Interdependence of the radioprotective effects of human recombinant interleukin 1 alpha, tumor necrosis factor alpha, granulocyte colony-stimulating factor, and murine recombinant granulocyte-macrophage colony-stimulating factor

Interleukin 1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF alpha), granulocyte-colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) are molecularly distinct cytokines acting on separate receptors. The release of these cytokines can be concomitantly induced by the same signal and from the same cellular source, suggesting that they may cooperate. Administered alone, human recombinant (hr)IL-1 alpha and hrTNF alpha protect lethally irradiated mice from death, whereas murine recombinant GM-CSF and hrG-CSF do not confer similar protection. On a dose basis, IL-1 alpha is a more efficient radioprotector than TNF alpha. At optimal doses, IL-1 alpha is a more radioprotective cytokine than TNF alpha in C57BL/6 and B6D2F1 mice and less effective than TNF alpha in C3H/HeN mice, suggesting that the relative effectiveness of TNF alpha and IL-1 alpha depends on the genetic makeup of the host. Administration of the two cytokines in combination results in additive radioprotection in all three strains. This suggests that the two cytokines act through different radioprotective pathways and argues against their apparent redundancy. Suboptimal, nonradioprotective doses of IL-1 alpha also synergize with GM-CSF or G-CSF to confer optimal radioprotection, suggesting that such an interaction may be necessary for radioprotection of hemopoietic progenitor cells.     

TNF-alpha induces phosphorylation of p47(phox) in human neutrophils: partial phosphorylation of p47phox is a common event of priming of human neutrophils by TNF-alpha and granulocyte-macrophage colony-stimulating factor

Phosphorylation of p47(phox) is a key event in NADPH oxidase activation. We examined the ability of proinflammatory cytokines such as TNFalpha, IL-1, and G-CSF to induce this process compared with GM-CSF. Only TNF-alpha and GM-CSF induced a clear p47(phox) phosphorylation. This phosphorylation was time dependent and reached its maximum at 20 min. Two-dimensional phosphopeptide mapping of p47(phox) phosphorylated in neutrophils primed with TNF-alpha revealed partial phosphorylation of p47(phox) on the same peptide as for GM-CSF. Neutrophil incubation with TNF-alpha and subsequent addition of the chemotactic peptide fMLP resulted in more intense phosphorylation of p47(phox) sites than with each reagent alone. A neutralizing Ab against the p55 TNF receptor, contrary to a neutralizing Ab against the p75 TNF receptor, inhibited TNF-alpha-induced p47(phox) phosphorylation. Neutrophil treatment with both TNF-alpha and GM-CSF resulted in more intense phosphorylation of the same p47(phox) peptide observed with each cytokine alone, suggesting that they engaged pathways converging on common serines. This additive effect was also obtained on the priming of NADPH oxidase activity. The use of protein kinase inhibitors pointed to the involvement of a protein tyrosine kinase, but not protein kinase C. These findings show that TNF-alpha, via its p55 receptor, induces a protein tyrosine kinase-dependent selective phosphorylation of p47(phox) on specific serines. The ability of TNF-alpha and GM-CSF, two different cytokines with two different receptors to induce this specific p47(phox) phosphorylation, suggests that this event could be a common element of the priming of neutrophils by TNF-alpha and GM-CSF.     

GM-CSF and TNFα modulate protein expression of human neutrophils visualized by fluorescence two-dimensional difference gel electrophoresis

Increased serum levels of TNFα and GM-CSF are found in various chronic inflammatory diseases and these cytokines affect the function of circulating and tissue neutrophils. TNFα- and GM-CSF-induced protein expression profiles could, therefore, serve as biomarker for the action of these cytokines in vivo. We stimulated human peripheral neutrophils with TNFα and GM-CSF in vitro and analyzed changes in their proteome by fluorescence two-dimensional difference gel electrophoresis (2D-DIGE). We report the differential expression of 3 and 18 protein spots following TNFα and GM-CSF stimulation, respectively. Differences in protein expression induced by TNFα were limited and did not show discriminatory power in a principal component analysis, whereas the profile induced by GM-CSF did. TNFα- and GM-CSF-induced both de novo IL-1β and sIL-1Ra protein expression as detected by Western blot analysis, which confirmed proper neutrophil activation by these cytokines in vitro. Mass spectrometry analysis of cytokine-regulated protein spots resulted in the identification of 8 proteins. Among the identified proteins, enolase 1 and annexin A1 might function as markers for peripheral neutrophil activation.In conclusion, a proteomic analysis of neutrophils by 2D-DIGE provides proof-of-principle that cytokine-induced protein profiles can serve as biomarkers for the action of individual cytokines in vivo.     

Actin reorganization and morphological changes in human neutrophils stimulated by TNF,GM-CSF,and G-CSF: the role of MAP kinases

Stimulation of human neutrophils with tumor necrosis factor- (TNF), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte CSF (G-CSF) resulted in decreased fluorescence intensity of FITC-phalloidin (actin depolymerization) and morphological changes. Cytokine-induced actin depolymerization was dependent on the concentration of cytokines used as stimuli. The maximal changes were detected at 10 min after stimulation with TNF or GM-CSF and at 20 min after stimulation with G-CSF. Cytokine-induced actin depolymerization was sustained for at least 30 min after stimulation. In contrast, N-formyl-methionyl-leucyl-phenylalanine (FMLP) rapidly (within 45 s) induced an increase in the fluorescence intensity of FITC-phalloidin (actin polymerization) and morphological changes. TNF- and GM-CSF-induced actin depolymerization and morphological changes, but not FMLP-induced responses, were partially inhibited by either PD-98059, an inhibitor of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase, or SB-203580, an inhibitor of p38 MAPK, and were almost completely abolished by these inhibitors in combination. G-CSF-induced responses were almost completely abolished by PD-98059 and were unaffected by SB-203580. These findings are consistent with the ability of these cytokines to activate the distinct MAPK subtype cascade in human neutrophils. Phosphorylated ERK and p38 MAPK were not colocalized with F-actin in neutrophils stimulated by cytokines or FMLP. Furthermore, FMLP-induced polarization and actin polymerization were prevented by cytokine pretreatment. These findings suggest that TNF, GM-CSF, and G-CSF induce actin depolymerization and morphological changes through activation of ERK and/or p38 MAPK and that cytokine-induced actin reorganization may be partly responsible for the inhibitory effect of these cytokines on neutrophil chemotaxis. neutrophil; actin reorganization; cytokines; mitogen-activated protein kinase; tumor necrosis factor-; granulocyte-macrophage colony stimulating factor     

The effects of cytokines, platelet activating factor, and arachidonate metabolites on C3B receptor (CR1, CD35) expression and phagocytosis by neutrophils

The cytokines tumor necrosis factor alpha (TNF alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and interleukin 1 (IL 1) all caused an upregulation of C3b receptors (CR1) on neutrophils that ranged from around 76% (G-CSF and IL 1) to 93% (TNF alpha and GM-CSF) of the upregulation obtained by pretreatment of the neutrophils with the chemotactic peptide FMLP. However, only TNF alpha and G-CSF caused a significant increase in phagocytosis of opsonized microspheres. Platelet derived growth factor, interleukin 2, and transforming growth factor beta had no effect on either of these parameters. The mediators platelet activating factor (PAF) and leukotriene B4 (LTB4) both caused a large upregulation of CR1 (93% and 80%, respectively, of the FMLP-mediated value); however, only PAF caused a significant enhancement of phagocytosis by the neutrophils. Prostaglandin E2 and thromboxane B2 had no effect on these parameters. Considerable individual variation was observed among some of the untreated and mediator-treated neutrophil preparations regarding CR1 expression and phagocytosis. The upregulation of CR1 and associated increase in phagocytic capacity of neutrophils caused by certain cytokines and other mediators may be important in host defense. Also the lack of enhancement of phagocytosis accompanying an upregulation of CR1 is unusual and may have important implications regarding the cellular mechanisms of phagocytosis by neutrophils.     

Granulocyte-macrophage colony stimulating factor induces both HLA-DR expression and cytokine production by human monocytes

The effect of recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) on the expression of HLA-DR, and the production of the cytokines interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha) by human peripheral blood monocyte-enriched populations was investigated. GM-CSF was shown to induce both the expression of HLA-DR and the cytokines IL-1 and TNF alpha in a dose-dependent manner. In contrast, interferon-gamma (IFN-gamma), which induced major histocompatibility complex (MHC) class II expression, did not induce IL-1 or TNF alpha production. However, IFN-gamma enhanced the cell surface expression of HLA-DR and the production of IL-1 and TNF alpha on monocyte-enriched cells stimulated by GM-CSF. By itself, GM-CSF did not induce surface class II expression on the human monocytic tumour cell line THP-1, whereas it synergized with IFN-gamma to induce surface expression. These cells responded to GM-CSF by producing IL-1 and TNF alpha; Northern blotting showed that mRNA levels of IL-1 and TNF alpha were transiently induced, similar to other cytokines. Our results indicate that GM-CSF is a major macrophage activating factor that is capable of inducing both the expression of HLA-DR and the cytokines involved in T-cell activation by macrophages; therefore, GM-CSF may be of importance in potentiating antigen presenting function.     

Comparison of granulocyte-macrophage colony stimulating factor and interleukin-1 production from human peripheral blood mononuclear cells as measured by specific radioimmunoassays.

Attention has focused on cytokine networks in which gene and protein expression of some cytokines is under the influence of other cytokines. In the present studies, we addressed the relationship between the synthesis of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-1 (IL-1) in human peripheral blood mononuclear cells (PBMC) stimulated with mitogens. Since bioassays for cytokines are sensitive to more than one of these factors, it was necessary to measure the amounts of IL-1 and GM-CSF independent of bioassays. A specific and sensitive (40 pg/ml) radioimmunoassay (RIA) was developed for human GM-CSF. The sensitivity of the RIA was greater when lysine residues were iodinated with Bolton-Hunter reagent than tyrosine residues using chloramine T. After stimulating PBMC with concanavalin A (Con A), the biological activity of GM-CSF was determined in bone marrow cultures and compared to immunoreactive GM-CSF; GM-CSF levels detected by bioassays and RIAs were highly correlated in two separate sets of experiments (r2 = 0.95 and 0.43). Incubation with Con A for 48 h induced more GM-CSF than stimulation by phytohemagglutinin (PHA) despite the fact that PHA stimulates large amounts of IL-1 alpha; indomethacin had no effect on Con A stimulated synthesis of GM-CSF or IL-1 alpha. In two separate studies, PBMC from 14 donors and a second group of 12 donors were incubated with Con A for 48 h and the total amount of immunoreactive IL-1 alpha and GM-CSF was determined in the same cell cultures. There was no correlation of the amount of either cytokines in these cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interleukin-1 beta- and tumor necrosis factor-alpha-independent monocyte stimulation of fibroblast collagenase activity

To investigate the mechanism of cyclosporine (Cs)-induced fibrous gingival enlargement, the indirect effects of Cs on fibroblast collagenolysis via the drug's effect on the synthesis of the fibroblast regulatory monokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF alpha) have been studied. Peripheral blood monocytes stimulated with lipopolysaccharide (LPS) for 48 h produced conditioned media (MCM-LPS) that contained 665 pg/ml IL-1 beta and 16 pg/ml TNF alpha and significantly (P less than 0.001) enhanced the collagenase activity of a fibroblast strain (GN 23) derived from a healthy individual with clinically normal gingiva. The concurrent addition of Cs (50, 100, or 150 ng/ml) with LPS to the monocytes (MCM-LPS-Cs) significantly diminished their ability to enhance GN 23 collagenase activity in a dose-dependent manner, with MCM-LPS-Cs (150 ng/ml) causing the greatest effect. Cs also significantly inhibited IL-1 beta and TNF alpha production. Although the greatest inhibition of both cytokines was at 50 ng/ml Cs, the corresponding MCM-LPS-Cs caused the least diminution (16%) of the collagenase stimulation caused by MCM-LPS (no Cs). This suggested that factor(s) other than or in addition to IL-1 beta and TNF alpha might be responsible for the stimulation of GN 23 collagenase activity. MCM-LPS depleted of IL-1 beta by affinity chromatography retained its stimulatory effect on GN 23 collagenolysis, and human recombinant IL-1 beta and TNF alpha, when tested alone or together at levels found in the stimulatory MCM-LPS and MCM-LPS-Cs, did not stimulate GN 23 collagenase activity as did the crude conditioned media. This evidence suggested that the conditioned media contained the complex mixture of cytokines necessary to stimulate collagenase activity of this fibroblast strain and that IL-1 beta and TNF alpha were not necessarily involved. Cs may alter the synthesis of other collagenase-stimulating cytokines, accounting for the diminished ability of Cs-treated monocytes to enhance collagenase activity of susceptible fibroblast strains. Decreased collagenase activity, therefore, resulting from Cs suppression of monokine production, may be an important factor in the development of fibrous gingival enlargement seen in some susceptible patients treated with Cs.     

Granulocyte-macrophage colony-stimulating factor and other cytokines regulate surface expression of the leukocyte adhesion molecule-1 on human neutrophils, monocytes, and their precursors.

There is increasing evidence that cytokines such as granulocyte-macrophage (GM)-CSF can profoundly affect the adhesion, aggregation, and mobility of neutrophils both in vitro and in vivo. However, the mechanisms whereby these factors might alter the adhesive properties of neutrophils are incompletely understood. A new family of cellular adhesion molecules has recently been identified by cDNA cloning. The members of this family include human leukocyte adhesion molecule-1 (LAM-1), the human endothelial-leukocyte adhesion molecule, and the mouse leukocyte homing receptor for high endothelial venules, MEL-14. LAM-1 is the human homologue of murine MEL-14, and is believed to mediate binding of leukocytes to human high endothelial venules. LAM-1 can be identified by mAb TQ-1, Leu 8, or anti-LAM1.1. The expression and regulation of LAM-1 on granulocytes, monocytes, and their precursors was investigated using flow cytometry and the anti-LAM-1.1 mAb. Neutrophils, eosinophils, monocytes, marrow myeloid cells, granulocyte/macrophage colony-forming unit, and burst-forming unit for erythroid cells were LAM-1+ by flow microfluorimetry. The regulation of LAM-1 expression was tested by treating various cell populations with cytokines or other stimuli for 0-90 min. Exposure of neutrophils, monocytes, and marrow myeloid cells to GM-CSF induced rapid and complete loss of LAM-1 from the cell surface, but had no effect on LAM-1 expression by lymphocytes. The loss of LAM-1 was temporally correlated with up-regulation of CD11b (Mo1), an adhesion molecule involved in neutrophil aggregation. Several other factors known to activate neutrophils also caused down-regulation of LAM-1 and up-regulation of CD11b, including TNF, FMLP, and leukotriene B4. Interestingly, granulocyte-CSF and IFN-gamma had minimal effects on neutrophil LAM-1 expression. Similar results were observed on monocytes and myeloid precursor cells. Thus, exposure of neutrophils to GM-CSF results in a profound change in surface expression of adhesion molecules, with coordinated up-regulation of CD11b and down-regulation of LAM-1. These changes in adhesion proteins are likely to alter aggregation and mobility of both mature myeloid cells and their precursors in patients receiving certain types of cytokine therapy.     

Granulocyte macrophage-colony stimulating factor stimulates the synthesis of membrane and nuclear proteins in murine neutrophils

The effect of granulocyte macrophage-colony stimulating factor (GM-CSF), a well-characterized hemopoietic regulator, on protein synthesis in murine bone marrow neutrophils is described. Bone marrow neutrophils in excess of 95% purity were obtained by fluorescence-activated cell sorting. While GM-CSF did not appear to slow the rate of dying of peritoneal exudate neutorphils or thymus cells, the viability of bone marrow neutrophils after 17 hr was enhanced (40%) by GM-CSF. GM-CFS had no effect on total 35S-methionine incorporation by thymocytes or peritoneal exudate neutrophils over a 17-hr incubation period; however, bone marrow neutrophils showed increased incorporation (approximately 10%) at all times between 5-17 hr. As viability and 35S-methionine incorporation of bone marrow neutrophils at 5 hr were minimally affected by GM-CSF, this time point was chosen to study the effect of GM-CSF on the synthesis of particular proteins. Two-dimensional polyacrylamide gels of 35S-methionine-labelled lysates were prepared from whole cells, isolated nuclei, and membranes. Quantitative analysis of the fluorograms obtained from the two-dimensional electropherograms by a computer-linked optical data digitiser indicated that out of a total of 180 proteins, the amount of label contained in 11 proteins was significantly higher in the presence of GM-CSF, while three proteins, apparently of cytoplasmic origin, contained less label than control cells. Eight of these proteins were identified as nuclear, and one was membrane derived. Attempts have been made to identify some of the inducible proteins and to correlate results with other studies of normal hemopoietic and leukemic cells. The significance and multiple functions of GM-CSF in hemopoiesis are discussed.     

Structural and Biochemical Characterization of Isolated Trichocysts of the Ciliate Pseudomicrothorax dubius1

ABSTRACT Trichocysts of Pseudomicrothorax dubius were ejected by 15% ethanol in phosphate-buffered culture medium (CM) and purified on discontinuous sucrose gradients, in which they concentrated in the lower part of the 27% phase and in the 57% phase. These phases were washed by 15% ethanol in CM, or by CM alone, and pooled. Ejected trichocysts observed by Nomarski interference contrast microscopy and after negative-staining for electron microscopy show a shaft with periodic cross-bands and four opened-out arms, sometimes with electron-dense droplets at both ends of each arm. On SDS-PAGE, trichocysts show ?20 protein bands. The major bands are at 31 and 30 kD (G₁), 27 and 26.5 kD (G₂), 25 kD, 23 kD, and six bands at 15–20 kD (G₃). Minor bands are observed above 30 kD, among them ciliary components which contaminate the trichocyst fraction. The trichocyst banding pattern was reproducible with different ejection media; however, the 30 kD disappeared when the buffered ejection medium contained no added Ca²⁺ or contained EDTA. When the trichocyst extract is solubilized in sample buffer without 2-mercaptoethanol, the major trichocyst bands are those of G₁ and bands at 32.5–35 kD and 41 kD, which appear to be dimers of a few of the G₃ proteins. On two-dimensional gels of trichocysts, ?40 acidic protein spots are resolved with pI's of 4.6–6.6. On Western blots of two-dimensional gels, glycoproteins were revealed by Concavalin A-peroxidase labeling in three spots of G₃, in two spots at 23 kD, in all five spots of G₁, and in seven spots over 35 kD.     

Cytokines modulate preimplantation development and pregnancy

Using two different models of pregnancy failure in mice, we show in this communication that preimplantation embryonic development can be controlled, in vivo, with cytokines. In one model, described by us previously, CSF-1 is shown to block gestations in plugged females due to its induction of defective early development. Pregnancies and preimplantation development are shown here to be restored to normal by TNF alpha or GM-CSF but not by TGF beta 1. In the second model, we reveal that CBA/J females plugged by various male strains produce spontaneously an extremely high proportion of abnormal precompaction embryos. Normal morulae and blastocysts, able to further develop and implant in vitro, are shown here to be induced by TNF alpha and to a lesser extent by GM-CSF and IL-1 alpha. These results suggest strongly that cytokines are potent modulators of early development. They may provide new and interesting insights into early events of mammalian embryonic development.     

Human articular cartilage and chondrocytes produce hemopoietic colony-stimulating factors in culture in response to IL-1.

The hemopoietic CSF, granulocyte-macrophage CSF (GM-CSF) and granulocyte CSF (G-CSF), are cytokines that mediate the clonal proliferation and differentiation of progenitor cells into mature macrophages and/or granulocytes. We have employed an all-human cell culture system, specific ELISA for GM-CSF and G-CSF, and Northern analysis to investigate whether chondrocytes are a potential source of CSF in rheumatoid disease. We report that human rIL-1 stimulated in a dose-dependent manner the production of GM-CSF and G-CSF by human articular cartilage and chondrocyte monolayers in organ and cell culture, respectively. Increased levels of the CSF Ag were detected after 2 to 8 h stimulation with IL-1, and the optimum dose of IL-1 was 10 to 100 U/ml (0.06 to 0.6 nM IL-1 alpha; 0.02 to 0.2 nM IL-1 beta); neither CSF was detectable in nonstimulated cultures nor in IL-1-stimulated cultures treated with actinomycin D or cycloheximide, indicating the requirement for de novo RNA and protein synthesis. The IL-1-mediated increase in GM-CSF could also be inhibited by the corticosteroid, dexamethasone, but not by the cyclo-oxygenase inhibitor, indomethacin. Although having little effect when tested alone, TNF-alpha and lymphotoxin (TNF-beta) could synergize with IL-1 for the production of GM-CSF. Basic fibroblast growth factor, platelet-derived growth factor, and IFN-alpha and IFN-gamma each had no effect on GM-CSF levels. Results obtained by Northern analysis of chondrocyte total RNA reflected those found for the CSF Ag, namely that CSF mRNA levels were elevated in response to IL-1, but not TNF, and that there was synergy between these two cytokines. We propose that chondrocyte CSF production in response to IL-1, and the concurrent destruction of cartilage by IL-1, could provide a mechanism for the chronic nature of rheumatoid disease.     

Identification of sporozoite surface proteins and antigens of Eimeria nieschulzi (Apicomplexa)

Sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting, lectin binding, and 125I surface labeling of sporozoites were used to probe sporozoites of the rat coccidian, Eimeria nieschulzi. Analysis of silver stained gels revealed greater than 50 bands. Surface iodination revealed about 14 well labeled, and about 10 weakly labeled but potential, surface proteins. The most heavily labeled surface proteins had molecular masses of 60, 53-54, 45, 28, 23-24, 17, 15, 14, 13, and 12 kD. Following electrophoresis and Western blotting, 2 of the 12 125I labeled lectin probes bound to two bands on the blots, which collectively indicated that two bands were glycosylated. Concanavalin A (ConA) specifically recognized a band at 53 kD, which may represent a surface glycoprotein, and a lectin derived from Osage orange (MPA) bound to a single band at 82-88 kD, that may also be a surface molecule. Immunoblotting using sera collected from rats inoculated orally with oocysts, as well as sera from mice hyperimmunized with sporozoites, revealed that many surface molecules appear to be immunogenic.     

Cytokine- and calcium ionophore A23187-mediated arachidonic acid metabolism in neutrophils

Arachidonic acid (AA) metabolism is implicated as an intracellular and/or intercellular second messenger system for the transmission of cytokine-initiated signals that affect neutrophils and mediate systemic toxicity. The purpose of the present study is to ascertain if cytokines that are known to affect neutrophil function in vivo and in vitro directly stimulate neutrophil AA metabolism in vitro. The recombinant human cytokines multi-colony stimulating factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 1, tumor necrosis factor (TNF), and interleukin 6 and the calcium ionophore A23187 were incubated with purified 14C-AA radiolabeled human peripheral blood neutrophils and the effects were assayed by one- and two-dimensional thin layer lipid chromatography. None of the cytokines appeared to induce the release of cell-incorporated AA or to increase the level of radiolabeled phosphatidic acid. TNF induces severe systemic toxicity that is inhibited by cyclooxygenase inhibitors, which suggests a role for AA metabolites in the pathophysiologic effects of TNF; we have confirmed that TNF and endotoxin act synergistically to induce indomethacin-inhibitable fatal shock in rats. However, when in 3H-AA radiolabeled human neutrophils were incubated with TNF in kinetic, cold-chase, and TNF preincubation experiments, TNF was not found to increase AA metabolism, although changes in the intracellular neutral lipid content were noted. GM-CSF, which has been reported by previous investigators to directly induce the release of AA, did not release neutrophil-associated 3H-AA. In conclusion, the direct release of AA from membrane-associated phospholipids does not appear to be a major second messenger pathway for cytokine-initiated activation of neutrophils.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tumour necrosis factor alpha production by polymorphonuclear neutrophils treated with mouse anti-human CD19 monoclonal antibody

Polymorphonuclear neutrophils (PMNs) play pivotal roles as phagocytic cells in immune defence against bacteria and parasites, exerting their effects by production of reactive oxygen species, several cytokines, chemokines and by phagocytotic reaction. In our investigation of properties of activated PMNs, we discovered that one of the two kinds of mouse anti-human CD19 monoclonal antibodies (mAbs) clone SJ25-C1, weakly binds to freshly prepared PMNs. Moreover, the treatment of freshly prepared PMNs with anti-CD19 mAb (clone SJ25-C1) at 37 degrees C for 6 h induces the production and the secretion of tumour necrosis factor alpha (TNF alpha) by PMNs in vitro which was detectable in culture supernatants by bioassay using mouse cell line L929 cells. The concentration of TNF alpha secreted into the culture supernatant of PMNs cultured in the presence of anti-CD19 mAb (clone SJ25-C1) was higher than those of PMNs treated at 37 degrees C for 6 h with various PMN activators, such as anti-CD24 mAb, granulocytes-macrophage colony stimulation factor (GM-CSF) or interferon gamma (IFN gamma). In contrast, another clone of anti-CD19 mAb, HD37, did not bind to freshly prepared PMNs and failed to produce TNF alpha. To confirm that anti-CD19 mAb (clone SJ25-C1)-treated PMNs definitely produce TNF alpha, we measured the levels of intracellular expression of TNF alpha in PMNs permeabilized by saponin. These cells were treated with fluorescence-conjugated mouse anti-human TNF alpha mAb for detection of intracellular TNF alpha expression. Consequently, large amounts of intracellular TNF alpha were detected in PMNs treated with anti-CD19 mAb (clone SJ25-C1) but not in those treated with anti-CD19 mAb (clone HD37).