Chimaeric Lym-1 monoclonal antibody-mediated cytolysis by neutrophils from G-CSF-treated patients: stimulation by GM-CSF and role of Fc gamma -receptors.

Chimaeric Lym-1 (chLym-1) is a monoclonal antibody generated by fusing the variable region genes of murine Lym-1 to human gamma1 and kappa constant regions. Owing to its selectivity and avidity for human malignant B cells, it is an attractive candidate for developing immune-interventions in B-lymphomas. In the attempt to identify rational bases for optimizing potential chLym-1 related therapeutic approaches, we studied the ability of this ch-mAb to trigger neutrophil-mediated Raji cell cytolysis in cooperation with two neutrophil-related cytokines, G-CSF and GM-CSF. ChLym-1 triggered low levels of cytolysis by normal neutrophils but induced consistent cytolysis in neutrophils from individuals treated with G-CSF. When exposed to GM-CSF, neutrophils from subjects treated with G-CSF became potent effectors, also leading to 75% lysis. By using mAbs specific for distinct FcgammaRs, normal neutrophils were inhibited by mAb IV.3, suggesting the intervention of FcgammaRII, constitutively expressed on the cells. On the other hand, neutrophils from patients treated with G-CSF were inhibited by mAb IV.3 plus mAb 197, a finding consistent with a cooperative intervention of FCgammaRII and G-CSF-induced FcgammaRI. The anti-FcgammaRIII mAb 3G8 promoted significant enhancement of the neutrophil cytolytic efficiency. Therefore, neutrophil FcgammaRIII behaves as a down-regulator of the cytolytic potential. The present findings suggest new attempts to develop mAb-based and G-CSF/GM-CSF combined immune-interventions in B lymphomas.

Immune complex stimulation of neutrophil apoptosis: investigating the involvement of oxidative and nonoxidative pathways.

Neutrophils are involved in the pathogenesis of various inflammatory diseases. One of the mechanisms by which neutrophilic inflammation is generated is immune complex (IC) deposition in tissue. As the clearance of apoptotic neutrophils from inflamed sites is considered a crucial determinant for the resolution of inflammation, we investigated the effects of IC-induced neutrophil activation on apoptosis and the mechanisms regulating neutrophil survival. Our results show that IC stimulated apoptosis efficiently. The percentage of apoptotic neutrophils was reduced by the anti-FcgammaRII mAb IV.3, but not by anti-FcgammaRIII mAb 3G8. The spontaneous apoptosis was completely inhibited by the antioxidant compound catalase, which in turn prevented only partially the apoptosis in presence of IC. The oxidative metabolism triggered by IC was inhibited only blocking both FcgammaRII and FcgammaRIII. Neutrophils from patients with chronic granulomatous disease, congenitally incapable of producing oxidants, showed low level of spontaneous apoptosis, but underwent a nearly 3-fold increment in the apoptosis rate when incubated with IC. In conclusion, neutrophil apoptosis appears to be a process governed by multiple pathways, some of which are strictly ROS-dependent, others acting in a nonoxidative manner. In particular, the herein shown FcgammaRII-dependent, ROS-independent, signal-inducing neutrophil apoptosis may uncover new pharmacological targets for the promotion of cell removal from sites of inflammation, thereby favoring the resolution of the inflammatory process.

Stromal cell-derived factor-1 as a chemoattractant for follicular center lymphoma B cells.

BACKGROUND: Follicular center lymphoma displays widespread lymph node involvement at diagnosis. The chemoattractants that control the locomotion of follicular center lymphoma B cells have not been established. Stromal cell-derived factor-1 (SDF-1) is a CXC-class chemokine that enhances the migration of normal human B cells and is expressed in peripheral lymphoid tissues. Here we have investigated 1) whether SDF-1 stimulates the in vitro locomotion of follicular center lymphoma B cells and of their presumed normal counterparts (i. e., germinal center B cells) and 2) whether the same cells express SDF-1 transcripts. METHODS: B cells were purified by immunomagnetic bead manipulation. Messenger RNA was detected by reverse transcription-polymerase chain reaction. Migration was assessed by the filter and collagen invasion assays. All P values were two sided. RESULTS: Follicular center lymphoma B lymphocytes showed a statistically significant migratory response to 300 ng/mL SDF-1, both in the filter and in the collagen assays (P =.002 for each). Such response was mediated by the SDF-1 receptor, CXCR4. CD40 monoclonal antibody (MAb) and tonsillar germinal center B cells treated with CD40 MAb and recombinant interleukin 4, but not freshly isolated, migrated statistically significantly faster in the presence than in the absence of SDF-1 (P =.002 in both filter and collagen assays). Freshly isolated follicular center lymphoma and germinal center B cells expressed SDF-1 transcripts. CONCLUSIONS: This study shows that SDF-1 substantially enhances the migration of follicular center lymphoma B cells but not the migration of freshly purified germinal center B cells. This difference may be related to the extended survival of follicular center lymphoma versus germinal center B cells. SDF-1 produced in follicular center lymphoma lymph nodes may play a role in the local dissemination of tumor cells.

rC5a directs the in vitro migration of human memory and naive tonsillar B lymphocytes: implications for B cell trafficking in secondary lymphoid tissues.

Human C5a is a potent chemoattractant for granulocytes, monocytes, and dendritic cells. In mice C5a has been shown to be chemotactic for germinal center (GC) B cells. To date, no information is available on the effects of C5a on human B cell locomotion. Here we demonstrate that rC5a increases polarization and migration of human tonsillar B cells. The locomotory response was due to both chemokinetic and chemotactic activities of rC5a. Moreover, memory and, at a lesser extent, naive B cell fractions from purified tonsillar populations displayed rC5a-enhanced migratory properties, whereas GC cells did not. Flow cytometry revealed C5aR (CD88) on approximately 40% memory and 10% naive cells, respectively, whereas GC cells were negative. Immunohistochemistry showed that a few CD88+ cells were of the B cell lineage and localized in tonsillar subepithelial areas, where the majority of memory B cells settle. Pretreatment of memory B cells with the CD88 mAb abolished their migratory responsiveness to rC5a. Finally, the C5 gene was found to be expressed in naive, GC, and memory B lymphocytes at both the mRNA and the protein level. This study delineates a novel role for C5a as a regulator of the trafficking of human memory and naive B lymphocytes and supports the hypothesis that the B cells themselves may serve as source of C5 in secondary lymphoid tissues.

Soluble Fas ligand is chemotactic for human neutrophilic polymorphonuclear leukocytes.

It has been recently shown that Fas ligand (FasL) expression on islet beta grafts results in neutrophilic infiltration and graft rejection. In this study, we show that human recombinant soluble FasL is endowed with potent chemotactic properties toward human neutrophilic polymorphonuclear leukocytes (neutrophils) at concentrations incapable of inducing cell apoptosis. Furthermore, neutrophils exposed to soluble FasL did not display detectable change of intracellular Ca2+ and did not undergo superoxide production or exocytosis of primary and secondary granules. Our results show that FasL is a potent chemoattractant for human neutrophils without evoking their secretory responses. This finding suggests a novel proinflammatory function for this ligand and may help to clarify the mechanism governing FasL-mediated graft rejection, thereby offering rational bases for controlling and modulating FasL-based immunotherapies.

Chemoattractant-induced release of elastase by tumor necrosis factor-primed human neutrophils: auto-regulation by endogenous adenosine.

OBJECTIVE AND DESIGN: In the present work, we studied the role of cell-derived adenosine in both the physiologic regulation and pharmacologic control of the exocytosis of azurophilic granules of neutrophils exposed to tumor necrosis factor alpha (TNF) and stimulated with some chemoattractants. MATERIAL AND METHODS: Human neutrophils were pre-incubated in the absence or presence of TNF. Thereafter, the appropriate chemoattractant was added to the cells. After incubation, the cell-free supernatant was collected for testing elastase activity and intracellular cAMP levels. Results, expressed as mean +/- 1 SD, were evaluated by unpaired, two-tailed Student's t-test and by analysis of variance followed by Student-Newman-Keuls multiple comparisons test. RESULTS: Neutrophil incubation with 10 ng/ml TNF or 0.1 micromol/l N-formyl-met-leu-phe (fMLP) failed to release elastase activity (NE) (NE in absence of stimulus: 23.1 +/- 5.7 nmol/h; TNF-induced NE: 26.4 +/- 14.4 nmol/h; fMLP-induced NE: 27.0 +/- 9.9 nmol/h). Neutrophils, pre-exposed to various amounts of TNF, released elastase in response to 0.1 micromol/l fMLP in a dose-dependent manner (NE in presence of 10 ng/ml TNF and 0.1 micromol/l fMLP: 133.7 +/- 24.0 nmoles/h). As compared with fMLP, C5a had lower activity (NE in presence of 10 ng/ml TNF and 0.1 micromol/l C5a: 66.4 +/- 25.1 nmoles/h), whereas interleukin-8, platelet activating factor and leukotriene B4 were ineffective. The secretory response of TNF-primed neutrophils to fMLP was inhibited by adenosine in a dose-dependent manner (IC50 = 5.18 +/- 7.1 micromol/l). The addition of adenosine deaminase (ADA) to TNF-primed neutrophils resulted in increased secretory response to fMLP (NE in absence and presence of 0.25 U/ml ADA: 71.5 +/- 11.0 and 107.3 +/- 18.6 respectively, P = 0.060). Moreover, two inhibitors of phosphodiesterase type IV (RO 20-1724 and nimesulide) reduced the elastase release only in the absence of ADA (RO 20-1724: percent inhibition in absence or presence of ADA = 20.2 +/- 15.0 and 4.4 +/- 5.1 respectively; nimesulide: percent inhibition in absence or presence of ADA = 22.2 +/- 19.6 and 0.8 +/- 3.0 respectively). Similarly, RO 20-1724 and nimesulide increased intracellular cAMP levels only in absence of ADA (RO 20-1724: percent cAMP increment in absence or presence of ADA = 215.4 +/- 97.5 and 47.3 +/- 53.3 respectively; nimesulide: percent cAMP increment in absence or presence of ADA = 177.7 +/- 19.0 and 19.5 +/- 29.3 respectively). CONCLUSIONS: Endogenous adenosine down-regulates the cell secretory response and is instrumental in uncovering the susceptibility of azurophilic granule exocytosis to control by inhibitors of phosphodiesterase type IV.

Recombinant tumor necrosis factor enhances the locomotion of memory and naive B lymphocytes from human tonsils through the selective engagement of the type II receptor.

Recent studies performed in mice knocked out for the tumor necrosis factor (TNF ), the lymphotoxin-alpha, or the type I TNF receptor (R), genes have shown that these animals display gross defects in germinal center (GC) formation, suggesting that members of the TNF and TNFR superfamilies are involved in the control of B-cell migration. Based on these premises, we have here investigated the effects of human recombinant (r) TNF on the polarization and locomotion of tonsillar B cells. rTNF increased the spontaneous polarization and locomotion of unfractionated tonsillar B lymphocytes in a dose-dependent manner by inducing a true chemotactic response. Memory (IgD-, CD38(-)) and naive (IgD+, CD38(-)), but not GC (IgD-, CD38(+)) B cells purified from total tonsillar B lymphocytes, showed a significantly higher locomotion in the presence than in the absence of rTNF. Accordingly, type I and II TNF receptors (TNFRs) were detected by flow cytometry on the surface of memory and naive, but not GC, B lymphocytes. Blocking experiments with monoclonal antibodies to type I or II TNFR showed that rTNF enhanced the spontaneous chemotaxis of memory and naive B cells through the selective engagement of type II TNFR. Finally, the TNF gene was found to be expressed in memory, naive and GC B lymphocytes; the cytokine was released in culture supernatants from the three B-cell subsets after stimulation. These data may support the hypothesis that human TNF is involved in the paracrine and perhaps autocrine control of B-cell migration in secondary lymphoid tissues.