Resistin is associated with overall survival in non-small cell lung cancer patients during nivolumab treatment.

PURPOSE: Since the role of resistin was evaluated only in patients with non-small cell lung cancer (NSCLC) not treated with immunotherapy, we aimed to evaluate levels of resistin during immunotherapy (nivolumab) and its prognostic role with regard to OS. METHODS/PATIENTS: From a cohort of 78 patients with advanced NSCLC enrolled in a prospective study at Ospedale Policlinico San Martino in Genoa (Italy), 43 patients have been considered for this sub-analysis because of the availability of samples. Before and during nivolumab administration, clinical information and blood samples were collected and resistin, matrix metalloproteinase (MMP)-8, MMP-9, and myeloperoxidase were evaluated by enzyme-linked immunosorbent assay (ELISA). RESULTS: Median age was 71 with a prevalence of males and former smokers. Median resistin levels presented a peak at cycle 2 and then dropped down until the last cycle. Resistin correlated with all neutrophil degranulation products at cycle 1 (except for MMP-9) and at cycle 2 as well as with white blood cells and neutrophils. By a ROC curve analysis, a resistin value at cycle 2 of 19 ng/mL was tested as the best cut-off point for OS. Kaplan-Meier analysis demonstrated that patients above the resistin cut-off experienced a reduced OS (median OS 242.5 vs. 470 days, p = 0.0073), as confirmed by Cox proportional hazards regression analysis. CONCLUSIONS: Resistin levels > 19 ng/mL at the time of the second cycle of nivolumab treatment independently predict a reduced OS in patients with advanced NSCLC.

Serum levels of osteopontin predict diabetes remission after bariatric surgery.

AIM: Bariatric surgery has been shown to effectively improve glycaemic control in morbidly obese subjects. However, the molecular bases of this association are still elusive and may act independently of weight loss. Here, our retrospective study has investigated the inflammatory molecule osteopontin (OPN) as a potential predictor of type 2 diabetes mellitus (T2DM) remission. METHODS: Baseline serum levels of OPN were analyzed in 41 T2DM patients who underwent bariatric surgery. Anthropometric measures and biochemical variables, including insulin sensitivity indices (HOMA2), were assessed at baseline and at 1 and 3 years after surgery. RESULTS: At baseline, patients who experienced T2DM remission had increased waist circumference, body weight and BMI, and higher serum OPN, compared with non-remitters. Patients with and without T2DM remission improved their lipid and glucose profiles, although insulin resistance indices were only improved in the T2DM remission group. In the overall cohort of both T2DM remission and non-remission patients, baseline circulating levels of OPN significantly correlated with reductions of body weight and BMI over time, and insulin sensitivity improved as well. However, only the HOMA2-%S remained independently associated with serum OPN on multivariate linear regression analysis (B: 0.227, 95% CI: 0.067-0.387, ß = 0.831; P = 0.010). Baseline values of OPN predicted 3-year T2DM remission independently of body weight loss, lower BMI and duration of diabetes (OR: 1.046, 95% CI: 1.004-1.090; P = 0.033). CONCLUSION: Although larger studies are still needed to confirm our preliminary results, pre-operative OPN serum levels might be useful for predicting 3-year T2DM remission independently of weight loss in patients undergoing bariatric surgery.

High serum levels of C-reactive protein (CRP) predict beneficial decrease of visceral fat in obese females after sleeve gastrectomy.

BACKGROUND & AIMS: Gender-related differences represent an emerging investigation field to better understand obesity heterogeneity and paradoxically associated cardiovascular (CV) risk. Here, we investigated if high-sensitivity C-reactive protein (hs-CRP) might differently affect adiposity and predict the clinical response to bariatric surgery in obese males and females. METHODS AND RESULTS: In 110 morbidly obese patients undergoing laparoscopic sleeve gastrectomy, hs-CRP as well as anthropometric assessment of adiposity, completed by electric bioimpedance and ultrasonography quantification of visceral fat area (VFA), were measured before and one year after surgery. As compared to males, obese female showed less severe overweight and prevalent subcutaneous fat deposition, but higher circulating hs-CRP. In obese females, hs-CRP was associated with VFA at baseline, independently of body mass index (BMI) and visceral adiposity index (OR 1.022 [95% CI 1.001-1.044]; p = 0.039). Based on decreases and increases in hs-CRP levels after surgery, two distinct subgroups of females were identified. Post-surgery decreases in hs-CRP was predominantly observed in patients with higher baseline levels of hs-CRP and associated with greater reduction of weight, BMI, fat and lean mass, VFA and visceral to subcutaneous fat ratio. Finally, we observed that high baseline values of hs-CRP were able to predict VFA reduction one-year after surgery, independently of BMI and visceral adiposity index (VAI) loss (OR 1.031 [95% CI 1.009-1.053]; p = 0.005). CONCLUSION: In obese females, hs-CRP levels might be a promising biomarker of visceral fat amount and dysfunction, in addition to predict the effectiveness of bariatric surgery in terms of loss of VFA one-year after surgery.

High baseline C-reactive protein levels predict partial type 2 diabetes mellitus remission after biliopancreatic diversion.

BACKGROUND AND AIMS: Several studies demonstrated that surgery can improve inflammation parameters, such as C-reactive protein (CRP). Few biomarkers have been investigated to potentially predict type 2 diabetes mellitus (T2DM) remission. We aimed at determining whether pre-surgery serum CRP levels could predict T2DM remission after 3 years in patients undergoing bariatric surgery, especially biliopancreatic diversion (BPD). METHODS AND RESULTS: This study was conducted from 2007 to 2009 at the Surgical Department of the University of Genoa, Italy. Forty-four patients with T2DM undergoing BPD (n = 38) or Roux-en-Y gastric bypass (n = 6) were enrolled. The primary endpoint was to evaluate whether pre-surgery CRP levels could predict T2DM partial remission at 3-year follow-up. Secondary endpoints were to assess whether glycaemic, lipid, and inflammatory parameters modified during the follow-up. At baseline, patients with T2DM ranged from overweight to morbid obesity, had mild dyslipidaemia, and a low-grade inflammation. Bariatric surgery improved body weight, lipid and glycaemic profile both at 1- and 3-year follow-up. Pre-surgery CRP levels progressively decreased at 1- and 3-year follow-up. Among inflammatory pre-surgery parameters, only high CRP levels were shown to predict T2DM partial remission after 3 years. Multivariate analysis confirmed the predictive value of pre-surgery CRP levels independently of age, gender, type of surgery, and body mass index. CONCLUSION: Bariatric surgery, in particular BPD, improved both metabolic and inflammatory biomarkers at 1- and 3-year follow-up. Pre-surgery high CRP levels predicted 3-year T2DM partial remission, indicating a promising target population to be especially treated with BPD.

Anti-apoA-1 auto-antibodies increase mouse atherosclerotic plaque vulnerability, myocardial necrosis and mortality triggering TLR2 and TLR4.

Auto-antibodies to apolipoprotein A-1 (anti-apoA-1 IgG) were shown to promote inflammation and atherogenesis, possibly through innate immune receptors signalling. Here, we aimed at investigating the role of Toll-like receptors (TLR) 2 and 4 on anti-apoA-1 IgG-induced atherosclerotic plaque vulnerability, myocardial necrosis and mortality in mice. Adult male apolipoprotein E knockout (ApoE)-/- (n=72), TLR2-/-ApoE-/- (n=36) and TLR4-/-Apo-/- (n=28) mice were intravenously injected with 50 µg/mouse of endotoxin-free polyclonal anti-apoA-1 IgG or control isotype IgG (CTL IgG) every two weeks for 16 weeks. Atherosclerotic plaque size and vulnerability were assessed by histology. Myocardial ischaemia and necrosis, respectively, were determined by electrocardiographic (ECG) changes assessed by telemetry and serum troponin I (cTnI) measurements. Impact on survival was assessed by Kaplan-Meier analyses. In ApoE-/- mice, anti-apoA-1 IgG passive immunisation enhanced histological features of atherosclerotic plaque vulnerability (increase in neutrophil and MMP-9 and reduction in collagen content), induced a substantial cTnI elevation (p=0.001), and increased mortality rate by 23 % (LogRank, p=0.04) when compared to CTL IgG. On a subgroup of ApoE-/- mice equipped with telemetry (n=4), a significant ST-segment depression was noted in anti-apoA-1 IgG-treated mice when compared to CTL IgG recipients (p< 0.001), and an acute ST-segment elevation myocardial infarction preceding mouse death was observed in one case. The deleterious effects of anti-apoA-1 IgG on atherosclerotic plaque vulnerability, myocardial necrosis and death were partially reversed in TLR2-/-ApoE-/- and TLR4-/-ApoE-/- backgrounds. In conclusion, anti-apoA-1 auto-antibodies seem to be active mediators of atherosclerotic plaque vulnerability, myocardial necrosis, and mortality in mice through TLR2- and TLR4-mediated pathways.

Chlorhexidine prevents hypochlorous acid-induced inactivation of alpha1-antitrypsin.

1. Chlorhexidine digluconate has been used as a topical antiseptic in the treatment of acne vulgaris and periodontitis. The acute phase of these diseases involves neutrophilic infiltration. Neutrophil activation and recruitment to inflammatory sites are crucial in both protection against bacterial infection and the induction of hystotoxic damage. Activated neutrophils release several enzymes, including elastase and myeloperoxidase (MPO), which contribute to tissue injury via direct toxic actions, the generation of oxidants and inactivation of protective factors, such as alpha1-antitrypsin (alpha1-AT). In the present study, we investigated whether chlorhexidine can modulate neutrophil-mediated histotoxicity. 2. Human primary neutrophils were isolated from healthy donors. Inactivation of alpha1-AT by neutrophils or hypochlorous acid (HOCl) was evaluated by spectrophotometry and sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis of its capacity to complex with porcine pancreatic elastase (PPE). Neutrophil generation of HOCl, superoxide anion and MPO release were assessed spectrophometrically. 3. Chlorhexidine (0, 0.5, 1, 5 and 10 micromol/L) dose-dependently prevented HOCl-induced inactivation of alpha1-AT and reduced HOCl recovery from phorbol myristate acetate (PMA)-treated human neutrophils, but did not inhibit superoxide anion and MPO release. Chlorhexidine directly inhibited HOCl recovery from neutrophils and HOCl-induced inactivation of alpha1-AT in a cell-free assay. Accordingly, chlorhexidine reversed HOCl-mediated inhibition of alpha1-AT capacity to complex with PPE. 4. These data suggest that chlorhexidine prevents neutrophil-induced alpha1-AT inactivation via a direct inhibitory action on HOCl. Although highly speculative, the present study indicates that chlorhexidine may protect inflamed tissues not only through its antimicrobial properties, but also via a direct anti-inflammatory effect on neutrophil toxic products.

Sulphasalazine accelerates apoptosis in neutrophils exposed to immune complex: Role of caspase pathway.

1. Neutrophils release several histotoxic molecules that cause tissue injury. Neutrophil apoptosis is a crucial process that governs the persistence of inflammatory disorders and tissue damage. Thus, in the present study, we investigated whether the anti-inflammatory drug sulphasalazine (SSZ) affects neutrophil apoptosis in the presence of insoluble immune complex (IC). 2. Neutrophils were obtained from healthy donors. Neutrophils were resuspended in incubation medium and incubated for 2-12 h with or without 10, 30 or 100 micromol/L SSZ and 25 microg/mL IC. In some experiments, cells were co-incubated with 20 micromol/L Z-IETD-fmk (a caspase 8 inhibitor) or 20 micromol/L Z-LEHD-fmk (a caspase 9 inhibitor). Apoptosis was evaluated morphologically on cytological preparations stained with May-Grünwald-Giemsa as well as by flow cytometry analysis of annexin V and propidium iodide staining. Caspase 3 activity was determined spectrophotometrically. 3. At 100 micromol/L, SSZ significantly accelerated IC-induced neutrophil apoptosis. Treatment of neutrophils with 20 micromol/L of the caspase 8 or 9 inhibitors Z-IETD-fmk or Z-LEHD-fmk, respectively, demonstrated that the SSZ-induced pro-apoptotic effect was mediated by a caspase 8- but not caspase 9-dependent pathway. The caspase 3 activity assay showed that treatment with 100 micromol/L SSZ increased caspase 3 activation. 4. In conclusion, the results of the present study indicate that it is possible that the molecular mechanism underlying SSZ protection against neutrophil-mediated tissue injury inflammatory disorders, such as rheumatoid arthritis and inflammatory bowel diseases, involves a caspase 8-dependent pathway.

Lymphoproliferative disorders and chemokines.

Chemokines are low molecular weight cytokines specialized in leukocyte recruitment. Recent studies have shown that tumor cells of hematopoietic and non hematopoietic origin express different chemokine receptors that may be involved in neoplastic cell growth, metastasis and angiogenesis. Human lymphoproliferative disorders arise from the malignant transformation of normal lymphoid cells frozen at discrete maturational stages. Studies performed with acute or chronic lymphoproliferative disorders have shown that CXCR4, the unique receptor for CXCL12, is up-regulated in many B and T cells malignancies and may be involved in metastatic localization of the neoplastic elements. Additional chemokine receptors are expressed in the individual lymphoproliferative disorders, but some of these are often non functional. Here we shall review the state of the art on chemokine receptor expression and function in human lymphoproliferative disorders, stressing the potential value of chemokines receptors as novel therapeutic targets. In this respect, small antagonistic peptides are being produced by pharmaceutical companies and hold great promise for clinical application.

Dexamethasone -induced apoptosis of human monocytes exposed to immune complexes. Intervention of CD95- and XIAP-dependent pathways.

Monocytes and macrophages play a key role in the initiation and persistence of inflammatory reactions. The possibility to interfere with the survival of these cells, once recruited and activated at sites of inflammation, is an attractive therapeutic option. Although resting monocytes are susceptible to pharmacologically induced apoptosis, no data are available about the possibility to modulate the survival of activated monocytes. The present work was planned to investigate if dexamethasone is able to promote apoptosis of human monocytes activated by immune complexes. When monocytes were cultured with immune complexes, a dose-dependent inhibition of apoptosis was observed. Dexamethasone stimulated apoptosis of resting and activated monocytes in a dose-dependent manner. Both the immune complex inhibitory activity and dexamethasone stimulatory properties depend on NF-kappaB/XIAP and Ras/MEK/ERK/CD95 pathways. In fact, the exposure of monocytes to immune complexes increased NF-kB activation and XIAP expression, which in turn were inhibited by dexamethasone. On the other hand, immune complex-stimulated monocytes displayed a reduced expression of CD95, which is prevented by dexamethasone, as well as by MEK inhibitor U0126. Furthermore, anti-CD95 ZB4 mAb prevented dexamethasone-induced apoptosis in immune complex stimulated monocytes. Similarly, ZB4 inhibited dexamethasone-mediated augmentation of caspase 3 activity. The present findings suggest that Fc triggering by insoluble immune complexes result in the activation of two intracellular pathways crucial for the survival of monocytes: 1. Ras/MEK/ERK pathway responsible for the down-regulation of CD95 expression; 2. NF-kappaB pathway governing the expression of XIAP. Both the pathways are susceptible to inhibition by monocyte treatment with pharmacologic concentrations of dexamethasone.

Synovial fluid from patients with rheumatoid arthritis inhibits neutrophil apoptosis: role of adenosine and proinflammatory cytokines.

OBJECTIVE: In synovial fluid (SF) from patients with rheumatoid arthritis (RA), neutrophils are exposed to proinflammatory mediators endowed with either anti-apoptotic or pro-apoptotic properties. We investigated neutrophil apoptosis in the presence of SF from 11 RA patients. METHODS: SF was obtained from affected knees of 11 patients with RA. Human neutrophil apoptosis was evaluated by light microscopic examination and flow-cytometric analysis of annexin V binding. Immune complex-induced neutrophil activation was evaluated as superoxide anion production. Adenosine levels in SF were detected by chromatographic analysis and cytokine levels were studied by enzyme-linked immunosorbent assay. RESULTS: Spontaneous and immune complex-triggered neutrophil apoptosis was reduced by SF from eight out of 11 patients. Immune complex-induced neutrophil activation was unaffected by SF. The cytokines tested had no role in promoting the anti-apoptotic activity of SF. On the contrary, the anti-apoptotic activity of SF was found to depend on the presence of adenosine. Adenosine levels detected in the various samples of SF correlated significantly with the anti-apoptotic activity of the fluids and with the number of apoptotic neutrophils detected in the articular exudate. CONCLUSION: The microenvironment of rheumatoid SF is a proinflammatory milieu responsible for the in loco persistence of activated and long-surviving neutrophils. Adenosine plays a crucial role in this phenomenon, which is related to anti-apoptotic activity.

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.

Spontaneous apoptosis in neutrophils is associated with downregulation of HLA Class I and is prevented by ligation of Class I.

In many types of cells, ligation of human leukocyte antigens (HLA) Class I molecules with specific mAbs results in the transduction of signals that trigger different cell functions. We have investigated the effects of Class I ligation in human neutrophils. After several hours in culture, neutrophils split spontaneously into two subpopulations, one with normal and the other with reduced levels of Class I. The latter subpopulation displayed high binding capacity for Annexin V, showed a hypodiploid peak, electrophoretic DNA fragmentation, and morphological features of apoptotic cells. The addition of drugs known to delay apoptosis (GM-CSF or cAMP) resulted in a reduction of Class I modulation. Furthermore, ligation of surface Class I with F(ab')2 fragments of the anti-Class I mAb W6/32 resulted in a delay in the progression of apoptosis. These data indicate that this surface Class I molecule is a marker of age-related apoptosis, and the ligation of these molecules results in the transduction of a signal that inhibits apoptosis. Thus, the downregulation of HLA Class I molecules in aging neutrophils prevents their halting the apoptotic process.

Monoclonal Lym-1 antibody-targeted lysis of B lymphoma cells by neutrophils. Evidence for two mechanisms of FcgammaRII-dependent cytolysis.

Human neutrophils incubated with the anti-HLA-DR mAb Lym-1, plus PMA, induced significant cytolysis of B lymphoma cells compared with Lym-1 and PMA alone. The effect of PMA was independent of the ability of the compound to stimulate neutrophil-respiratory burst. In fact, first, neutrophils from a patient with chronic granulomatous disease were cytolytically effective in spite of their inability to produce oxidants. Second, various kinase inhibitors exerted different effects on the PMA-stimulated cytolytic system and neutrophil-oxidative burst. Previous studies have shown the involvement of the FcgammaRII, CD11b-CD18 integrins, and CD66b glycoproteins in the Lym-1 mAb-dependent cytolysis by GM-CSF-stimulated neutrophils. The present PMA-stimulated system was inhibited by the anti-FcgammaRII mAb IV.3, the anti-CD18 mAb MEM 48, and the anti-CD11b mAb 2LPM19c but not by the anti-CD66b mAb 80H3 and N-acetyl-D-glucosamine. Furthermore, the PMA- and GM-CSF-stimulated cytolysis was insensitive and sensitive to inhibition by pertussis toxin, respectively. Thus, the use of PMA and GMCSF as neutrophil stimulants uncovers the existence of distinct mechanisms of Lym-1 mAb-mediated cytolysis.

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.

Cefoperazone prevents the inactivation of alpha(1)-antitrypsin by activated neutrophils.

At sites of neutrophilic inflammation, tissue injury by neutrophil elastase is favored by phagocyte-induced hypochlorous acid-dependent inactivation of the natural elastase inhibitor alpha(1)-antitrypsin. In the present study, cefoperazone prevented alpha(1)-antitrypsin inactivation by neutrophils and reduced the recovery of hypochlorous acid from these cells. Moreover, the antibiotic reduced the free elastase activity in a neutrophil suspension supplemented with alpha(1)-antitrypsin without affecting the cells' ability to release elastase. These data suggest that the drug inactivates hypochlorous acid before its reaction with alpha(1)-antitrypsin, thereby permitting the antiprotease-mediated blockade of released elastase. In conclusion, cefoperazone appears to have the potential for limiting elastase-antielastase imbalances, attenuating the related tissue injury at sites of inflammation.

FMLP- and TNF-stimulated monoclonal Lym-1 antibody-dependent lysis of B lymphoblastoid tumour targets by neutrophils.

Human neutrophils, incubated with Cr51-labelled B lymphoblastoid Raji cells in the presence of the anti-target monoclonal antibody (mAb) Lym-1 plus formyl-methionyl-leucyl-phenylalanine (FMLP) or tumour necrosis factor alpha (TNF-alpha), were found to induce significant C51 release, i.e. significant cytolysis. The lytic process was inhibited by mAb IV.3, specific for the Fcgamma receptor (FcgammaR) type II. The mAb 3G8, which reacts with FcgammaR type III, was ineffective. Moreover, the lysis was inhibited by the anti-CD18 mAb MEM-48. These data suggest that FMLP/Lym-1 as well as TNF-alpha/Lym-1 cytolytic systems strictly require FcgammaRII and CD18 integrins. As the lysis induced by TNF-alpha/Lym-1 was prevented by pertussis toxin (PT), PT-sensitive G-proteins are likely to intervene in post-FcgammaRII signal transduction. Both the FMLP- and the TNF-alpha-dependent systems were also found to be equally susceptible to inhibition by various inhibitors of kinases (genistein, staurosporin, 1-(5-isoquinolinnylsulphonyl)-2-methylpiperazine and wortmannin). On the contrary, an inhibitor of protein kinase C (bis-indolyl-maleimide, BIM) was effective only in the FMLP/Lym-1 cytolytic system. Therefore, it appears that signals delivered by FMLP or TNF-alpha, BIM-sensitive and insensitive respectively, converge and synergize with those from G-protein-coupled FcgammaRII and, probably, CD18-integrins to promote the expression of the neutrophil cytolytic potential.

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.