogenic assays, in semisolid agar and methylcellulose supplemented with IL-7. No lymphoid colonies were observed, while in parallel cultures of bone marrow-derived cells we obtained the expected number of colonies (Table 2). In contrast, both cell populations were able to form myeloid colonies, when stimulated with M-CSF.

B lymphopoiesis in liver granulomas 513

Fig. 3. B220 and c-kit expression on cells in the lymphocyte gate, obtained from the normal bone marrow (A), from granulomas of mice with the acute (B) and chronic schistosomal infection (C). B220 and CD43 expression on cells from granulomas obtained from mice with chronic schistosomiasis (D), and the CD43 (E) and c-kit (F) expression on the double-positive cells (full lines, and negative controls represented by dotted lines).

Table 1. Quantitation of B2201 c-kit1 cells in the bone marrow of normal C3H/HeN adult mice, and in liver granulomas of mice infected by S. mansoni, in the acute and the chronic phases of the disease (mean 6 SE) B2201 c-kit1 cells Granulomas

Total no. of cells per liver

Percent among non-adherent cells

Percent in the lymphocyte gate

No. of cells per liver

Acute phase Chronic phase

4.93107 4.83107

0.53 6 0.2 1.04 6 0.2

3.5 6 1.3 5.9 6 1.3

1.9 6 0.33105 3.7 6 0.63105

Bone marrow

Total No of cells

Percent among dispersed cells

Percent in the lymphocyte gate

No. of cells per femur

Femur

1.53107

2.4 6 0.5

7.4 6 3.0

3.7 6 0.53105

The absence of pre-B cells raised the question of the ability of the granuloma connective stroma to sustain the progression from the pro-B to the pre-B stages of the B lymphopoiesis. Connective tissue stromas were prepared from bone marrow and from granulomas obtained from mice with either acute or chronic schistosomiasis, under the Whitlock–Witte conditions, and inoculated with lymphocyte precursors obtained from Whitlock–Witte cultures established 6–10 weeks prior to the experiment. Both types of stromas were able to sustain the long-term production of B cells (Fig. 5). As expected, the same cells plated in plastic dishes

were not able to proliferate or establish secondary stromas (data not shown). The ability to sustain B lymphopoiesis requires an active production of IL-7 in the stroma layer. RTPCR analysis of the granuloma-derived stroma has shown that these cells express constitutively the IL-7 (Fig. 6). Conversely, the granuloma-derived non-adherent cells plated over the Whitlock–Witte stroma could not initiate a productive culture of B lymphocytes, although in the first weeks a shortterm relative increase of B2201 sIg– cells was detected and small lymphocyte-like colonies were observed (Fig. 7). This observation suggested that the connective tissue stroma

514 B lymphopoiesis in liver granulomas derived from granulomas was able to sustain B lymphopoiesis, but its full accomplishment was inhibited in the granuloma microenvironment. In the methylcellulose clonogenic assays, the supernatant of granulomas was able to inhibit completely the pre-B colony formation (Fig. 8). Similarly, in the Whitlock–Witte cultures, addition of the granuloma supernatant inhibited the proliferation of B cell production and stimulated the proliferation of myeloid lineages (Fig. 9). Both results are consistent with the presence of factor(s) inside the schistosomal granulomas,

Fig. 4. RT-PCR, followed by agarose gel analysis and ethidium bromide staining, of the expression of RAG-1 in (1) adult thymus, (2) bone marrow, (3) granulomas from mice with chronic schistosomiasis, (4) normal mouse liver tissue and (5) mock cDNA from granulomas. Expression of λ5 in (2) bone marrow, (3) granulomas from mice with chronic schistosomiasis and (4) normal mouse liver; (1) and (5) the mock cDNA. Expression of β-actin in thymus (1), bone marrow (2), granulomas (3), normal liver (4) and the mock cDNA.

Fig. 5. Lymphocyte proliferation in long-term cultures. Non-adherent cells from a Whitlock–Witte culture of bone marrow-derived lymphocytes were harvested and plated onto a GR cell stroma or onto a new bone marrow stroma prepared under Whitlock–Witte culture conditions. The non-adherent cells produced in vitro were counted weekly.

which can actively inhibit the lymphopoiesis from the pre-B stage on. Discussion Schistosomal infection elicits a strong inflammatory response in the host, which is particularly exacerbated after the beginning of the oviposition and the production of potent egg-derived antigens. This hyperergic reaction elicits florid tissue inflammatory infiltrates and a very high production of myeloid lineages at the systemic level. At this stage, .30% of the bone marrow is involved in the production of eosinophils (5,25). The evolution from the acute to the chronic phase of the disease is associated with an overall down-regulation of the host’s immune response and a modified pattern of tissue reaction to parasites. A variety of mechanisms have been proposed to explain this down-regulation, involving T cells,

Fig. 6. RT-PCR analysis of IL-7 expression in mouse skin fibroblasts (1), in connective tissue cells obtained from granulomas of mice with chronic schistosomiasis in an early (2) and late (3) passage, and in 3T3 cells transfected with IL-7 (4). Expression of β-actin in mouse skin fibroblasts (1), in connective tissue cells obtained from granulomas of mice with chronic schistosomiasis in an early (2) and late (4) passage, with the respective controls using the mock cDNA (3) and (5), and in 3T3 cells transfected with IL-7 (6).

Fig. 7. (A) B220 and sIg expression on non-adherent cells obtained from granulomas of schistosome-infected mice along 3 weeks of cultures under Whitlock–Witte conditions. (B) Lymphocyte-like cells tightly adherent to the bone marrow-derived stroma under the same culture conditions (phase contrast microscopy, 3485).

B lymphopoiesis in liver granulomas 515 anti-idiotypic antibodies and B cells (26–28). The overall lymphocyte participation in the granulomatous periovular reactions was reported to remain high or only slightly decreased along the evolution of the disease. In our study, the relative quantity of lymphocytes increased in the chronic phase, but this was essentially due to the decrease of

Fig. 8. Clonogenic assays for pre-B cell c.f.u., in methylcellulose cultures stimulated with IL-7, using non-adherent bone marrow cells in the presence of 10 or 20% granuloma culture supernatant. Data represent individual results of three independent experiments done in triplicate.

Fig. 9. (A) Non-adherent cells produced in Whitlock–Witte cultures of bone marrow cells, in the presence of 10% supernatant of cultures of the granulomas obtained from mice with acute or chronic schistosomiasis. Total cell numbers (T) and cells identified as lymphocytes (L) or cells of the myeloid lineages (M) on cytosmears ¨ stained by May–Grunwald/Giemsa stains are shown (mean 6 SE of two experiments done in triplicate, representing the relative increase/ decrease as compared to the control cultures maintained in the standard culture medium). (B) B220 expression on non-adherent cells produced in cultures in the presence of chronic granuloma supernatant (10%) (full line) and the control (dotted line).

eosinophils (29–31). In the chronic phase, the myelograms returned to the values close to the normal ones, suggesting that the systemic hyperplasia of the inflammatory cell lineages is accounted for by the extramedullar mobilization of immature hematopoietic cells and their peripheral proliferation inside the inflamed tissues (32). We have shown in previous studies that schistosomal granulomas contained pluripotent hematopoietic precursors and that the liver of a mouse with chronic schistosomiasis contained the quantity of pluripotent colony forming cells (CFC) roughly equivalent to 50% of a femur (7). We have now addressed the question of the potential involvement of these precursors in the local production of lymphoid lineages. Our results indicated that the liver granuloma environment was either permissive or able to induce the proliferation and differentiation of the CFC along all the hematopoietic pathways, including the B lymphopoiesis. The early B cell precursors, the pro-B cells, were identified by the presence of the pan-B antigen B220, together with the expression of CD43. This population can include precursors of other lineages, such as NK cells (33,34). In liver, ‘pit-cells’ belong to the NK lineage, and their participation in the host response to schistosomal infection or their potential activation and mobilization into granulomas is not clear. Therefore, the B2201 CD431 cells were further characterized by the presence of c-kit whose expression is associated with the clonogenic capacity of B cell precursors in vitro (35,36). Similar to the hematopoietic pluripotent cells and myeloid precursors, the B cell precursors were slightly increased in the chronic phase of the disease. The total quantity of cells expressing the pro-B phenotype per one schistosomeinfected liver was equivalent to their quantity in one femur, indicating that the potential production of B cells associated with liver granulomas may be relevant. Besides the expression of specific surface markers, early B cells proceed to the rearrangement of Ig genes, for which they express the RAG-1 recombinase. This enzyme is normally produced in the fetal but not in the adult liver, and in the adult bone marrow, associated with B lymphopoiesis. It is also expressed in the thymus, where it mediates the TCR rearrangement, as well as in the central nervous system (21). The expression of RAG-1 in the adult liver, in granulomatous inflammatory reactions, suggested the active production of B cell precursors expressing their normal activity related to the Ig gene rearrangement. This result was confirmed by the expression of the λ5 gene, coding the surrogate light chain expressed only in early B cell stages prior to the light chain gene rearrangement (reviewed in 37). The presence of early B cell precursors raised the question of whether the granulomatous stroma was able to sustain lymphopoiesis. The granuloma-derived myofibroblasts expressed the IL-7, which is an essential factor for B lymphopoiesis (38,39) and previous studies have shown that they expressed also the stem cell factor (kit ligand), which is not essential for lymphopoiesis, but can be synergetic with the IL-7 (40–43). Consequently, the granulomatous stroma can offer the required physical contact and adequate growth stimulation to the pro-B cells during the early stages of their differentiation. Since the pre-B cells that grow in clonogenic assays when stimulated with IL-7 could not be isolated from

516 B lymphopoiesis in liver granulomas Table 2. Clonogenic assays for pre-B cells and macrophage c.f.u. in cells harvested from the normal bone marrow, and granulomas from the acute and the chronic phase of schistosomiasis

Bone marrow Granuloma (acute) Granuloma–(chronic)

rm IL-7

IL-7 (conditioned medium)

M-CSF (conditioned medium)

360 6 30 0 0

380 6 85 0 0

560 6 110 32 6 25 122 6 57

Results show mean 6 SD of six experiments, in each of which nine semi-solid medium cultures were done, and are expressed per million cells used to initiate the cultures.

schistosomal liver granulomas, we addressed the question of an active inhibition of B lymphopoiesis at the pre-B stage in granulomas. The granuloma supernatant inhibited formation of both the pre-B cell colonies and was able to fully inhibit the B lymphopoiesis in Whitlock–Witte cultures of bone marrow cells. The same supernatant stimulated the myelopoiesis in long-term cultures, as well as in the soft-agar clonogenic assays (5), showing that the lymphopoiesis inhibition was specific and not due to the cytotoxicity of the assayed supernatant. The granulomas are produced around the schistosome eggs, a potent source of antigens, and they are rich in T and B lymphocytes, macrophages, eosinophils, and mast cells, which secrete a vast array of cytokines, several of which have been shown to inhibit lymphopoiesis in vitro and in vivo. IL-1, IL-3, IL-4, IFN, transforming growth factor-β, tumor necrosis factor-α and vasoactive intestinal peptide (VIP), were shown both to be present in granulomas, and to be able to induce an inhibition of the lymphopoiesis at the pre-B stage, either directly or indirectly (16,18,44,45). Several of these factors are known to induce apoptosis and the inhibition of lymphopoiesis at this stage may be related to an active interruption of this differentiation pathway. Alternatively, the simultaneous increased myelopoiesis in vitro and in vivo, and in particular the intense production of macrophages in situ (46,47), may suggest the diversion of early B cell precursors towards the differentiation of macrophages. Recent reports on the putative precursors that maintain the double capacity of differentiation into macrophages and B cells can give support to this hypothesis (48,49). The abortion of extramedullar B lymphopoiesis may be necessary, since B cell production has to be rigorously controlled in terms of selection of the clones that can efficiently interact with antigens while maintaining the tolerance to the self. We have now shown that the liver may contain large quantities of B cell precursors. An extensive hepatic production of new B cells in the site where the potent parasite antigens are present, in association with intense inflammatory reactions and high cytokine levels, as well as with tissue destruction and cicatrization, might be harmful to the systemic equilibrium of the B cell populations. The interruption of B cell production and differentiation in granulomas may prevent the generation and peripheral release of B cell clones without their appropriate selection that normally occurs in the bone marrow hematopoietic environment. The terminal differentiation of B cells in schistosomal granulomas was reported to be dependent upon the production of the

neuropeptides such as substance-P and VIP inside the granulomas (50). VIP inhibits the early B lymphopoiesis while the substance-P has no effect (45). The fine-tuning of both early and late B cell production in schistosomal granulomas may be relevant to the overall kinetics of the immune tissue response to schistosomes.

Acknowledgements This study was done with the financial support from PRONEX, FINEP, PADCT and CNPq grants of the Brazilian Ministry of Science and Technology. Dr Paul W. Kincade is gratefully acknowledged for helpfull suggestions, and for suplying cell lines and cell culture supernatants used in this study. Alvaro Bertho and Marta Santiago, Instituto Oswaldo Cruz have given valuable support in the cytofluorometric analysis of cell populations.

Abbreviations CFC M-CSF PE VIP SRBC

colony forming cells macrophage colony stimulating factor phycoerythrin vasoactive intestinal peptide sheep red blood cells

References 1 McCarthy, D. M. 1985. Fibrosis of the bone marrow: content and causes. Br. J. Haematol. 59:1. 2 Deimann, W. and Fahimi, H. D. 1980. Induction of focal hemopoiesis in adult rat liver by glucan, a macrophage activator. Lab. Invest. 42:217. 3 Perez, S. A. C., Silva, P. M. R., Martins, M. A., El-Cheikh, M. C., Coedeiro, R. S. B. and Borojevic, R. 1993. Eosinophil granulocyte proliferation induced by an intermediate factor generated in the pleural cavity of PAF-acether-injected rats. Int. Arch. Allergy Immunol. 102:368. 4 Borojevic, R., Stocker, S. and Grimaud, J. A. 1981. Hepatic eosinophil granulocitopoiesis in murine experimental schistosomiasis. Br. J. Exp. Pathol. 62:480. 5 Borojevic, R., Pinto, C. G., El-Cheikh, M. C. and Dutra, H. S. 1989. Experimental murine schistosomiasis mansoni: hyperplasia of the mono-macrophage cell lineage and stimulation of myeloid proliferation by peripheral macrophages. Braz. J. Med. Biol. Res. 22:579. 6 Maruyama, H., Higa, A., Asami, M., Owashi, M. and Nawa, Y. 1990. Extramedullary eosinopoiesis in the liver of Schistosoma japonicum-infected mice, with reference to hemopoietic stem cells. Parasitol. Res. 76:461.

B lymphopoiesis in liver granulomas 517 7 Dutra, H. S., Rossi, M. I. D., Azevedo, S. P., El-Cheikh, M. C. and Borojevic, R. 1997. Haematopoietic capacity of colony-forming cells mobilized in hepatic inflammatory reactions as compared to that of normal bone marrow cells. Res. Immunol. 148:437. 8 Moore, D. L., Grove, D. I. and Warren, K. S. 1977. The Schistosoma mansoni egg granuloma: quantitation of cell populations. J. Pathol. 121:41. ` 9 Boloukhere, M., Baldo-Correa, E. and Borojevic, R. 1993. Experimental schistosomiasis mansoni: characterization of connective tissue cells in hepatic periovular granulomas. J. Submicroscop. Cytol. Pathol. 25:505. 10 Lenzi, H. L., Lenzi, J. A., Rosman, F. C., Pelajo-Machado, M., Mota, E. M., Panasco, M. S. and Oliveira, D. N. 1995. Extramedullary hematopoiesis in murine schistosomiasis mansoni. Mem. Inst. Oswaldo Cruz. 90:169. 11 Stall, A. M., Wells, S. M. and Lam, K. P. 1996. B1 cells: unique origin and function. Semin. Immunol. 8:45. 12 Borojevic, R., Nicola, M. H. and Santos-da Silva, C. 1984. Modulation of macrophage and polymorphonuclear granulocyte inflammatory reactions in experimental Schistosomiasis mansoni. Cell. Mol. Biol. 30:37. 13 Alvarez-Silva, M., Silva, L. C. and Borojevic, R. 1993. Cell membrane-associated proteoglycans mediate extramedullar myeloid proliferation in granulomatous inflammatory reactions to schistosome eggs. J. Cell Sci. 104:477. 14 Whitlock, C. A., Robertson, D. and Witte, O. N. 1984. Murine B cell lymphopoiesis in long term culture. J. Immunol. Methods 67:353. 15 Kincade, P. W., Paige, C. J., Parkhouse, M. E. and Lee, G. 1978. Characterization of murine colony-forming B cells. I. Distribution, resistance to anti-immunoglobulin antibodies, and expression of Ia antigens. J. Immunol. 120:1289. 16 Lee, G., Namen, A. E., Gillis, S., Ellingsworth, L. R. and Kincade, P. W. 1989. Normal B cell precursors responsive to recombinant murine IL-7 and inhibition of IL-7 activity by Transforming Growth Factor-β. J. Immunol. 142:3875. 17 Suda, T., Okada, S., Suda, J., Miura, Y., Ito, M., Sudo, T., Hayashi, S.-I., Nishikawa, S.-I. and Nakaouchi, H. 1989. A stimulatory effect of recombinant murine interleukin-7 (IL-7) on B cell colony formation and an inhibitory effect of IL-1α. Blood 74:1936. 18 Lee, M. Y., Fevold, K. L., Dorshkind, K., Fukunaga, R., Nagata, S. and Rosse, C. 1993. In vivo and in vitro suppression of primary B lymphopoiesis by tumor-derived and recombinant granulocyte colony-stimulating factor. Blood 82:2062. 19 O’Garra, A., Stapleton, G., Dhar, V., Pearce, M., Schumacher, J., Rugo, H., Barbis, D., Stall, A., Cupp, J., Moore, K., Vieira, P., Mosmann, T., Whitmore, A., Arnold, L., Haughton, G. and Howard, M. 1990. Production of cytokines by mouse B cells: B lymphomas and normal B cells produce interleukin 10. Int. Immunol. 2:821. 20 Alonso, S., Minty, A., Bourlet, J. and Buckingham, M. 1986. Comparison of three actin-coding sequences in the mouse; evolutionary relationships between the actin genes of warmblooded vertebrates. J. Mol. Evol. 23:11. 21 Chun, J. J. M., Schatz, D. G., Oettinger, M. A., Jaenisch, R. and Baltimore, D. 1991. The recombination activating gene-1 (RAG1) transcript is present in the murine central nervous system. Cell 64:189. 22 Kudo, A., Sakaguchi, N. and Melchers, F. 1987. Organization of the murine Ig-related lambda 5 gene transcribed selectively in pre-B lymphocytes. EMBO J. 6:103. 23 Hardy, R. R., Carmack, C. E., Shinton, S. A., Kemp, J. D. and Hayakama, K. 1991. Resolution and characterization of pro-B and pre-pro-B cell stages in normal mouse bone marrow. J. Exp. Med. 173:1213. 24 Hunte, B. E., Hudak, S., Campbell, D., Xu, Y. and Rennick, D. 1995. flk2/flt3 ligand is a potent cofactor for the growth of primitive B cell progenitors. J. Immunol. 156:489. 25 El-Cheikh, M. and Borojevic, R. 1990. Extramedullar proliferation of eosinophil granulocytes in chronic Schistosomiasis mansoni is mediated by a factor secreted by inflammatory macrophages. Infect. Immun. 58:816. 26 Abe, T. and Colley, D. G. 1984. Modulation of Schistosoma mansoni egg-induced granuloma formation. III. Evidence for anti-

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

idiotypic, I-J-restricted, soluble T suppressor factor. J. Immunol. 132:2084. Flores Villanueva, P. O., Zheng, X. X., Storm, T. B. and Stadecker, M. J. 1996. Recombinant IL-10 and IL-10/FC treatment downregulate egg antigen-specific delayed hypersensitivity reactions and egg granuloma formation in schistosomiasis. J. Immunol. 156:3315. Jankovic, D., Cheever, A. W., Kullberg, M. C., Wynn, T. A., Yap, G., Caspar, P., Lewis, F. A., Clynes, R., Ravetch, J. V. and Sher, A. 1998. CD41 T cell-mediated granulomatous pathology in schistosomiasis is down-regulated by B cell-dependent mechanism requiring Fc receptor signaling. J. Exp. Med. 187:619. Boros, D. L., Amsden, A. F. and Hood A. T. 1982. Modulation of granulomatous hypersensitivity. IV. Immunoglobulin and Ab production by vigorous and immunomodulated liver granulomas of Schistosoma mansoni-infected mice. J. Immunol. 128:1050. ´ El-Cheikh, M. C., Dutra, H. S., Minoprio, P. and Borojevic, R. 1994. Increase of B lymphocyte number and activity during experimental murine Schistosomiasis mansoni. Braz. J. Med. Biol. Res. 27:1605. ´ Rathore, A., Sacristan, C., Ricklan, D. E., Flores Villanueva P. O. and Stadecker, M. J. 1996. In situ analysis of B7-2 costimulatory, major histocompatibility complex class II, and adhesion molecule expression in schistosomal egg granulomas. Am. J. Pathol. 149:187. Borojevic, R. 1992. Experimental murine schistosomiasis mansoni: establishment of the chronic phase of the disease. Mem. Inst. Oswaldo Cruz 87/S-IV:171. Li, S.-Y., Wasserman, R., Hayakawa, K. and Hardy, R. R. 1996. Identification of the earliest B lineage stage in mouse bone marrow. Immunity 5:527. Rolink, A., ten Boekel, E., Melchers, F., Fearon, D. T., Krop, I. and Andersson, J. 1997. A subpopulation of B2201 cells in murine bone marrow does not express CD19 and contains natural killer cell progenitors. J. Exp. Med. 183:187. Era, T., Nishikawa, S., Sudo, T., Fu-Ho, W., Ogawa, M., Kunisada, T., Hayashi, S.-I. and Nishikawa, S.-I. 1994. How B precursor cells are driven to cycle. Immunol. Rev. 137:35. Rolink, A., Haasner, D., Nishikawa, S.-I. and Melchers, F. 1993. Changes in frequencies of clonable Pre B cells during life in different lymphoid organs of mice. Blood 81:2290. Melchers, F., Haasner, D., Grawunder, U., Kalberer, C., Karasuyama, H., Winkler, T. and Rolink, A. 1994. Roles of IgH and L chains and of surrogate H and L chains in the development of cells of the B lymphocyte lineage. Annu. Rev. Immunol. 12:209. Namen, A. E., Lupton, S., Hjerrild, K., Wignall, J., Mochizuki, D. Y., Schmierer, A., Mosley, B., Masch, C.,J., Urdal, D., Gillis, S., Cosman, D. and Goodwin, R. G. 1988. Stimulation of B cell progenitors by cloned murine interleukin-7. Nature 333:571. Freeden-Jeffrey, U., Vieira, P., Lucian, L. A., McNeil, T., Burdach, S. E. G., Murray, R. 1995. Lymphopenia in interleukin (IL)-7 genedeleted mice identifies IL-7 as a nonredundant cytokine. J. Exp. Med. 181:1519 McNiece, I. K., Langley, K. E. and Zsebo, K. M. 1991. The role of recombinant stem cell factor in early B cell development. J. Immunol. 146:3785. Billips, L. G., Petitte, D., Dorshkind, K., Narayanan, R., Chiu, C.P. and Landreth, K. S. 1992. Differential roles of stromal cells, interleukin-7, and kit-ligand in the regulation of B lymphopoiesis. Blood 79:1185. Funk, P. E., Varas, A. and Witte, P. L. 1993. Activity of stem cell factor and IL-7 in combination on normal bone marrow B lineage cells. J. Immunol. 150:748. Brito, J. M. and Borojevic, R. 1997. Liver granulomas in schistosomiasis: mast cell-dependent induction of SCF expression in hepatic stellate cells is mediated by TNF-α. J. Leuk. Biol. 62:389. Wang, J., Lin, Q., Langston, H. and Cooper, M. D. 1995. Resident bone marrow macrophages produce type 1 interferons that can selectively inhibit interleukin-7-driven growth of B lineage cells. Immnunity 3:475. Shimozato, T. and Kincade, P. W. 1997. Indirect suppression of

518 B lymphopoiesis in liver granulomas IL-7-responsive B cell precursors by Vasoactive Intestinal Pepetide. J. Immunol. 158:5178. 46 Stadecker, M. J. and Wright, J. A. 1984. Distribution and kinetics of mononuclear phagocytes in granulomas elicited by eggs of Schistosoma mansoni. J. Pathol. 116:245. 47 Clark, C. R., Chen, B. D. M. and Boros, D. L. 1988. Macrophage progenitor cell and colony-stimulating factor production during granulomatous Schistosomiasis mansoni in mice. Infect. Immun. 56:2680.

48 Cumano, A., Paige, C. J., Iscove, N. N. and Brady, G. 1992. Bipotential precursors of B cells and macrophages in murine fetal liver. Nature 356:612. 49 Kee, B. L. and Paige, C. J. 1996. In vitro tracking of IL-7 responsiveness and gene expression during commitment of bipotent B cell/macrophage progenitors. Curr. Biol. 6:1159. 50 Neil, G. A., Blum, A. and Weinstock J. V. 1991. Substance P but not vasoactive intestinal peptide modulates immunoglobulin secretion in murine schistosomiasis. Cell. Immunol. 135:394.