Proinflammatory activity of TWEAK on human dermal fibroblasts and synoviocytes: blocking and enhancing effects of anti-TWEAK monoclonal antibodies

Human tumour necrosis factor (TNF)-like weak inducer of apoptosis (hTWEAK) and two anti-hTWEAK mAbs were tested for their ability to elicit or block inflammatory responses in cultured human dermal fibroblasts and synoviocytes. Incubation with hTWEAK increased the production of prostaglandin E2, matrix metalloproteinase-1 (MMP-1), IL-6, and the chemokines IL-8, RANTES (regulated on activation, normal T expressed and secreted) and interferon-gamma-inducible protein-10 (IP-10) in culture supernatant of fibroblasts and synoviocytes. In combination with TNF or IL-1beta, hTWEAK further stimulated the secretion of prostaglandin E2, MMP-1, IL-6 and IL-8 up to fourfold, and IP-10 and RANTES up to 70-fold compared to TNF or IL-1beta alone. An anti-hTWEAK mAb, BCB10, blocked the effects of hTWEAK, whereas hTWEAK crosslinked by the anti-hTWEAK mAb, BEB3, further stimulated the inflammatory response of fibroblasts and synoviocytes. The anti-hTWEAK mAbs were ineffective in blocking or increasing the responses of TNF or IL-1beta and blocking anti-TNF mAb was ineffective in preventing the responses to TWEAK. These results were also confirmed at the RNA level for MMP-1, macrophage chemoattractant protein-1, RANTES, macrophage inflammatory protein-1alpha, IP-10 and IL-8. TWEAK in synergism with IL-1 and TNF may be an additional cytokine that plays a role in destructive chronic arthritic diseases.     

Construction and characterization of single-chain antibodies against human insulin-like growth factor-I receptor from hybridomas producing 1H7 or 3B7 monoclonal antibody

Recombinant antibody consisting of the single-chain variable fragment (scFv) of 1H7 monoclonal antibody against insulin-like growth factor-I receptor (IGF-IR) and human IgG(1) Fc domain, scFv-Fc, has been found to exhibit inhibitory effects on breast cancer growth in vitro and in vivo [Li et al. (2000) Cancer Immunol. Immunother. 49, 243; Sachdev et al. (2003) Cancer Res. 63, 627]. Various types of scFvs from hybridomas producing 1H7 or 3B7 mAb were constructed using conventional phage display technology to further characterize the specificity and affinity of anti-IGF-IR mAbs. Binding studies performed using either phage antibodies or soluble scFv proteins to IGF-IR or insulin receptor (IR) and IGF-IR pre-incubated with mAbs suggested that (i) 1H7 and 3B7 bind to IGF-IR but do not bind to its structurally related IR, (ii) either the VL-VH or VH-VL sequence order does not apparently affect specificity for IGF-IR and (iii) 1H7 and 3B7 bind the independent epitopes, located in or near the N-terminal (440-514) and C-terminal (62-184) domains of the alpha subunit, respectively. This study not only revealed new information on binding regions for two anti-IGF-IR mAbs, but also provided the scFv genes as tools for further manipulation of the affinity or development of new IGF-IR-targeted cancer therapeutics.     

Construction and characterization of two anti‐sweetener single chain antibodies using radioligand binding,fluorescence and circular dichroism spectroscopy

Two single‐chain antibodies (scFv) that bind the superpotent sweetener ligand, NC‐174, were generated from mouse monoclonal antibodies (mAb) NC6.8 (IgG, κ) and NC10.14 (IgG, λ). These scFv were constructed by cloning the variable region sequences of the mAb, connecting them in tandem with a 25‐amino‐acid polypeptide linker, and expressing them in E. coli using the pET‐11a system. The recombinant proteins were purified using Ni²⁺–NTA–agarose by virtue of a hexahistidine sequence introduced to the C‐terminus of the heavy chain variable region during the cloning process. The secondary structure and ligand binding properties of the two scFv, the parent mAbs and proteolytically derived Fab fragments were examined using radioligand binding, circular dichroism (CD) and fluorescence spectroscopy. The far‐UV CD spectra of both scFv possessed predominantly β character, as did those of the Fab, and the near‐UV CD spectral data for scFvNC10.14, NC6.8 and NC10.14 Fab indicated that chromophore perturbation occurred upon ligand binding. The affinity constants determined for the two scFv, Fab and mAb were nearly equivalent. Copyright © 1999 John Wiley & Sons, Ltd.     

Proinflammatory activity of TWEAK on human dermal fibroblasts and synoviocytes: blocking and enhancing effects of anti-TWEAK monoclonal antibodies

Human tumour necrosis factor (TNF)-like weak inducer of apoptosis (hTWEAK) and two anti-hTWEAK mAbs were tested for their ability to elicit or block inflammatory responses in cultured human dermal fibroblasts and synoviocytes. Incubation with hTWEAK increased the production of prostaglandin E₂, matrix metalloproteinase-1 (MMP-1), IL-6, and the chemokines IL-8, RANTES (regulated on activation, normal T expressed and secreted) and interferon-γ-inducible protein-10 (IP-10) in culture supernatant of fibroblasts and synoviocytes. In combination with TNF or IL-1β, hTWEAK further stimulated the secretion of prostaglandin E₂, MMP-1, IL-6 and IL-8 up to fourfold, and IP-10 and RANTES up to 70-fold compared to TNF or IL-1β alone. An anti-hTWEAK mAb, BCB10, blocked the effects of hTWEAK, whereas hTWEAK crosslinked by the anti-hTWEAK mAb, BEB3, further stimulated the inflammatory response of fibroblasts and synoviocytes. The anti-hTWEAK mAbs were ineffective in blocking or increasing the responses of TNF or IL-1β and blocking anti-TNF mAb was ineffective in preventing the responses to TWEAK. These results were also confirmed at the RNA level for MMP-1, macrophage chemoattractant protein-1, RANTES, macrophage inflammatory protein-1α, IP-10 and IL-8. TWEAK in synergism with IL-1 and TNF may be an additional cytokine that plays a role in destructive chronic arthritic diseases.     

Brucella abortus inhibits IFN-γ-induced FcγRI expression and FcγRI-restricted phagocytosis via toll-like receptor 2 on human monocytes/macrophages

The strategies that allow Brucella abortus to persist for years inside macrophages subverting host immune responses are not completely understood. Immunity against this bacterium relies on the capacity of IFN-γ to activate macrophages, endowing them with the ability to destroy intracellular bacteria. We report here that infection with B. abortus down-modulates the expression of the type I receptor for the Fc portion of IgG (FcγRI, CD64) and FcγRI-restricted phagocytosis regulated by IFN-γ in human monocytes/macrophages. Both phenomena were not dependent on bacterial viability, since they were also induced by heat-killed B. abortus (HKBA), suggesting that they were elicited by a structural bacterial component. Accordingly, a prototypical B. abortus lipoprotein (L-Omp19), but not its unlipidated form, inhibited both CD64 expression and FcγRI-restricted phagocytosis regulated by IFN-γ. Moreover, a synthetic lipohexapeptide that mimics the structure of the protein lipid moiety also inhibited CD64 expression, indicating that any Brucella lipoprotein could down-modulate CD64 expression and FcγRI-restricted phagocytosis. Pre-incubation of monocytes/macrophages with anti-TLR2 mAb blocked the inhibition of the CD64 expression mediated by HKBA and L-Omp19. These results, together with our previous observations establish that B. abortus utilizes its lipoproteins to inhibit the monocytes/macrophages activation mediated by IFN-γ and to subvert host immunonological responses.     

FcγRI blockade and modulation for immunotherapy

 Splenectomy and corticosteroids are the treatment of choice for patients with immune thrombocytopenic purpura (ITP). However, for the 10%–15% of patients who do not respond to conventional therapy, high-dose i.v. IgG can induce life-saving transient responses. The benefits of i.v. IgG have been attributed to Fc receptor blockade; however, the involvement of the individual Fc receptors for IgG (FcγR) in ITP remain to be more completely defined. Recently a mAb, designated mAb H22, which recognizes an epitope on FcγRI (CD64) outside the ligand-binding domain, was humanized. Because mAb H22 is a human IgG1 and FcγRI has a high affinity for human IgG1 antibodies, we predicted that mAb H22 would bind to the FcγRI ligand-binding site through its Fc domain and to its external FcγRI epitope through both Fab domains. These studies demonstrate that mAb H22 blocked FcγRI-mediated phagocytosis of opsonized red blood cells more effectively than an irrelevant IgG. Moreover, cross-linking FcγRI with mAb H22 down-modulated FcγRI expression on monocytes, an effect seen within 2 h. Accepted: 14 October 1997     

Effects of interleukin-18 on natural killer cells: costimulation of activation through Fc receptors for immunoglobulin

The antitumor activity of monoclonal antibodies is mediated by effector cells, such as natural killer (NK) cells, that express Fc receptors for immunoglobulin. Efficacy of monoclonal antibodies, including the CD20 antibody rituximab, could be improved by agents that augment the function of NK cells. Interleukin (IL)-18 is an immunostimulatory cytokine that has antitumor activity in preclinical models. The effects of IL-18 on NK cell function mediated through Fcγ receptors were examined. Human NK cells stimulated with immobilized IgG in vitro secreted IFN-γ as expected; such IFN-γ production was partially inhibited by blocking CD16 with monoclonal antibodies. IL-18 augmented IFN-γ production by NK cells stimulated with immobilized IgG or CD16 antibodies. NK cell IFN-γ production in response to immobilized IgG and/or IL-18 was inhibited by chemical inhibitors of Syk and several other kinases involved in CD16 signaling pathways. IL-18 augmented antibody-dependent cellular cytotoxicity (ADCC) of human NK cells against rituximab-coated Raji cells in vitro. IL-18 and rituximab acted synergistically to promote regression of human lymphoma xenografts in SCID mice. Inasmuch as IL-18 costimulates IFN-γ production and ADCC of NK cells activated through Fc receptors in vitro and augments antitumor activity of rituximab in vivo, it is an attractive cytokine to combine with monoclonal antibodies for treatment of human cancer.     

IL-12 enhances the antitumor actions of trastuzumab via NK cell IFN-γ production

The antitumor effects of therapeutic mAbs may depend on immune effector cells that express FcRs for IgG. IL-12 is a cytokine that stimulates IFN-γ production from NK cells and T cells. We hypothesized that coadministration of IL-12 with a murine anti-HER2/neu mAb (4D5) would enhance the FcR-dependent immune mechanisms that contribute to its antitumor activity. Thrice-weekly therapy with IL-12 (1 μg) and 4D5 (1 mg/kg) significantly suppressed the growth of a murine colon adenocarcinoma that was engineered to express human HER2 (CT-26(HER2/neu)) in BALB/c mice compared with the result of therapy with IL-12, 4D5, or PBS alone. Combination therapy was associated with increased circulating levels of IFN-γ, monokine induced by IFN-γ, and RANTES. Experiments with IFN-γ-deficient mice demonstrated that this cytokine was necessary for the observed antitumor effects of therapy with IL-12 plus 4D5. Immune cell depletion experiments showed that NK cells (but not CD4(+) or CD8(+) T cells) mediated the antitumor effects of this treatment combination. Therapy of HER2/neu-positive tumors with trastuzumab plus IL-12 induced tumor necrosis but did not affect tumor proliferation, apoptosis, vascularity, or lymphocyte infiltration. In vitro experiments with CT-26(HER2/neu) tumor cells revealed that IFN-γ induced an intracellular signal but did not inhibit cellular proliferation or induce apoptosis. Taken together, these data suggest that tumor regression in response to trastuzumab plus IL-12 is mediated through NK cell IFN-γ production and provide a rationale for the coadministration of NK cell-activating cytokines with therapeutic mAbs.     

Functional signaling of membrane-bound TL1A induces IFN-γ expression

TL1A, a TNF member implicated in autoimmune diseases, is a transmembrane protein that is processed to release soluble TL1A (TL1A-S). TL1A-S induces a Th1 response, although the functional significance of membrane-bound TL1A (TL1A-M) remains unknown. We generated TL1A-M expression in HEK-293 cells capable of binding DR3-Fc. Co-incubating IL-12/IL-18-primed CD4⁺ T cells with HEK-293 cells expressing TL1A-M induced 3-fold increase in IFN-γ that was blocked by anti-TL1A Ab. These results demonstrate that TL1A-M can bind death domain receptor 3 (DR3) through cell-cell contact to induce downstream IFN-γ secretion enhancement. Anti-TL1A antibodies designed to treat immune diseases should be verified to block both endogenous TL1A forms.     

A novel synthetic na?ve human antibody library allows the isolation of antibodies against a new epitope of oncofetal fibronectin

Human monoclonal antibodies (mAbs) can routinely be isolated from phage display libraries against virtually any protein available in sufficient purity and quantity, but library design can influence epitope coverage on the target antigen. Here we describe the construction of a novel synthetic human antibody phage display library that incorporates hydrophilic or charged residues at position 52 of the CDR2 loop of the variable heavy chain domain, instead of the serine residue found in the corresponding germline gene. The novel library was used to isolate human mAbs to various antigens, including the alternatively-spliced EDA domain of fibronectin, a marker of tumor angiogenesis. In particular, the mAb 2H7 was proven to bind to a novel epitope on EDA, which does not overlap with the one recognized by the clinical-stage F8 antibody. F8 and 2H7 were used for the construction of chelating recombinant antibodies (CRAbs), whose tumor-targeting properties were assessed in vivo in biodistribution studies in mice bearing F9 teratocarcinoma, revealing a preferential accumulation at the tumor site.Key words: human antibody library, phage display, oncofetal fibronectin, vascular tumor targeting, scFv antibody fragments, chelating recombinant antibody (CRAb)     

siRNA沉默t-bet基因对CD4、CD8T淋巴细胞亚群IFN-γ产生的影响

利用化学合成针对人t-bet基因的小干扰RNA（siRNA）,转染体外培养的人外周血单个核细胞,利用流式细胞仪分选纯化CD4^＋、CD8^＋T淋巴细胞,半定量RT-PCR检测CD4^＋、CD8^＋T淋巴细胞中t-betmRNA的表达变化,流式细胞术检测转染前后IFN-γ产生的变化情况。探讨转录因子t-bet对人CD4^＋、CD8^＋T淋巴细胞亚群IFN-γ产生的调控作用。与对照组相比,转染后的人CD4^＋、CD8^＋T淋巴细胞t-betmRNA表达水平明显下降;转染siRNA后,CD4＋T淋巴细胞中IFN-γ＋细胞比例为（18.46±6.86）%,与对照组（50.20±5.91）%比较有显著性差异（P〈0.01）;CD8＋T淋巴细胞IFN-γ＋细胞比例为（74.18±9.33）%,和对照组（76.51±6.49）%比较差异不明显（P〉0.05）。体外转录合成的siRNA可有效降低人CD4^＋、CD8^＋T淋巴细胞t-bet的基因表达;转录因子t-bet对人不同淋巴细胞亚群IFN-γ的产生所起作用不同。     

Neutrophil adhesion to TNF alpha-activated endothelial cells potentiates leukotriene B4 production.

Since adhesion of neutrophils (PMN) to endothelial cells may influence PMN activation responses, we examined whether adhesion of PMN to TNF alpha-activated human umbilical vein endothelial cells (HUVEC) stimulates leukotriene B4 (LTB4) production. Endothelial adhesivity towards PMN increased after HUVEC pretreatment with TNF alpha for 4 h. LTB4 production increased markedly in response to stimulation with arachidonic acid (20 microM) when PMN were added to the hyperadhesive HUVEC. In contrast, stimulation of PMN in suspension did not potentiate LTB4 production. LTB4 production persisted when PMN were applied to TNF alpha-pretreated HUVEC fixed with 1% paraformaldehyde excluding the possibility that metabolic activity of endothelium participates in this response. PMN adhesion to plastic and gelatin also enhanced LTB4 indicating that adhesion was a critical event in inducing LTB4 production. We used monoclonal antibodies (mAb) to adhesion molecules on endothelial cells (i.e., endothelial leukocyte adhesion molecule-1 (ELAM-1) and intercellular adhesion molecule-1 (ICAM-1)) or on PMN (CD18) to assess the role of PMN adhesion to the activated endothelium on LTB4 potentiation. Both anti-ELAM-1 mAb and anti-ICAM-1 mAb inhibited PMN adhesion (by 55 and 41%, respectively) as well as LTB4 production (by 65 and 50%, respectively). Anti-CD18 mAb also reduced the adhesion (65%) and the LTB4 production (66%). Furthermore, combination of anti-ELAM-1 mAb (H18/7) and anti-ICAM-1 mAb (RR1/1) or of anti-ELAM-1 mAb (H18/7) and anti-CD18 mAb (IB4) had an additive effect in inhibiting both PMN adhesion as well as LTB4 production. PMN adherence to immobilized recombinant soluble rELAM-1 or rICAM-1 also increased LTB4 production, which was prevented with relevant mAbs. However, neither rELAM-1 nor rICAM-1 stimulated LTB4 production of PMN in suspension. We conclude that PMN adhesion to TNF alpha-stimulated endothelial cells enhances LTB4 production by PMN, a response activated by binding of PMN to expressed endothelial cell surface adhesion molecules.     

Preparation and diagnostic use of a novel recombinant single-chain antibody against rabies virus glycoprotein

Rabies virus (RABV) causes a fatal infectious disease, but effective protection may be achieved with the use of rabies immunoglobulin and a rabies vaccine. Virus-neutralizing antibodies (VNA), which play an important role in the prevention of rabies, are commonly evaluated by the RABV neutralizing test. For determining serum VNA levels or virus titers during the RABV vaccine manufacturing process, reliability of the assay method is highly important and mainly dependent on the diagnostic antibody. Most diagnostic antibodies are monoclonal antibodies (mAbs) made from hybridoma cell lines and are costly and time consuming to prepare. Thus, production of a cost-effective mAb for determining rabies VNA levels or RABV titers is needed. In this report, we describe the prokaryotic production of a RABV-specific single-chain variable fragment (scFv) protein with a His-tag (scFv98H) from a previously constructed plasmid in a bioreactor, including the purification and refolding process as well as the functional testing of the protein. The antigen-specific binding characteristics, affinity, and relative affinity of the purified protein were tested. The scFv98H antibody was compared with a commercial RABV nucleoprotein mAb for assaying the VNA level of anti-rabies serum samples from different sources or testing the growth kinetics of RABV strains for vaccine manufactured in China. The results indicated that scFv98H may be used as a novel diagnostic tool to assay VNA levels or virus titers and may be used as an alternative for the diagnostic antibody presently employed for these purposes.     

ASC, a novel 22-kDa protein, aggregates during apoptosis of human promyelocytic leukemia HL-60 cells

The cytoskeletal and/or nuclear matrix molecules responsible for morphological changes associated with apoptosis were identified using monoclonal antibodies (mAbs). We developed mAbs against Triton X-100-insoluble components of HL-60 cells pretreated with all-trans retinoic acid. In particular, one mAb recognized a 22-kDa protein that exhibited intriguing behavior by forming an aggregate and appearing as a speck during apoptosis induced by retinoic acid and other anti-tumor drugs. Cloning and sequencing of its cDNA revealed that this protein comprises 195 amino acids and that its C-terminal half has a caspase recruitment domain (CARD) motif, characteristic of numerous proteins involved in apoptotic signaling. We referred to this protein as ASC (apoptosis-associated speck-like protein containing a CARD). The ASC gene was mapped on chromosome 16p11.2-12. The antisense oligonucleotides of ASC were found to reduce the expression of ASC, and consequently, etoposide-mediated apoptosis of HL-60 cells was suppressed. Our results indicate that ASC is a novel member of the CARD-containing adaptor protein family.     

T Cell-Dependent Activation of Macrophages and Enhancement of Their Phagocytic Activity in the Lungs of Mice Inoculated with Heat-Killed Cryptococcus neoformans: Involvement of IFN-γ and Its Protective Effect against Cryptococcal Infection

Previous investigations have demonstrated that macrophages play a critical role in the first-line cellular defense mechanism against infection with Cryptococcus neoformans. In the present study, to elucidate the way in which anticryptococcal activity of macrophages is regulated at the site of infection, pulmonary intraparenchymal macrophages were directly analyzed for expression of their surface molecules and their phagocytic activities against the organism, and the effects of depletion of T cells and endogenous IFN-γ in vivo on these parameters were examined. In the lungs of mice intratracheally inoculated with heat-killed C. neoformans, macrophages were activated, as indicated by augmented expression of MHC class II, intercellular adhesion molecule-1 (ICAM-1) and Fc receptor (FcR), and about two-thirds of macrophages were found to have ingested an average of 3.77 ± 0.12 yeast cells per macrophage. In mice depleted of both CD4⁺ and CD8⁺ T cells by injecting the specific monoclonal antibodies (mAbs) or anti-IFN-γ mAb, not only augmentation of the expression of macrophage activation markers but also phagocytosis of C. neoformans was significantly reduced. These results suggest that anticryptococcal activity of macrophages is regulated by IFN-γ endogenously produced by T cells. Additionally, treatment with IFN-γ were shown to significantly prolong the survival time of mice infected with viable C. neoformans. Additionally, preimmunization with heat-killed C. neoformans significantly prolonged the survival time of mice which received the following infection.     

Targeting CD64 mediates elimination of M1 but not M2 macrophages in vitro and in cutaneous inflammation in mice and patient biopsies

Macrophages are key players in controlling the immune response that can adapt to microenvironmental signals. This results in distinct polarization states (classical M1 or alternative M2), that play a differential role in immune regulation. In general, the M1 contribute to onset of inflammation, whereas the M2 orchestrate resolution and repair, whereby failure to switch from predominantly M1 to M2 reinforces a pro-inflammatory environment and chronic inflammation. Here, we show selective elimination of M1 macrophages in vitro by a range of CD64-targeted immunotoxins, including H22(scFv)-ETA'. After re-polarization of already polarized macrophages, still only M1 polarization showed sensitivity toward CD64-directed immunotoxins. The selectivity for M1 was found linked to reduced endosomal protease activity in M1 macrophages as demonstrated by inhibition of endosomal proteases. Using the H22(scFv)-ETA' in a transgenic mouse model for chronic cutaneous inflammation, the M1 specificity was confirmed in vivo and a beneficial effect on inflammation demonstrated. Also ex vivo on skin biopsies from atopic dermatitis and diabetes type II patients with chronically-inflamed skin, a clear M1 specific effect was found. This indicates the potential relevance for human application. Our data show that targeting M1 macrophages through CD64 can be instrumental in developing novel intervention strategies for chronic inflammatory conditions.     

CD22 ligand binding regulates normal and malignant B lymphocyte survival in vivo

The CD22 extracellular domain regulates B lymphocyte function by interacting with alpha2,6-linked sialic acid-bearing ligands. To understand how CD22 ligand interactions affect B cell function in vivo, mouse anti-mouse CD22 mAbs were generated that inhibit CD22 ligand binding to varying degrees. Remarkably, mAbs which blocked CD22 ligand binding accelerated mature B cell turnover by 2- to 4-fold in blood, spleen, and lymph nodes. CD22 ligand-blocking mAbs also inhibited the survival of adoptively transferred normal (73-88%) and malignant (90%) B cells in vivo. Moreover, mAbs that bound CD22 ligand binding domains induced significant CD22 internalization, depleted marginal zone B cells (82-99%), and reduced mature recirculating B cell numbers by 75-85%. The CD22 mAb effects were independent of complement and FcRs, and the CD22 mAbs had minimal effects in CD22AA mice that express mutated CD22 that is not capable of ligand binding. These data demonstrate that inhibition of CD22 ligand binding can disrupt normal and malignant B cell survival in vivo and suggest a novel mechanism of action for therapeutics targeting CD22 ligand binding domains.     

Tumor necrosis factor alpha stimulates sphingomyelinase through the 55 kDa receptor in HL-60 cells.

Tumor necrosis factor alpha (TNF alpha) stimulated rapid (seconds) hydrolysis of sphingomyelin in HL-60 cells, formation of phosphocholine (PCho) and a decrease in choline. The response to TNF alpha was concentration dependent with a maximal effect at 3-10 nM. The monoclonal antibody (mAb), htr-9, which behaves as an agonist at the 55 kDa subtype of the TNF receptor, also stimulated sphingomyelin hydrolysis in intact cells. In contrast, the mAb, utr-1, which behaves as an antagonist at the 75 kDa receptor subtype, had no effect on sphingomyelin hydrolysis either on its own or in the presence of TNF alpha. In addition, htr-9 or TNF alpha stimulated hydrolysis of sphingomyelin in a membrane fraction of HL-60 cells. These results are consistent with a role of sphingomyelin hydrolysis as an early event in the signalling mechanism of TNF alpha, and suggest that this pathway is activated through the 55 kDa subtype of the TNF receptor.