Rituximab treatment in patients with active Graves' orbitopathy: effects on proinflammatory and humoral immune reactions.

In active Graves' orbitopathy (GO), proinflammatory cytokines predominate. Circulating thyroid stimulating hormone (TSH)-receptor antibodies (TRAb) have been correlated with GO clinical activity and severity. In preliminary studies rituximab (RTX), an anti-CD 20 monoclonal antibody, has induced clinical improvement of active GO without a change in serum anti-thyroid antibodies. We have studied whether RTX in GO acts by affecting proinflammatory cytokines and thyroid and orbital-directed antibodies. Ten patients with GO were treated with RTX, administered twice intravenously (i.v.) (1000 mg) at days 1 and 15, and 20 with methylprednisolone, administered weekly i.v. (500 mg), for 16 weeks. Patients were studied before treatment, at B cell depletion and at 4, 8, 16, 20, 30 and 50 weeks. Peripheral lymphocytes, serum interleukin (sIL)-6, sIL-6r, chemokine (C-X-C motif) ligand 10 (CXCL10), TRAb and stimulating antibodies (TSAb) and autoantibodies against orbital calsequestrin, collagen XIII and flavoprotein subunit of succinate dehydrogenase (FP-SDH) were measured at baseline and after treatment. Serum IL-6 and sIL-6R concentrations did not change after RTX [P = not significant (n.s.)]. Serum CXCL10 increased after RTX at B cell depletion and at 30 weeks (P < 0·003). Serum TSAb did not change in relation to TRAb, nor did antibodies against orbital antigens (P = n.s.). In conclusion, this study shows that RTX in GO does not affect humoral reactions. The observed increase of serum CXCL10 concentrations at B cell depletion may result from cell lysis. We suggest that RTX may exert its effect in GO by inhibiting B cell antigen presentation.

Characterization of the T-cell epitopes of the major peach allergen Pru p 3.

BACKGROUND: Pru p 3 is the major peach allergen recognized by more than 90% of peach-allergic individuals of the Mediterranean area. Identification of the dominant Pru p 3 T-cell epitopes can improve our understanding of the immune responses against this protein and could be helpful in the development of hypoallergenic immunotherapy. For this purpose, we examined the phenotypes, specificities and cytokine secretion profiles of proliferating T cells in response to Pru p 3 in peach-allergic individuals. METHODS: Peripheral blood mononuclear cells from 15 peach-allergic patients were incubated with Pru p 3. The proliferation of antigen-specific T-cell lines (TCLs) was assessed by tritiated methylthymidine incorporation. T-cell epitopes were identified by analyzing the reactivity of TCLs against 8 overlapping peptides spanning the entire length of Pru p 3. We characterized the phenotype of Pru-p-3-specific TCLs by flow cytometry and analyzed their production of interleukin (IL) 4 and gamma-interferon (IFN-gamma) by ELISA. RESULTS: Ninety-two Pru-p-3-specific TCLs were isolated (stimulation index > or =5). These TCLs proliferated mainly in response to Pru p 3(12-27) and Pru p 3(57-72). Pru-p-3-specific TCLs were mainly CD4+ (81%) and expressed cell surface CD30. In addition, TCLs produced high levels of IL-4 and low levels of IFN-gamma, indicating a Th2 phenotype. CONCLUSIONS: Two immunodominant T-cell-reactive regions of Pru p 3 were identified: Pru p 3(12-27) and Pru p 3(57-72). These peptides showed a differential ability to elicit a Th2 response. Taken together, our results provide a better understanding of the immunological T-cell reactivity against Pru p 3.

Rituximab induces distinct intraorbital and intrathyroidal effects in one patient satisfactorily treated for Graves' ophthalmopathy.

Hyperthyroid Graves' disease (GD) is a B-cell-mediated disease caused by antibodies stimulating the thyroid stimulating hormone (TSH) receptor (TRAb). A proportion of patients (40-60%) present with an associated ophthalmopathy (TAO), a progressive inflammatory autoimmune disease of the retroorbital tissue. We thought that the anti-CD20 monoclonal antibody rituximab (RTX), by inducing transient B-cell depletion, may potentially modify the active inflammatory phase of TAO. One patient with GD and TAO in its active phase and unresponsive to steroid, was treated with RTX. Whereas the ophthalmopathy responded to RTX therapy and a decrease in the clinical activity score from 5 to 2 was observed during the B-cell depletion, serum antithyroid antibodies, and in particular serum TRAb, were not affected by therapy. When the patient underwent total thyroidectomy, we found B-cells in the thyroid tissue specimens. The eye disease remained stable (clinical activity score=2) and the patient subsequently underwent orbital decompression to correct proptosis of the eye. At that time we did not find lymphocytes in any of the orbital tissue specimens. We believe that RTX therapy in GD may cause amelioration of ophthalmopathy by depleting total lymphocyte population in the orbit, but not lymphocyte depletion in thyroid tissue with consequent unchanged serum TRAb levels.

Unusual presentation of large B cell lymphoma: a case report and review of literature.

Diffuse large B cell lymphoma (DLBCL) is the largest subtype of non-Hodgkin's lymphomas (NHLs) and is characterized by relatively frequent extranodal presentation. In these cases, the most common extranodal localizations are stomach, CNS, bone, testis and liver. Simultaneous detection of multiple extranodal involvement at presentation is quite uncommon, with the majority of these cases characterized by gastric or intestinal disease localization. Retrospective analysis concerning multifocal extranodal NHLs never pointed out disease features such as those described here. We report a patient with an unusual presentation of DLBCL, characterized by adrenal and renal involvement, associated with symptoms and signs of the cold agglutinin disease and a hypercoagulable state. Subsequently, computed tomography (CT) and fluorodeoxyglucose-positron emission tomography (FDG-PET) scanning disclosed a rapidly extensive spread to nodes and bones. Cytofluorimetric analysis of a renal specimen showed medium-to-large lympho-monocytoid elements positive for CD20 with monoclonal expression of immunoglobulin kappa light chain. Histopathological examination confirmed a renal CD20 positive DLBCL localization.

[Case report: rheumatoid arthritis and large granular lymphocytes syndrome]. / Artrite reumatoide e leucemia a "large granular lymphocytes": descrizione di un caso clinico.

Felty's syndrome (FS) is a rare complication (less than 1%) of rheumatoid arthritis (RA), with the clinical feature of splenomegaly and neutropenia. Approximately 10-40% of FS patients have an expansion of peripheral blood large granular lymphocytes (LGL). This cell population mainly consists of two subsets: cytotoxic T cells (CD8+, CD57+) and natural killer cells (CD3-,CD8-,CD56+). It has been hypothesised that LGL expansion could be responsible for neutropenia by suppressing neutrophil precursors in the bone marrow, but various mechanisms have been proposed to explain this association. We report a case of a 60-year-old woman with rheumatoid factor positive RA who developed LGL expansion responsible for splenomegaly, but without neutropenia. In conclusion, LGL expansion is an uncommon complication of RA and may be responsible for both FS and clinical pictures resembling FS.

Novel mode of action of iloprost: in vitro down-regulation of endothelial cell adhesion molecules.

Iloprost is a stable prostacyclin analog commonly employed in the treatment of peripheral vascular disease and also indicated in the treatment of patients affected by systemic sclerosis (SSc) in the presence of severe Raynaud's phenomenon (RP). Several mechanisms of action of the drug other than vasodilation and antiplatelet effect have been demonstrated that may be involved in the exertion of its clinical efficacy. Aim of the present study was to investigate whether iloprost down-regulated lymphocyte adhesion to endothelium through a modulation of adhesion molecule expression on the surface of endothelial cells. In the presence of iloprost, both lymphocyte adhesion and IL-1 stimulated expression of ICAM-1 and ELAM-1 exhibited a significant reduction, while unstimulated adhesion molecule expression was not significantly affected. Our results confirm that iloprost is able to down-regulate lymphocyte adhesion to endothelial cells and indicate that endothelium itself could be target of iloprost administration. Attenuation of the inflammatory response through modulation of cellular interactions could be suggested as a potential mechanism of action of iloprost, when used in the treatment of pathological conditions characterized by endothelial activation.

Lymphocyte-endothelium interaction in systemic sclerosis and Raynaud's phenomenon.

OBJECTIVE: To investigate interactions of immune cells with vascular endothelium in patients with systemic sclerosis (SSc) and in patients with idiopathic or autoimmune Raynaud's phenomenon (RP). METHODS: Lymphocytes obtained from 11 patients with SSc, 9 with RP and 14 control subjects were pre-stimulated in vitro with alloantigens and cultured together with human umbilical vein endothelial cells (HUVECs). Lymphocyte adhesion and induction of endothelial HLA-class 11 molecules were measured by flow cytometry. Lymphocyte cytotoxicity against HUVECs was also evaluated. In some cases cells were cultured under experimental conditions of hypoxia and reoxygenation. RESULTS: Lymphocyte adhesion and induction of endothelial cell expression of HLA-DR molecules were similar in controls and SSc patients, but significantly lower in RP (p < 0.05 and p < 0.03, respectively). Cytotoxic activity of lymphoblasts against endothelial cells was negligible in all patient groups. Under experimental conditions of hypoxia and reoxygenation lymphocyte adhesion was significantly greater than in normoxic conditions in SSc patients, while it was similar to normoxia in control subjects and RP patients. CONCLUSION: These results suggest that in RP patients there may be regulatory mechanisms of lymphocyte response able to control the processes that lead to lymphocyte adhesion and endothelial HLA-DR molecule induction. These mechanisms could play an important role in RP, and might possibly be lost in clinically evident SSc.

Development of phagocytic function of cultured human monocytes is regulated by cell surface IL-10.

Monocytes differentiating in vitro into macrophages increase their capacity to ingest particles via FcgammaR and CR3. Because human recombinant IL-10 is a potent up-regulator of phagocytosis in human monocytes, we investigated whether spontaneously produced IL-10 could be a signal for the modulation of phagocytosis by cultured monocytes. We show here that culture of monocytes in the presence of anti-IL-10 mAb completely abolished up-regulation of phagocytosis of both EIgG and EIgMC3bi, suggesting a role for spontaneously produced IL-10 in the modulation of phagocytosis by cultured human monocytes. The inhibition exerted by anti-IL-10 mAb on the development of FcgammaR-mediated ingestion was dependent on the concomitant inhibition of FcgammaRIII induction in cultured cells. On the other hand, a similar down-regulation of CR3 expression was not involved in the inhibitory effect exerted by anti-IL-10 mAb on the development of CR3-mediated ingestion. Monocytes secreted detectable levels of IL-10 when cultured in medium but the concentrations of IL-10 in the supernatants decreased with length of time in culture, the decrease being completely reversed by anti-IL-10 mAb. In addition, we showed that monocytes expressed immunoreactive IL-10 on their surface and this expression increased during differentiation into macrophages. Whether this IL-10 was bound to specific membrane receptors or it was an integral membrane protein remains to be determined; however, this latter possibility is consistent with our observations that IL-10 did not elute with acid treatment and exogenous IL-10 did not increase surface staining of monocytes. Our data indicate that human mononuclear phagocytes express IL-10 on their membrane and suggest that this cytokine may represent an autocrine signal for the increased phagocytic function observed during differentiation of monocytes into macrophages.

High frequency of T-cell lines responsive to immunodominant epitopes of thyrotropin receptor in healthy subjects.

In this study we analyzed the proliferative response to the extracellular domain of thyrotropin receptor (TSHR-ECD) of T-cell lines raised from healthy subjects. We found high frequencies of cell lines reactive to TSHR-ECD, ranging from 12% to 37%. The response of the cell lines to a set of overlapping peptides of TSHR-ECD showed that the most recognized epitopes by T lymphocytes are on the C-terminal portion. In particular, the regions of residues 360-396 and 258-277 are immunodominant in T-lymphocyte reactivity. A group of cell lines specific for the peptides of TSHR-ECD lost the response to the peptides during time in culture. However, these lines were still responsive to TSHR extracellular domain. The cloning of one of these lines showed three types of T-cell clones: (1) CD4+ clones (n = 4) highly responsive to the TSHR-ECD; (2) CD4+ clones (n = 4) low responsive to TSHR-ECD; (3) CD8+ clones (n = 9) not responsive to TSHR-ECD. The first group of clones was stable during time in culture, while the second group was characterized by the loss of the specific response to TSHR-ECD after some weeks from the first analysis. The observation of a spontaneous anergy in the second group of CD4+ clones suggests that mechanisms of control of the lymphocyte response to TSHR-ECD could be activated in vitro.

Flow cytometric analysis of bronchoalveolar lavage and venous blood lymphocyte phenotype for the diagnosis of acute graft rejection in lung transplant patients. The Lung Transplant Group, Ospedale Maggiore di Milano.

OBJECTIVE: To investigate modifications of phenotype in bronchoalveolar lavage (BAL) and venous blood lymphocytes as markers of acute organ rejection in lung transplant patients. STUDY DESIGN: Five consecutive patients receiving successful single lung transplants between March 1991 and April 1992 were followed for two years; serial bronchoscopies with BAL and transbronchial biopsies (TBBs) were performed. BAL and venous blood lymphocyte cytofluorimetry was performed at every procedure, and an index, (blood T4/T8)/(BAL T4/T8), was computed. RESULTS: The index was always > or = 3 in the two patients who did not have graft rejection and always < 3 in the two patients who had repeated episodes of acute rejection (even when no rejection was apparent). The index was frequently < 3 when cytomegalovirus infection was diagnosed. CONCLUSIONS: Since BAL is far less invasive and carries lower risks than TBB, the index might be considered, if our results are confirmed, for screening patients at high risk of acute rejection. TBB could be used as a confirmatory tool for patients who have an index < 3.

Relationship between anti-phospholipid and anti-endothelial cell antibodies III: beta 2 glycoprotein I mediates the antibody binding to endothelial membranes and induces the expression of adhesion molecules.

OBJECTIVE: To investigate the role of antibodies reacting with beta 2 glycoprotein I (beta 2GPI) in the antiendothelial cell binding activity present in sera from patients with the anti-phospholipid syndrome. METHODS: Sera positive for anti-phospholipid, anti-endothelial and anti-beta 2 GPI antibodies were studied for their binding activity on endothelial monolayers cultured in the presence or absence of media containing bovine serum as a source of beta 2GPI. Anti-endothelial activity was also evaluated on endothelial cells cultured without serum and supplemented with exogenous human purified beta 2GPI. Affinity purified anti-beta 2 GPI antibodies were investigated under the same experimental conditions. Finally, the effect of the incubation of these affinity purified fractions on the expression of adhesion molecules (ELAM-1) was studied. RESULTS: The reactivity of the sera decreased on endothelial cells incubated in serum-free medium, while endothelial cell binding was restored in a dose dependent manner after the addition of exogenous purified human beta 2 GPI. Affinity purified anti-beta 2 GPI antibodies obtained from the same sera retained their endothelial cell binding and were able to activate endothelial cells by inducing the ex novo surface expression of adhesion molecules (ELAM-1). CONCLUSIONS: These findings indicate that the close association between anti-endothelial and anti-phospholipid antibodies is sustained by antibodies which recognize beta 2 GPI adhering to the endothelial cells, and can promote their activation.

Fc receptors expression and function in mononuclear phagocytes from AIDS patients: modulation by IFN-gamma.

Fc-receptor (FcR)-mediated phagocytosis and FcR (FcRI, FcRII and FcRIII) membrane expression was studied on freshly separated and cultured monocytes (Mo) from 20 AIDS patients and 20 healthy controls. Both Mo and Mo-derived macrophages from AIDS patients presented a significant defect in their capacity to ingest IgG-coated erythrocytes (EA) compared to control cells. This functional defect did not depend on a decline in the number of FcR+ cells or on a decrease in the expression of FcR on their surface. In fact, the percentages of phagocytes reacting with anti-FcRI MoAb (32.2) or anti-FcRII MoAb (IV.3) were similar for controls and AIDS patients, while the percentage of FcRIII-positive Mo (MoAb 3G8) was higher in the AIDS population than in controls, though this difference was not seen on cultured Mo. The level of FcRI expression, evaluated as mean fluorescence intensity (MFI), was higher on freshly separated Mo from AIDS patients than from controls but this difference disappeared also with differentiation of Mo to Mo-derived macrophages in vitro. Parallel analysis of FcRII and FcRIII on phagocytes revealed no differences in the MFI between the AIDS and control groups. Some observations suggested that this functional defect might be secondary to phagocyte priming by circulating IFN-gamma: (1) in vitro stimulation of Mo with hrIFN-gamma, which increased FcRI expression, actually reduced phagocytosis of IgG-coated particles; and (2) IFN-gamma concentrations were increased in AIDS patients' plasma. In spite of these findings, no significant correlation was found between plasma IFN-gamma concentrations and FcR-mediated ingestion in AIDS patients, making the hypothesis uncertain. Even if the basis for the impaired FcR-mediated phagocytosis in AIDS patients remains unclear, this functional defect may have a role in the immunopathogenesis of AIDS, constituting a component cause of the immunodeficiency.

Increased expression of IgG Fc receptor type I on neutrophils and monocytes from HIV-infected subjects.

Interferon-gamma (IFN-gamma) induces de novo expression of IgG Fc receptor type I (FcRI) on neutrophils and significantly raises the level of these receptors on monocytes. Since increased concentrations of IFN-gamma have been observed in sera from patients with HIV infection, FcRI expression might also be increased on these subjects' phagocytes. FcRI expression was assessed by indirect immunofluorescence staining of phagocytes in whole blood from 40 healthy controls and 55 HIV+ subjects, 24 belonging to CDC class III and 31 to CDC class IV; 42 were intravenous drug abusers (IVDA) and 13 were homosexual men. Plasma levels of IFN-gamma were measured using a modified immunoradiometric assay. The mean linear fluorescence intensity, used as a relative measure of receptor expression, was significantly higher on unseparated neutrophils from HIV+ subjects in CDC classes III (P < 0.001) and IV (P < 0.0001) than from controls. Similar changes in FcRI expression were observed on monocytes from HIV+ subjects. While no differences were observed between IVDA and homosexual HIV+ patients, there was a significant association between FcRI expression and the patients' CDC stage, those in class IV having the highest FcRI levels. Plasma IFN-gamma concentrations were significantly higher in HIV+ patients than in controls and a positive correlation with the stages of HIV infection was again observed. FcRI expression was also increased on freshly purified neutrophils from five HIV+ patients in CDC class IV but did not increase further after 18 h incubation with IFN-gamma, a treatment that up-regulated FcRI expression on control neutrophils. These data suggest that: (i) FcRI evaluation may be a sensitive marker for the biological activity of IFN-gamma in vivo; (ii) phagocytes from HIV+ subjects are activated in vivo by IFN-gamma, expressing increased levels of FcRI; (iii) these IFN-gamma-activated cells may play a role in the pathogenesis of AIDS.

Membrane expression and function of complement receptors CR1 and CR3 on neutrophils from HIV-infected subjects: modulation by rTNF-alpha and rGM-CSF.

We evaluated membrane expression and function of complement receptors CR1 and CR3 on neutrophils from 27 HIV-positive (HIV+) subjects (14 in the CDC class III and 13 class IV) as well as their modulation in vitro by recombinant tumour necrosis factor-alpha (rTNF-alpha) and granulocyte-macrophage colony stimulating factor (rGM-CSF). While CR1 was expressed at similar levels on neutrophils from controls and HIV+ subjects, CR3 expression was significantly higher in CDC class IV subjects than in healthy controls. CR1 and CR3 expression was significantly increased after treatment of neutrophils with both cytokines, without differences between controls and HIV+ subjects. Similarly, the superoxide anion (O2-) production in response to C3-coated zymosan (C3zy) was significantly enhanced on neutrophils from CDC class IV subjects when compared with controls. rGM-CSF and rTNF-alpha treatment significantly enhanced the spontaneous as well as C3zy-stimulated O2- production by neutrophils from controls and CDC class III subjects, and induced an upward trend in the CDC class IV group. These results indicate that the neutrophils of HIV+ patients are preactivated in vivo but they also indicate that these cells may correctly respond to a subsequent particulate stimulus as well as to activating cytokines. Our findings suggest that desensitization or functional exhaustion of complement receptors are not implicated in the abnormalities observed on neutrophils from HIV+ patients.

The effect of cytokines on human neutrophil Fc receptor-mediated phagocytosis.

The recombinant (r) cytokines interferon-gamma (rIFN-gamma), granulocyte/macrophage-colony stimulating factor (rGM-CSF) and tumor necrosis factor-alpha (rTNF-alpha) all activate neutrophils. The aim of this work was to determine the effect of these cytokines on neutrophil Fc-receptor (FcR)-mediated phagocytosis and membrane expression of FcR, particularly FcRII and FcRIII. A short treatment (greater than or equal to 15 min) of neutrophils with rGM-CSF and rTNF-alpha at concentrations greater than or equal to 62.5 U/ml significantly increased their ability to bind and phagocytize IgG-coated erythrocytes (EA). Both cytokines also showed more enhancing activity when suboptimally sensitized EA were used for binding and ingestion assays. A similar treatment of neutrophils with rIFN-gamma at doses up to 500 U/ml was ineffective. The effect of rGM-CSF and rTNF-alpha was blocked by a monoclonal anti-GM-CSF antibody and by a polyclonal anti-TNF-alpha antibody respectively, thus establishing that the cytokines were responsible for the activity of the recombinant preparations. The cytokine-induced enhancement of FcR-mediated phagocytosis did not correlate with an enhancement of FcRII membrane expression on treated neutrophils; rGM-CSF significantly increased FcRIII expression, but rTNF-alpha and rIFN-gamma were both ineffective in this respect. Since different roles of FcRII and FcRIII have been reported on ligand binding and ingestion, we also studied the effect of rGM-CSF and rTNF-alpha on the functional properties of these FcR, using specific monoclonal antibodies (mAbs). In the blocking experiments the pretreatment of neutrophils with rGM-CSF and rTNF-alpha did not modify the blocking properties of either anti-FcRII or anti-FcRIII mAbs, suggesting that cytokine-pretreatment does not affect the individual contribution of each type of FcR to ligand binding and internalization. Our data point to a new activity for both rGM-CSF and rTNF-alpha in augmenting FcR-mediated phagocytosis on neutrophils, but the mechanism of this enhancement remains to be elucidated.

Increased expression of C3b and C3bi receptors on human neutrophils and monocytes induced by a glycoprotein extract from Klebsiella pneumoniae (RU41740).

RU41740 is a glycoprotein extract from Klebsiella pneumoniae with immunomodulating properties under different experimental conditions. In particular the compound is able to stimulate several functions of human phagocytes in vitro and ex vivo. Using monoclonal antibodies and flow cytometry, in this work we assessed the effect of RU41740 on surface expression of receptors for C3b (CR1) and C3bi (CR3) in human phagocytic cells in vitro. The incubation of whole blood with varying RU41740 concentrations led to a dose-dependent increase in surface expression of CR1 and CR3 on both neutrophils and monocytes when compared with control samples incubated in buffer alone. The maximal drug-induced enhancement of complement receptors was: 291% +/- 13.4% for CR1 and 265% +/- 8.5% for CR3 in neutrophils; 117% +/- 4.5% for CR1 and 98% +/- 4.1% for CR3 in monocytes. These peak effects were observed using RU41740 at a final concentration of 10 micrograms/ml and were similar to those induced by optimal concentrations of the activating compound N-formyl-methionyl-leucyl-phenylalanine (10(-7)M). Polymyxin B did not modify the RU41740-induced enhancement of CR1 and CR3 expression on phagocytes, suggesting no role for endotoxin in this activity. These results define, at least in part, the mechanism of action of RU41740 on human phagocytes in vitro and could be relevant to in vivo events during RU41740 treatment.