Growth and immortalization of human myeloma cells in immunodeficient severe combined immunodeficiency mice: a preclinical model.

Human multiple myeloma (MM) purified tumour cells readily undergo apoptosis in vitro. Interleukin 6 (IL-6), a main growth factor of tumour cells, has enabled the development of IL-6-dependent MM cell lines. Recently, we developed anti-gp130 monoclonal antibodies (mAbs), two of which (B1 + I2) were able to dimerize gp130 and replace IL-6 in vitro. We show here that the injection of B1 + I2 IL-6 agonistic mAbs via the inguinal subcutaneous (SC) route efficiently produced tumours in severe combined immunodeficiency (SCID) mice grafted with IL-6-dependent myeloma cell lines compared with either the intraperitoneal (IP) or abdominal surgical bursa (SB) routes. The SC tumour graft, together with Matrigel and vascular endothelial growth factor (VEGF), leads to a strong vascularization and early detection of serum human immunoglobulins (huIgs). SCID mice treated with B1 + I2 mAbs were injected with fresh MM cells from five patients, four of whom had consistent levels of huIgs, and tumour growth was present in two. For one patient, tumour plasma cells that were passed several times subcutaneously in new SCID mice, still expressed their initial markers after several months. They remained unable to grow in vitro in the presence of B1 + I2 or IL-6. The nature of the SCID factors involved and the triggered genes are under investigation.

Plasma bioavailable interleukin-6 is elevated in human immunodeficiency virus-infected patients who experience herpesvirus-associated immune restoration disease after start of highly active antiretroviral therapy.

This study compared plasma bioavailable interleukin (IL)-6 levels in 3 groups: human immunodeficiency virus (HIV)-infected patients who had a human herpesvirus (HHV)-associated immune restoration disease (IRD) during highly active antiretroviral therapy (HAART); patients who experienced an IRD initiated by Mycobacterium avium complex, hepatitis C virus, or human papillomavirus; and control patients who had uneventful immune reconstitution. Total IL-6, soluble IL-6 receptor (sIL-6R), and soluble gp130 were measured by ELISA, and levels of free IL-6 and sIL-6/IL-6R complex were modeled mathematically. Persons who had an HHV-associated IRD had increased plasma bioavailable IL-6 before HAART, compared with patients who experienced a non-HHV-associated IRD and with control patients, and their plasma bioavailable IL-6 increased progressively over 3-4 years of treatment. Increased IL-6 production may be a feature of HAART-induced restoration of immune responses to HHV infections and may have long-term immunopathologic consequences.

Optimizing therapeutic strategies to inhibit circulating soluble target molecules with monoclonal antibodies: example of the soluble IL-6 receptors.

Therapeutic targeting of soluble molecules such as cytokines can be achieved with monoclonal antibodies (mAb). Anti-IL-6 mAb have been shown to form circulating complexes, resulting in the increase of the half-life of the cytokine in vivo. In IL-6-related diseases, the soluble human IL-6 receptors (shIL-6R), which have been shown to possess strong agonist activity, circulate in the plasma at a high concentration and must be neutralized. Their clearance was studied in mice that had been made to express circulating shIL-6R after i.p. grafting of mouse thymoma cells transfected with a gene coding for shIL-6R, treated with various anti-shIL-6R mAb recognizing different epitopes of the molecule. Injection of one anti-hIL-6R mAb stabilized the short-lived hIL-6R and led to their accumulation. The same result was observed when two mAb directed against two different epitopes of the hIL-6R were used. Clearance of the receptors was only achieved when three mAb specific for three different epitopes were injected. A permanent clearing of the hIL-6R could be obtained by repeated injections of the clearing mixture. No correlation was found between the ability of the mAb to clear the sIL-6R and to immunoprecipitate them in agarose gel. The F(ab')2 fragments lost the clearing ability of the intact mAb. These results clearly show that therapeutic clearance of sIL-6R by mAb need at least three mAb directed against three different epitopes of the molecule, a conclusion which is likely to apply for clearing any soluble target molecule.

Insulin-like growth factor induces the survival and proliferation of myeloma cells through an interleukin-6-independent transduction pathway.

Multiple myeloma (MM) is a B-cell neoplasia that is associated with an increased level of bone resorption. One important mediator of bone remodelling, insulin-like growth factor (IGF-I), has been shown to stimulate the proliferation of human myeloma cells. However, the mechanisms of action of IGF-I in these cells have not been determined. Using interleukin (IL)-6-dependent myeloma cell lines, we show IGF-I to be as potent a survival and proliferation factor as IL-6. We demonstrated that IGF-I functions independently of the IL-6 transducer gp130 and that these two cytokines have additive effects. Moreover, inhibition of the IGF-I pathway did not modulate the proliferative effect of IL-6. Accordingly, we found that IL-6 and IGF-I activated distinct downstream signalling molecules: IL-6 activated STAT3 phosphorylation, whereas IGF-I treatment resulted in the phosphorylation of IRS-1. Interestingly, these signalling pathways appear to converge as both cytokines activated the ras/MAPK pathway. Thus, IGF-I acts as a potent survival and proliferation factor for myeloma cells by stimulating an IL-6-independent signalling cascade. These data, together with the finding that, in vivo, IGF-I is normally expressed in close proximity to myeloma cells within the bone matrix, strongly suggest a role for this cytokine in the pathophysiology of multiple myeloma.

Interleukin-6 receptor signaling. II. Bio-availability of interleukin-6 in serum.

Interleukin-6 (IL-6) is used as a growth factor by various tumor cells. It binds to a gp80 specific receptor (IL-6R) and then to a gp130 transducing chain. Both receptor chains are released as soluble functional proteins which circulate in biological fluids. With a view to studying the physiological role of these soluble receptors, both proteins were purified from human plasma. Surface plasmon resonance was used to measure the kinetic constants of equilibria between IL-6 and natural sIL-6R, and between the IL-6/sIL-6R complex and soluble gp130. Kd values were found to be 0. 9 and 2.3 nM respectively. Soluble natural IL-6R and gp130 were also found to interact with a Kd of 2.8 nM in the absence of IL-6. By using these Kd values, a mathematical simulation predicted that 1) within a large range of IL-6, sIL-6R and sgp130 concentrations, free IL-6 represents 30% of the total circulating cytokine, 2) sIL-6R overconcentrations lead to dramatic changes of the concentration of free IL-6, 3) increased concentrations of sgp130 should produce an efficient buffering effect on the IL-6/sIL-6R complex without incidence on the level of free IL-6. According to this model, the IL-6/sIL-6R complex appears to be an important support of IL-6 signaling in the most commonly encountered in vivo situations. The concentration of this complex is directly under the control of the concentration of sIL-6R; its bio-availability should be efficiently buffered by increased sgp130 concentrations.

Interleukin-6 receptor signaling. I. gp80 and gp130 receptor interaction in the absence of interleukin-6.

Interleukin-6 (IL-6) is used as a growth factor by various tumor cells. It binds to a gp80 specific receptor (IL-6R) and then to a gp130 transducing chain. Both receptor chains are released as soluble functional proteins which circulate in biological fluids. To study the physiological role of these soluble receptors, both proteins were purified from human plasma and the kinetic constants of equilibria between IL-6 and its natural soluble IL-6R (sIL-6R) and gp130 receptor (sgp130) were measured using surface plasmon resonance analysis. Unexpectedly, natural sIL-6R and natural sgp130 were found to interact (Kd = 2.8 nM) in the absence of IL-6. No interaction was seen between the recombinant soluble receptors or between either natural soluble receptor and its recombinant partner. This binary complex was not due to copurification of IL-6 and was detected in human plasma of healthy donors. It results from either direct interaction between the two natural soluble receptors or indirect binding mediated by a yet unidentified copurified plasma molecule playing the role of an IL-6 antagonist. Once formed, the binary complex was found to be unable to bind IL-6. Soluble gp130 had already been shown to inhibit IL-6 signaling by inactivating the IL-6/IL-6R complex. In addition we show that, in the absence of IL-6, circulating natural sgp130 is able to inhibit directly the circulating sIL-6R that is a strong synergic molecule of IL-6 signaling.

Tumor necrosis factor is a survival and proliferation factor for human myeloma cells.

Interleukin-6 (IL-6) is a major survival factor for malignant plasma cells. In patients with multiple myeloma (MM), cell lines whose survival and proliferation are dependent upon addition of exogenous IL-6 have been obtained. We show here that tumor necrosis factor-alpha (TNF-alpha) is also a survival factor for myeloma cell lines, although less potent than IL-6. The survival activity of TNF-alpha is not affected by anti-IL-6 or anti-gp130 monoclonal antibodies (mAbs). TNF-alpha also induces myeloma cells in the cell cycle and promotes the long-term growth of malignant plasma cell lines. As TNF-alpha is produced in patients with MM and associated with a poor prognosis, these results suggest that anti-TNF-alpha therapies could be useful in this disease.

The Mi15 monoclonal antibody (anti-syndecan-1) is a reliable marker for quantifying plasma cells in paraffin-embedded bone marrow biopsy specimens.

Plasmocyte selective monoclonal antibodies (MAb) recognizing syndecan-1 have recently been described. They belong to a new cluster, CD138. Using the MAb MI15, we investigated the expression of syndecan-1 in routinely paraffin-embedded tissues. Nontumoral lymph nodes (25 cases) and bone marrow biopsy specimens (63 cases) showed strong membrane staining of plasma cells only, allowing accurate analysis of the nuclear structure. The MI15 positivity correlated with kappa and lambda light chain expression in the cytoplasm. The percentages of plasma cells calculated in bone marrow biopsy specimens after MI15 staining were, respectively, 2.1% (range, 1% to 4%) in normal bone marrows, 8.5% (range, 5 to 17) in reactive plasmocytosis, and 4.66% in monoclonal gammapathy of undetermined significance (MGUS) patients (range, 1 to 13), in the same range but slightly higher than those obtained on smears or on hematoxylin and eosin (H&E)-stained sections. In multiple myeloma (40 cases), all plasma cell types were marked, and Mi15 MAb gave additional information in 8 of 40 (20%) patients. In lymph nodes, Mi15 MAb reacted with Reed-Sternberg cells of classical Hodgkin's disease in 23 of 31 cases (74%) with variable intensity. In contrast, nodular lymphocyte predominance Hodgkin's disease (10 cases), most B cell lymphomas (88 of 107 cases) and all T cell lymphomas (30 cases) were negative. In B cell lymphomas, plasmocytomas (8 cases), plasmocytic lymphomas (2 cases), and 5 of 13 cases of immunoblastic lymphoma with plasmocytoid differentiation were stained. In lymphoplasmocytoid lymphomas (4 lymph nodes and 20 bone marrow biopsy specimens), only mature plasma cells were positive. Moreover, a wide distribution of syndecan-1 was observed in normal and tumoral epithelial tissues. Finally, Mi15 MAb appears to be a reliable marker for identifying and quantifying normal and tumoral plasma cells in paraffin-embedded bone marrow and lymph node samples.

IFN-alpha is a survival factor for human myeloma cells and reduces dexamethasone-induced apoptosis.

IFN-alpha is used as a maintenance therapy in patients with multiple myeloma, but its benefit is a matter of controversy. In vitro studies show that IFN-alpha can both stimulate and inhibit myeloma cell proliferation. We have tested the effect of IFN-alpha on the survival of myeloma cell lines and primary plasma cells. IFN-alpha significantly reduced the apoptosis induced by removal of IL-6 in four IL-6-dependent myeloma cell lines. It also reduced the level of apoptosis induced by dexamethasone in these cell lines as well as in purified primary myeloma cells from seven patients. IFN-alpha promoted the survival of myeloma cells, which, following removal of IL-6, were blocked in G1 and died. However, unlike IL-6, IFN-alpha-treated cells remained mainly blocked in the G1 phase of the cycle. While the effects of IL-6 are mediated through stimulation of its gp130 receptor subunit, the IFN-alpha-induced survival of myeloma cells was independent of gp130 transducer activation (as demonstrated using a neutralizing anti-gp130 Ab). However, the signal transduction cascades activated by these two cytokines share at least some common elements, since stimulation with either IFN-alpha or IL-6 resulted in STAT3 phosphorylation. These results indicate that IFN-alpha promotes the survival, but not the proliferation, of myeloma cells, preventing the apoptosis induced by removal of IL-6 or addition of dexamethasone. This survival factor activity may explain the conflicting reports on the effects of IFN-alpha on myeloma cell proliferation.

A gp130 interleukin-6 transducer-dependent SCID model of human multiple myeloma.

Agonist antihuman gp130 transducer monoclonal antibodies (MoAbs) were used in SCID mice to grow myeloma cells whose survival and proliferation is dependent on gp130 transducer activation. The agonist anti-gp130 MoAbs neither bound to murine gp130 nor activated murine cells and, as a consequence, did not induce interleukin-6 (IL-6)-related toxicities in mice. They have a 2-week half-life in vivo when injected in the peritoneum. The agonist antibodies made possible the in vivo growth of exogenous IL-6-dependent human myeloma cells as well as that of freshly explanted myeloma cells from 1 patient with secondary plasma cell leukemia. Tumors occurred 4 to 10 weeks after myeloma cell graft and weighed 3 to 5 g. They grew as solid tumors in the peritoneal cavity and metastasized to the different peritoneal organs: liver, pancreas, spleen, and intestine. Tumoral cells were detected in blood and bone marrow of mice grafted with the XG-2 myeloma cells. Tumoral cells grown in SCID mice had kept the phenotypic characteristics of the original tumoral cells and their in vitro growth required the presence of IL-6 or agonist anti-gp130 MoAbs. Myeloma cells from 4 patients with medullary involvement persisted for more than 1 year as judged by detectable circulating human Ig. However, no tumors were detected, suggesting a long-term survival of human myeloma cells without major proliferation. These observations paralleled those made in in vitro cultures as well as the tumor growth pattern in these patients. This gp130 transducer-dependent SCID model of multiple myeloma should be useful to study various therapeutical approaches in multiple myeloma in vivo.

The myeloma cell antigen syndecan-1 is lost by apoptotic myeloma cells.

Syndecan-1 is a cell membrane proteoglycan that binds extracellular matrix components and various growth factors. It is expressed only on malignant plasma cells in bone marrow samples from patients with multiple myeloma (MM). Several reports have suggested that syndecan-1 was present only on a part of the myeloma cells. By using either IL-6-dependent myeloma cell lines or primary myeloma cells stained by annexin V, we report here that syndecan-1 was rapidly lost by myeloma cells undergoing apoptosis. In the same experimental conditions, expression of other cell membrane antigens such as CD38, HLA class-I or CD49d on apoptotic myeloma cells was not affected. In addition, we show that syndecan-1 loss was independent of activation of the gp130 IL-6 transducer. Dexamethasone induced a strong apoptosis of myeloma cells associated with the loss of syndecan-1. Finally, by using freshly-explanted tumoural samples, we show that syndecan-1 rapidly disappeared from myeloma cells in association with induction of apoptosis. In conclusion we showed that syndecan-1 is a marker for viable myeloma cells which is rapidly lost by apoptotic cells. These results emphasize the usefulness of anti-syndecan-1 antibodies to purge tumoural cells from haemopoietic grafts or to purify these cells for further manipulations for immuno or gene therapies.

Functional interaction of the gp80 and gp130 IL-6 receptors in human B cell malignancies.

IL-6, or cytokines of the IL-6 family using gp130 as transducer chain receptor, have been suggested to play a role in certain B lymphoid neoplasia. The presence of cell membrane gp80 and gp130 IL-6 receptors was studied in 98 patients with various leukaemia and non-Hodgkin's malignant lymphoma using flow cytofluorometry and immunohistology. Except neoplasia of immature B cells which expressed neither of the receptors, the majority of B cell tumours expressed one or both of them, mantle cell lymphoma being found to express the highest density of receptors. Using IL-6-dependent XG myeloma cell lines and mAb recognizing various gp80 and gp130 functional epitopes, it has been shown that IL-6 activation leads to a modified expression of some epitopes. In particular, the decrease or the disappearance of a gp130 epitope called A1 signed gp130 dimerization which is the first step of the gp130 activation pathway. Gp80 and gp130 epitope analysis was achieved in 17 of the patients. In four, an epitope phenotype compatible with a cytokine-induced activation was found. The cells of five B-CLL patients which expressed both gp80 and gp130 receptors were incubated with IL-6 to induce activation. In three of the cases they were found to rearrange their receptors in activated forms but not in the two others, showing that cells able to be activated or not can be found. These results confirm that gp130 signalling might play an important role in the pathogenesis of certain B cell neoplasia.

Dimerization and activation of the common transducing chain (gp130) of the cytokines of the IL-6 family by mAb.

The objective of our research was to study the mechanisms of activation of mAb against the gp130 transducer chain common to the IL-6 cytokine family. It has been found that among the 56 anti-gp130 available worldwide, none was able to activate the growth of IL-6-dependent myeloma cell lines. When certain of them were associated in pairs they allowed the cells to grow; alone, they were inhibitory. The same activation was also obtained by cross-linking certain anti-gp130 mAb on the cell membrane with a goat anti-mouse Ig antiserum. A bispecific mAb was prepared by the somatic fusion of two hybridomas secreting two mAb whose association was able to activate gp130 signaling; the bispecific mAb was inactive. The activating mAb were able to support long-term proliferation of the IL-6-dependent myeloma cell lines, which indicates that they are potential valuable growth factors of tumor cells and hematopoietic stem cells. When they were injected into SCID mice, they allowed human IL-6-dependent myeloma cell lines to grow, develop tumors and metastasize. By studying the functional epitopes of the cell membrane gp130 receptors, it was shown that the activating mAb induced gp130 dimerization and STAT3 activation, as did IL-6.

Large scale and clinical grade purification of syndecan-1+ malignant plasma cells.

For cancer immunotherapy, it is usually necessary to obtain a large number of tumor cells from patients. We have previously reported that syndecan-I is present only on malignant plasma cells in samples from patients with multiple myelomatosis. We report here that this antigen is cleaved by chymopapain. This makes it possible to develop a rapid and clinical grade procedure to purify large numbers of myeloma cells using anti-syndecan-1 mAb, magnetic beads and chymopapain.

Specific inhibition of IL-6 signalling with monoclonal antibodies against the gp130 receptor.

A family of cytokines [IL-6, IL-11, oncostatin M (OM), leukaemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF) and cardiotrophin-1] involved in various inflammatory or tumoral diseases share the same gp130 signal transducer chain. The complex formed with their specific receptors associates with a common transducing gp130 membrane protein (gp130) resulting in the formation of high avidity receptor and activation of tyrosine kinases. With the view of identifying gp130 domains specifically involved in IL-6 signalling, the authors prepared 37 new anti-gp130 mAb and analysed the structure-function relationship of the molecule. By cross-competition ELISA, the mAb were classified in 10 subgroups called A to J. By ELISA and BIAcore analysis, the mAb were found to recognize at least 18 antigenic specificities of the gp130 chain. The mAb reacted against the soluble and the membrane forms of gp130 as well. Their ability to inhibit the proliferation of the human myeloma cell line XG-4 of which the growth is strictly dependent on the presence of either exogenous IL-6, or LIF, or OM, or CNTF was studied. Besides mAb with no evident neutralizing effect (G and H) and mAb which neutralized equally well the activity of all tested cytokines (all mAb of groups A, I and J), some showed a selective effect. Those of group F inhibited also the proliferation induced by the 4 cytokines, but more specifically that dependent on the CNTF. mAb of groups B and E specifically inhibited the growth induced by IL-6, whereas those of group C inhibited that induced by LIF and OM. These results show the presence of different gp130 epitopes specifically involved in the signaling induced by the cytokines of the gp130 family. In ELISA, only mAb of group B and E were found to inhibit the binding of the IL-6-IL-6R complex to gp130, showing that they identified one or two domains of gp130 involved in its interaction with the IL-6-IL-6R complex. Precise identification of this(ese) epitope(s) would be useful to better understand the mechanisms of the IL-6 signalling.

Activation of the gp130 signaling pathway by monoclonal antibodies directed against the gp130 molecule.

Six cytokines of the interleukin (IL)-6 family involved in various inflammatory or tumoral diseases share the same gp130 signal transducer chain. We made a panel of anti-gp130 monoclonal antibodies (mAb) to study the structure and function of the gp130 molecule. These mAb recognized different epitopes of the gp130 that we called A to J. Most of the mAb were found to be inhibitors and we studied whether some of them could also induce gp130 activation. When used alone, none of them was able to initiate the proliferation of IL-6-dependent cell lines. However, some particular associations of the mAb were able to induce a proliferative response. mAb B1 could activate the lines in association with F1 or with I2 but not with I1, which in ELISA was similar to I2. In contrast mAb B2, which in ELISA appeared to be very similar to B1, was able to activate the cells in association with I1 but not with F1 or I2. Two other mAb belonging to specificities A and C were found to be activators either in association with I1 only, or with I1 or B2, respectively. These associations of mAb appeared to be nearly as potent activators as IL-6 itself. Although we still have no precise idea of the mechanisms involved, they are interesting tools to study the molecular interactions leading to gp130 activation and, from a practical point of view, valuable growth factors of hematopoietic stem cells.

Major role of the soluble interleukin-6/interleukin-6 receptor complex for the proliferation of interleukin-6-dependent human myeloma cell lines.

Interleukin-6 (IL-6) is a proinflammatory cytokine which possesses a central growth factor activity for certain tumor cells such as plasma cells in multiple myeloma (MM). Upon binding of IL-6, soluble IL-6 receptor (sIL-6R) has been shown to retain its affinity for IL-6 and to associate with the signal-transducing gp130 chain. Therefore, contrary to the majority of soluble cytokine receptors, it plays an agonist role in IL-6 signaling. In order to test its physiological importance as compared to that of its membrane counterpart, we studied cells from two myeloma cell lines which need exogenous IL-6 to proliferate and release sIL-6R into their culture supernatant. Using a new culture system where the supernatant recirculated permanently through an anti-IL-6R affinity column, all sIL-6R was removed from the culture medium throughout the culture period. Under these conditions IL-6-dependent cells were unable to grow in the presence of physiological concentrations of IL-6, showing the major role of the sIL-6R for sustaining the proliferation of these cell lines. Increasing IL-6 concentrations well over the physiological values allowed the cells to proliferate again. No effect was seen when sIL-6R was removed from the supernatant of an IL-6-independent myeloma cell line. These results show that the levels of circulating sIL-6R (and thus those of IL-6/sIL-6R complex) are worth looking at in pathologies involving IL-6 hyperactivity.

Expression of the interleukin 6 receptor in primary renal cell carcinoma.

AIMS: Interleukin 6 (IL-6) is expressed in the majority of renal cell carcinomas and has an important role in the proliferation of some renal cell carcinoma cell lines. This action is mediated by two membrane proteins, gp80 (the IL-6 receptor; IL-6R), which binds IL-6, and gp130, which transduces the signal. The soluble form of gp80 (sIL-6R) is able to activate gp130 when complexed to the IL-6 molecule. These considerations prompted an investigation of IL-6R expression in this malignancy. IL-6, C reactive protein (CRP), and sIL-6R were also measured in serum and correlated to clinical and pathological features. METHODS: Immunostaining was performed on cryostat sections from renal cell carcinoma tumours with M91, an anti-IL-6R monoclonal antibody, using the alkaline phosphatase antialkaline phosphatase technique. The proliferation index was measured using the KI-67 monoclonal antibody. CRP, IL-6, and sIL-6R were measured in serum before nephrectomy, using an immunoenzymatic or immunoradiometric assay. RESULTS: There were significant differences in survival in patients with tumours larger than 8 cm, metastasis at diagnosis, high nuclear grade tumours, detectable serum concentrations of IL-6 (correlated to CRP serum concentration), more than 4% proliferating cells, and the presence of the IL-6R in situ. Furthermore, the serum IL-6 concentration correlated with tumour size and stage. The mean serum sIL-6R concentration was not significantly different from that observed in 40 normal subjects. Tumour IL-6R expression was present in 10 samples. There was a significant association between the presence of the IL-6 receptor in tumours and tumour stage, nuclear grade, proliferation index, and serum IL-6. CONCLUSIONS: This study revealed the importance of IL-6/CRP and IL-6R expression in situ as potential new prognostic factors and opens the way to new therapeutic strategies in renal cell carcinoma.

Mononuclear cell retention in rheumatoid synovial tissue engrafted in severe combined immunodeficient (SCID) mice is up-regulated by tumour necrosis factor-alpha (TNF-alpha) and mediated through intercellular adhesion molecule-1 (ICAM-1).

The aim of this study was to assess regulation of mononuclear cell (MNC) traffic to human synovial tissue by TNF-alpha and IL-1 and the involvement of ICAM-1 in MNC retention in rheumatoid synovial tissue. Human rheumatoid arthritis synovium was engrafted subcutaneously in 6-8 week-old SCID/CB17 mice. Three weeks later, we injected 20 x 10(6) human peripheral blood mononuclear cells (PBMC) previously labelled with 111indium intraperitoneally into mice containing control or cytokine-injected grafts. Total body scintigraphy was performed 72 h postinjection. The graft was removed and immunochemical analysis carried out to assess ICAM-1, vascular cell adhesion molecule-1 (VCAM-1) and E-selectin expression. In some experiments, mice were treated intravenously with 500 micrograms MoAb anti-ICAM-1 (BIRR-1) or an isotype-matched control MoAb before introduction of MNC. TNF-alpha, but not IL-1 alpha, enhanced MNC retention in the rheumatoid synovial graft 72 h post-injection (graft activity 989 +/- 1227 ct/min per 200 pixels or 3.36 +/- 4.16% of initial injected activity versus 411 +/- 157 ct/min per 200 pixels or 1.13 +/- 0.45% in controls; P < 0.03). TNF-alpha enhanced ICAM-1 expression by synovial cells and endothelial cells, whereas VCAM-1 or E-selectin expression was not enhanced on either cell type. After MoAb treatment of ICAM-1, synovial lymphocyte recruitment of TNF-alpha-treated mice decreased significantly to levels below that of control mice (160 +/- 97 ct/min per 200 pixels, 0.54 +/- 0.33%; P < 0.01). Mononuclear cell retention in rheumatoid synovial tissue engrafted into SCID mice was up-regulated by TNF-alpha and blocked by MoAb to ICAM-1. These results suggest that ICAM-1 is involved in mononuclear cell retention in rheumatoid synovium.