Cocaine increases natural killer cell activity.

The administration of epinephrine to humans increases natural killer (NK) cell activity and numbers. If endogenous catecholamines regulate NK cells, then their activity should be increased by cocaine, an agent that potentiates endogenous catecholamines. We investigated the in vivo effect of cocaine on NK cell activity and on the distribution of lymphocyte subsets, including NK cells. Intravenous cocaine (0.6 mg/kg) produced a three- to fourfold increase in NK cell activity in peripheral blood. The increase was accompanied by a marked and selective increase in circulating NK cells, as identified by the Fc receptor (Leu-11). Normal saline and benzoylecgonine, a major metabolite of cocaine, had little effect on NK cell activity or on levels of Leu-11+ cells. Other lymphocyte subpopulations were not increased by cocaine. The time course of the alterations in NK cell numbers and activity paralleled plasma levels of cocaine. In vitro cocaine did not increase NK cell activity. Our results indicate that cocaine selectively alters the activity and distribution of the NK lymphocyte subset. Because cocaine increases the activity of endogenous catecholamines, these findings suggest that human NK cells are selectively regulated by the sympathetic nervous system.

Expression of the chemokine receptor CXCR2 in normal and neoplastic neuroendocrine cells.

BACKGROUND: Chemokines effect their proinflammatory and growth regulatory roles through interaction with serpentine receptors. One such receptor, CXCR2, binds multiple CXC chemokines, including interleukin 8, GRO-alpha, GRO-beta, GRO-gamma, and NAP-2. We have previously identified CXCR2 expression on myeloid cells, notably mature granulocytes, and projection neurons. OBJECTIVE: To determine the expression of CXCR2 by cells of the neuroendocrine system. DESIGN: Archival specimens from normal neuroendocrine tissues and their malignant counterparts were analyzed by immunohistochemistry with monoclonal antibodies specific for CXCR1 and CXCR2. RESULTS: Immunohistochemical analysis revealed high-level expression of CXCR2 by cells in the pituitary, adrenal medulla, pancreatic islets, thyroid C cells, scattered Kulchitsky cells in the bronchi, and counterpart neuroendocrine cells in the stomach, small bowel, colon, and appendix. Neuroendocrine neoplasms that demonstrated high-level CXCR2 expression included (1) primary carcinoids localized to the stomach, small bowel, colon, appendix, fallopian tube, ovary, and lung; (2) atypical carcinoids of the lung; (3) metastatic carcinoids; (4) pituitary adenomas; (5) pheochromocytomas; and (6) medullary carcinomas of the thyroid. Small cell lung carcinomas, large cell neuroendocrine carcinomas of the lung, small cell carcinoma of the cervix, Merkel cell carcinomas, neuroblastomas, and malignant melanomas lacked evidence of CXCR2 expression. CONCLUSIONS: The expression of CXCR2 by normal neuroendocrine cells and neoplastic counterparts that have retained phenotypic features of this differentiation program suggests that chemokines may play an important role in functions that are characteristic of this cell type. In addition, this raises the possibility that chemokines may modulate secretion of biologically active products of these cells and their neoplastic counterparts.

Characterization of novel neutralizing monoclonal antibodies specific to human neurturin.

Neurturin (NTN) a structural and functional relative of glial cell line-derived neurotrophic factor, was originally identified based on its ability to support the survival of sympathetic neurons in culture. Similar to glial cell line-derived neurotrophic factor (GDNF), Neurturin has been shown to bind to a high affinity glycosylphosphatidylinositol (GPI)-linked receptor (GFRalpha2) and induce phosphorylation of the tyrosine kinase receptor Ret, resulting in the activation of the mitogen activated protein kinase (MAPK) signalling pathway. A panel of six novel murine monoclonal antibodies (MAbs) specific to human Neurturin has been developed and characterized. Four of the MAbs tested inhibit, to varying degrees, binding of NTN to the GPI-linked GFRalpha2 receptor. Three MAbs cross-react with the murine homolog. These antibodies have been shown to be useful reagents for Western blotting, immunohistochemistry, and also for the development of a sensitive, quantitative enzyme-linked immunosorbent assay (ELISA) for human NTN. Novel, specific MAbs with varying epitope specificities and blocking activity will be valuable tools for both the in vitro and in vivo characterization of NTN and its relationship to the GFRalpha2 and Ret receptors.

Regulation of the expression of IL-8 receptor A/B by IL-8: possible functions of each receptor.

We investigated the regulatory mechanism of the expression of IL-8R, IL-8R type A (IL-8RA), and IL-8R type B (IL-8RB) on human neutrophils by IL-8. The expression of IL-8RA/B was analyzed by flow cytometry using mAb specific for each receptor. IL-8 down-modulated > 90% of IL-8RA and IL-8RB expression within 5 min. A related C-X-C chemokine, melamona growth stimulatory activity, down-modulated IL-8RB but not IL-8RA. It required 7 to 13 times more IL-8 to down-modulate IL-8RA than IL-8RB, as determined by the half-maximal effective concentration of IL-8. Scatchard analysis showed that the affinity of IL-8RA for IL-8 was lower than that of IL-8RB. The possible functions of each IL-8R were explored by comparing 1) the expression levels of IL-8RA/B on migrated neutrophils during in vitro chemotaxis assay and 2) the recovery rate of IL-8RA/B expression after down-modulation by IL-8. Results obtained from the in vitro chemotaxis show that the expression level of IL-8RB, but not IL-8RA, on neutrophils that migrated into the chamber containing low concentrations (< 1 nM) of IL-8 was significantly reduced compared with the control level. This suggests that IL-8RB may play as active role in the initiation of neutrophil migration distant from the site of inflammation, where the concentration of IL-8 is at the picomolar level. After down-modulation by 119 nM IL-8, the expression of IL-8RA fully recovered within 1.5 h, while the recovery rate of IL-8RB expression was slow and never reached more than 40% of the control level during a 3-h culture period. The rapid reexpression of IL-8RA suggests that the low affinity IL-8RA may play a more active role in mediating IL-8 signal at the site of inflammation, where the concentration of IL-8 is high.

Blocking monoclonal antibodies to alpha V beta 3 integrin: a unique epitope of alpha V beta 3 integrin is present on human osteoclasts.

Integrins are a family of cell surface glycoproteins that promote cell adhesion. The integrin alpha V beta 3, vitronectin receptor, is a major integrin expressed by osteoclasts. To further investigate the role of alpha V beta 3 in cell adhesion, we generated and characterized monoclonal antibodies to alpha V beta 3 by immunizing BALB/c mice with purified alpha V beta 3 protein. Three monoclonal antibodies (mAbs), 9D4.9.1, 9G2.1.3, and 10C4.1.3, from a total of more than 1100 positive cultures which bound alpha V beta 3, were characterized extensively: mAbs 9G2.1.3 and 10C4.1.3 recognize the alpha V beta 3 complex whereas mAb 9D4.9.1 reacts with the beta 3-chain shared between the alpha V beta 3 complex and gpIIbIIIa. Further epitope mapping using flow microfluorometry analysis and histochemical staining of various tissues showed that 9D4.9.1 and 10C4.1.3 recognized distinct epitopes. Ligand-binding studies using cell-bound and purified alpha V beta 3 demonstrated that all three mAbs blocked fibrinogen binding. Vitronectin binding was blocked by mAb 9D4.9.1 and, less effectively, by mAb 10C4.1.3; mAb 9G2.1.3 was without effect. All three mAbs recognized osteoclasts from human tissues; mAb 9G2.1.3 also stained osteoclasts from a wide range of nonhuman species. Monoclonal antibodies 9D4.9.1 and 9G2.1.3 bound to a panel of cultured cell lines and various tissues. In contrast, mAb 10C4.1.3 bound only weakly or not at all to tissues expressing alpha V beta 3 with the exception of osteoclasts. Thus, mAb 10C4.1.3 showed a very narrow tissue specificity being restricted to high-level expression on human osteoclasts.

A negative regulatory region in the intracellular domain of the human interferon-alpha receptor.

Interferon-alpha (IFN-alpha)-mediated intracellular signaling is initiated by ligand-induced receptor dimerization, tyrosine phosphorylation of the Tyk2 and Jak1 tyrosine kinases, and subsequent phosphorylation of the Stat1 and Stat2 proteins. The IFN-alpha receptor consists of at least two distinct subunits. One subunit, IFNAR1, has low affinity binding for interferon yet is required for signal transduction. We introduced mutations in the cytoplasmic domain of human IFNAR1 in order to identify residues involved in the mediation of biological responses. We took advantage of the species specificity of the interferon receptors by analyzing human IFN-alpha-induced major histocompatibility complex class I antigen expression in mouse L929 cells stably transfected with mutant human receptors. The membrane proximal 60-amino acids were insufficient to signal a biological response even though within these residues Tyk2 and Stat2 binding sites have been identified. IFN-alpha-induced receptor tyrosine phosphorylation was not critical for signaling because mutation of Tyr residues to Phe did not prevent the biological response to IFN-alpha. The deletion of a 16-amino acid region highly homologous between species created a receptor which signals an enhanced response. Tyrosine dephosphorylation is a component of this enhanced response as mutation of the Tyr residues within this region to Phe resulted in a receptor with increased sensitivity to IFN. The known signaling molecules that interact with IFNAR1 are positive regulators of IFN-alpha function. The presence of this domain in the COOH-terminal region suggests that the receptor may interact with signaling molecules that negatively regulate interferon responses.

Interleukin-8 (IL-8), melanoma growth-stimulatory activity, and neutrophil-activating peptide selectively mediate priming of the neutrophil NADPH oxidase through the type A or type B IL-8 receptor.

The capacity of neutrophils to generate superoxide (O-2) can be enhanced by prior exposure to "priming" agents such as interleukin-8 (IL-8), melanoma growth-stimulatory activity (MGSA), and neutrophil-activating peptide (ENA-78). The biological effects of these chemokines are mediated by at least two distinct receptors: type A (IL-8-RA) and type B (IL-8-RB). Using neutralizing monoclonal antibodies to IL-8-RA and IL-8-RB, we have investigated the contribution each receptor makes to the priming response. Preincubation with IL-8, MGSA, or ENA-78 enhanced the ability of neutrophils to generate O-2 following stimulation with the bacterial peptide formyl-Met-Leu-Phe. The priming effect of IL-8 was eliminated by an anti-IL-8 monoclonal antibody (mAb) that is known to bind IL-8 with high affinity and prevent receptor occupancy. Incubation of neutrophils with a neutralizing mAb specific for IL-8-RA blocked IL-8-induced priming but had no effect on priming by MGSA or ENA-78. In contrast, treatment with a neutralizing mAb specific for IL-8-RB failed to inhibit the priming effect of IL-8 but blocked both MGSA and ENA-78-induced priming. These observations indicate that the priming effect of IL-8 on the neutrophil respiratory burst is predominantly mediated via IL-8-RA, whereas priming by MGSA and ENA-78 is mediated by IL-8-RB.

Neutralizing monoclonal antibodies to human IL-8 receptor A map to the NH2-terminal region of the receptor.

mAbs against human IL-8 receptor A (IL-8R-A) were generated by immunizing mice with either: 1) peptides corresponding to various extracellular domains of IL-8R-A or 2) transfected 293 cells expressing IL-8R-A (293-71 cells). Among the seven peptides used for immunization, only the peptide corresponding to residues 2-19 of IL-8R-A produced Abs capable of recognizing native IL-8R-A on 293-71 cells. We screened for hybridomas secreting mAbs capable of binding strongly to peptide 2-19 in an ELISA and capable of recognizing native IL-8R-A in flow cytometry experiments with 293-71 cells. Two clones secreting mAbs capable of recognizing native IL-8R-A were selected for further characterization. None of these mAbs were able to block the binding of 125I-IL-8 to 293-71 cells. We also screened hybridomas derived from mice immunized with the intact receptor expressed on transfected 293-71 cells and identified several clones secreting mAbs capable of recognizing native IL-8R-A in flow cytometry experiments. Two of these mAbs were capable of blocking the binding of 125I-IL-8 to 293-71 cells. The epitopes, recognized by these blocking mAbs and by the other nonblocking mAbs derived from the peptide immunization, mapped to the NH2-terminal region of IL-8R-A using an ELISA against synthetic peptides: the two blocking mAbs mapped to residues 2-14 of IL-8R-A, whereas the nonblocking mAbs mapped to residues 2-11. Furthermore, flow cytometry analysis of IL-8 receptor mutants showed that Asp6 plays an important role in the binding of the blocking mAbs but not in the binding of the nonblocking mAbs. Conversely, a mutation of Asp11 to Lys disrupts the binding of one of the nonblocking mAbs (4C8) but has no effect on recognition by others. Analysis of the affinity of these mAbs for IL-8R-A demonstrated that blocking mAbs have affinities at least sevenfold higher than the nonblocking mAbs.

Partial functional mapping of the human interleukin-8 type A receptor. Identification of a major ligand binding domain.

We have previously demonstrated that a basic amino acid residue of interleukin (IL)-8, namely Arg-6, is critical for the binding of IL-8 to its receptor. We reasoned that this residue is likely to be poised to directly interact with a counterpart acidic residue on the receptor. To identify this key residue, we systematically mutated to Ala all acidic residues present on the ligand accessible surface of IL-8 receptor type A. Using this strategy, we demonstrate that two residues which are present in extracellular loop 3 of the receptor, namely Glu-275 and Arg-280, are critical for ligand binding. In addition, we show that although Asp-11 is critical for ligand binding, a conservative mutation of Asp-11 to Glu or a substitution of Asp-11 with Lys (the residue found at position 11 in IL-8 receptor type B) does not affect the Kd of the receptor/ligand interaction. These data suggest that Lys-11 recruits a new and favorable interaction with IL-8 (analogous to that of IL-8 receptor type B with IL-8) or that the cavity created by mutating Asp-11 to Ala is particularly deleterious. Finally, we discuss fluorescence-activated cell sorter staining data which support the hypothesis that the N-terminal region and the extracellular loop 3 of the receptor may lie in close proximity of one another and constitute a major binding domain for IL-8.

Ontogeny of the secretory immune system: maturation of a functional polymeric immunoglobulin receptor regulated by gene expression.

In the rat, secretion of polymeric IgA from serum into bile is dependent upon the presence of a functional polymeric immunoglobulin receptor (pIgR) that acts as a hepatocyte plasma membrane receptor for ligand binding and as a transcellular transport molecule. The objective of this study was to document the developmental maturation and regulation of functionally intact rat liver pIgR. An adult pattern of IgA secretion was not detected until after day 23 postpartum (dPP), by using intravenously injected 125I-labeled dimeric IgA. Radioactive dimeric IgA was not detectable in hepatocyte transport vesicles until 21 dPP by electron microscopy autoradiographic analysis. By using a rabbit polyclonal antibody against the rat secretory component domain of the pIgR, Western blot analysis demonstrated that the plasma-membrane-bound pIgR levels in hepatocytes from rats aged 19-22 dPP increased 10-fold during this period. To determine whether or not this increase in membrane-bound pIgR reflected increased pIgR gene expression, we probed Northern blots of total cellular RNA extracted from neonatal rat liver with pIgR cDNA [GORF-1; Banting, G., Brake, B., Braghetta, P., Luzio, J.P. & Stanley, K. K. (1989) FEBS Lett. 254, 177-183]. The pIgR RNA levels between 19 and 22 dPP rose more than 20-fold and paralleled the increased membrane-bound pIgR protein during this same interval. These data demonstrate a developmentally regulated process that controls the ontogeny of biliary dimeric IgA secretion at the termination of the third week postpartum. The process appears to depend on the up-regulation of pIgR gene expression.

Monoclonal antibodies detect different distribution patterns of IL-8 receptor A and IL-8 receptor B on human peripheral blood leukocytes.

mAbs previously reported to be specific for IL-8R type A (IL-8R-A) and mAbs specific for IL-8R type B (IL-8R-B), which are described in this paper, were used to investigate the expression of each receptor on various types of cells. We generated mAbs specific for IL-8R-B, 4D1, and 10H2 by immunizing mice with 293 cells that expressed IL-8R-B and by selecting hybridoma cell lines that secreted mAbs that bind to human neutrophils. Flow cytometry showed that mAbs 4D1 and 10H2 were specific for IL-8R-B, as determined by their exclusive binding to 293-27 cells that expressed IL-8R-B, but not to 293-71 cells that expressed IL-8R-A. Epitopes recognized by these IL-8R-B-specific mAbs were shown to be within the N-terminal residues 1-18 of the IL-8R-B on the basis of their binding to various N-terminal peptides, as measured by ELISA. These IL-8R-B-specific mAbs were able to inhibit up to 90 and 50% of the 125I-labeled IL-8 binding to 293-27 cells and human neutrophils, respectively. The combination of mAb 9H1 (anti-IL-8R-A) and mAb 10H2 (anti-IL-8R-B) inhibited approximately 70% of 125I-labeled IL-8 binding to human neutrophils. Flow cytometry showed a wide range of donor variation in the expression levels of IL-8R-A and IL-8R-B on various human peripheral blood leukocytes. All neutrophils, all monocytes, and 5 to 25% of total lymphocytes (CD8+ T cells and CD56+ NK cells) expressed IL-8R. Neutrophils expressed the highest level of both IL-8R-A and IL-8R-B, at an approximately equal ratio, whereas monocytes and IL-8R+ lymphocytes expressed higher levels of IL-8R-B than IL-8R-A. Double-color flow cytometric analysis showed that 7 to 42% of CD8+ T cells and 39 to 76% of CD56+ NK cells, but no CD 20+ B cells or CD4+ T cells, expressed IL-8R.

Characterization and humanization of a monoclonal antibody that neutralizes human leukocyte interferon: a candidate therapeutic for IDDM and SLE.

We have developed a panel of murine monoclonal antibodies that recognize human interferon alpha. One of these mononclonal antibodies binds and neutralizes, with high affinity, all of seven tested recombinant human interferon alphas. This mononclonal antibody also neutralizes the interferon activity present in two independent pools of interferon alphas prepared following stimulation of human peripheral blood leukocytes. The complementary determining regions from this murine mononclonal antibody were transferred to a human IgG2 heavy chain and to a human kappa1 light chain. In addition, six (heavy chain) and two (light chain) amino acids were transferred from the framework regions. This generated a humanized mononclonal antibody that retained the specificity of the mouse parent. The humanized anti-interferon alpha antibody is a candidate therapeutic for those diseases, such as insulin-dependent diabetes, systemic lupus erythematosis, psoriasis and Crohn's disease, which are all characterized by pathological expression of interferon alpha.

Determination of residues involved in ligand binding and signal transmission in the human IFN-alpha receptor 2.

The human IFN-alpha receptor (hIFNAR) is a complex composed of at least two chains, hIFNAR1 and hIFNAR2. We have performed a structure-function analysis of hIFNAR2 extracellular domain regions using anti-hIFNAR2 mAbs (1D3, 1F3, and 3B7) and several type I human IFNs. These mAbs block receptor activation, as determined by IFN-stimulated gene factor 3 formation, and block the antiviral cytopathic effects induced by type I IFNs. We generated alanine substitution mutants of hIFNAR2-IgG and determined that regions of hIFNAR2 are important for the binding of these blocking mAbs and hIFN-alpha2/alpha1. We further demonstrated that residues E78, W101, I104, and D105 are crucial for the binding of hIFN-alpha2/alpha1 and form a defined protrusion when these residues are mapped upon a structural model of hIFNAR2. To confirm that residues important for ligand binding are indeed important for IFN signal transduction, we determined the ability of mouse L929 cells expressing hIFNAR2 extracellular domain mutants to mediate hIFN signal. hIFN-alpha8, previously shown to signal a response in L929 cells expressing hIFNAR1, was unable to signal in L929 cells expressing hIFNAR2. Transfected cells expressing hIFNAR2 containing mutations at residues E78, W101, I104, or D105 were unresponsive to hIFN-alpha2, but remained responsive to hIFN-beta. In summary, we have identified specific residues of hIFNAR2 important for the binding to hIFN-alpha2/1 and demonstrate that specific regions of the IFNAR interact with the subspecies of type I IFN in different manners.

A neutralizing monoclonal antibody specific for the dimer interface region of IL-8.

We have generated two mAbs, 6G4.2.5 and A5.12.14, that are similarly capable of neutralizing the biologic activity of wild-type IL-8. To characterize these antibodies further, their reactivity against a series of engineered IL-8 monomer and dimer variants was examined using a neutrophil degranulation assay. While 6G4.2.5 was found to block effectively the biologic activity of all variants regardless of their dimerization status, the results for A5.12.14 differed dramatically. A5.12.14 fully inhibited the agonist activity of one of the monomer variants, partially blocked the activity of another, and had no effect on the activity of two other variants. These results suggested that the binding epitope of A5.12.14 was being affected by the particular amino acid substitutions introduced into the dimer interface region of the variants to disfavor dimerization. If A5.12.14 indeed binds to the dimer interface region of IL-8, it could be predicted that this mAb would be unable to inhibit the activity of dimeric IL-8. This was confirmed in studies which showed that A5.12.14 had no demonstrable effect on the activity of a constitutively dimeric IL-8 variant. These studies represent the first example of a mAb specific for the dimerization status of IL-8.

Isotype-dependent inhibition of tumor growth in vivo by monoclonal antibodies to death receptor 4.

To explore an approach for death receptor targeting in cancer, we developed murine mAbs to human death receptor 4 (DR4). The mAb 4H6 (IgG1) competed with Apo2L/TNF-related apoptosis-inducing ligand (DR4's ligand) for binding to DR4, whereas mAb 4G7 (IgG2a) did not. In vitro, both mAbs showed minimal intrinsic apoptosis-inducing activity, but each triggered potent apoptosis upon cross-linking. In a colon tumor nude mouse model in vivo, mAb 4H6 treatment without addition of exogenous linkers induced apoptosis in tumor cells and caused complete tumor regression, whereas mAb 4G7 partially inhibited tumor growth. An IgG2a isotype switch variant of mAb 4H6 was much less effective in vivo than the parent IgG1-4H6, despite similar binding affinities to DR4. The same conclusion was obtained by comparing other IgG1 and IgG2 mAbs to DR4 for their anti-tumor activities in vivo. Thus, the isotype of anti-DR4 mAb may be more important than DR4 binding affinity for tumor elimination in vivo. Anti-DR4 mAbs of the IgG1 isotype may provide a useful tool for investigating the therapeutic potential of death receptor targeting in cancer.

Apo2L/TRAIL-dependent recruitment of endogenous FADD and caspase-8 to death receptors 4 and 5.

Fas (APO-1/CD95) and tumor necrosis factor receptor 1 (TNFR1) trigger apoptosis by recruiting the apoptosis initiator caspase-8 through the adaptor FADD. Fas binds FADD directly, whereas TNFR1 binds FADD indirectly, through TRADD. TRADD alternatively recruits the NF-kappaB-inducing adaptor RIP. The TNF homolog Apo2L/TRAIL triggers apoptosis through two distinct death receptors, DR4 and DR5; however, receptor over-expression studies have yielded conflicting results on the ligand's signaling mechanism. Apo2L/TRAIL induced homomeric and heteromeric complexes of DR4 and DR5 and stimulated recruitment of FADD and caspase-8 and caspase-8 activation in nontransfected cells. TRADD and RIP, which bound TNFR1, did not bind DR4 and DR5. Thus, Apo2L/TRAIL and FasL initiate apoptosis through similar mechanisms, and FADD may be a universal adaptor for death receptors.

Interleukin-8 is a major component of pleural liquid chemotactic activity in a rabbit model of endotoxin pleurisy.

Gram-negative endotoxin induces production of the potent chemotactic factor interleukin-8 (IL-8) in vitro; however, the importance of IL-8 in endotoxin-induced inflammation in vivo is unknown. We asked whether IL-8 is an important contributor to chemotactic activity in acute inflammatory liquids formed in response to endotoxin, and, if present, what concentrations of IL-8 antigen are generated. For these studies, we cloned and expressed rabbit recombinant IL-8 (rrIL-8), developed specific anti-rabbit IL-8 monoclonal antibodies (mAb), and then used these reagents to develop assays to detect rabbit IL-8 bioactivity and measure rabbit IL-8 antigen. Escherichia coli endotoxin (20 ng/ml, n = 4, or 2,000 ng/ml, n = 4) was instilled into the pleural space of eight rabbits for 6 h. Rabbit IL-8 bioactivity in the endotoxin pleurisy samples was assayed by measuring the migration of rabbit neutrophils toward the pleural liquid under two different conditions: 1) after addition of an anti-IL-8 neutralizing mAb and 2) after desensitization of the neutrophils to rrIL-8. Addition of the anti-IL-8 mAb decreased neutrophil migration toward the pleural liquid by 65 +/- 13 and 75 +/- 22% (mean +/- SE, after 20 and 2,000 ng/ml endotoxin, respectively; P < 0.01 compared with a control mAb). Desensitization of neutrophils to rrIL-8 decreased their migration toward the pleural liquid by 72 +/- 5% (P = 0.03, compared with exposure of neutrophils to buffer alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Neutralization of IL-8 inhibits neutrophil influx in a rabbit model of endotoxin-induced pleurisy.

Although the potent neutrophil chemotaxin, IL-8, is a known product of endotoxin-stimulated cells in vitro, the contribution of IL-8 to neutrophil recruitment in Gram-negative endotoxin inflammation in vivo is unknown. To determine whether neutralization of IL-8 would decrease endotoxin-induced neutrophil influx, we generated neutralizing mAbs to rabbit rIL-8 for use in our rabbit model of endotoxin-induced pleurisy. One mAb, ARIL8.2, specifically inhibited both rabbit rIL-8-induced chemotactic activity and activation of the rabbit IL-8 receptor transfected in 293 cells. Anesthetized rabbits with in-dwelling pleural catheters received either neutralizing mAb (ARIL8.2; 1 mg/kg) or irrelevant isotype-matched mAb (anti-HIV gp120) i.v. 1 h before as well as intrapleurally (20 micrograms/ml) at the time of intrapleural instillation of Escherichia coli endotoxin (200 ng bilaterally). ARIL8.2 blocked 77% of endotoxin-induced neutrophil influx (21 +/- 2 (SE) x 10(6) (ARIL8.2) vs 91 +/- 15 x 10(6) (anti-gp120) (p < 0.0001)). By Western analysis, a band corresponding to rabbit IL-8 was detected in the pleural liquid of rabbits in both groups. By ELISA, however, the concentration of free, unbound IL-8 in the pleural liquid was significantly less in the ARIL8.2 group than in the anti-gp120 group for at least 4 h, confirming that ARIL8.2 bound the IL-8 generated in vivo during that time. We conclude that neutralization of IL-8 profoundly inhibits neutrophil recruitment in endotoxin-induced pleurisy indicating that IL-8 is a major chemotactic factor in this model of acute inflammation.

Involvement of APO2 ligand/TRAIL in activation-induced death of Jurkat and human peripheral blood T cells.

The interaction of Fas with Fas ligand (FasL) mediates activation-induced cell death (AICD) of T hybridomas and of mature T lymphocytes. The TNF/TNF receptor system also plays a significant role in AICD of mature T cells and in the maintenance of peripheral tolerance. We previously demonstrated that in human Jurkat leukemia cells, AICD is triggered mainly by the rapid release of preformed FasL upon TCR stimulation. In the present work, we show that the cytotoxic cytokine APO2 ligand (APO2L; also known as TRAIL) is constitutively expressed as an intracytoplasmic protein in Jurkat T cells and derived sublines. APO2L is also detected in fresh human peripheral blood mononuclear cells (PBMC) from a significant number of donors, and the amount of both FasL and APO2L substantially increases upon blast generation. A neutralizing anti-APO2L monoclonal antibody (mAb) partially suppresses the cytotoxicity induced by supernatants of phytohemagglutinin (PHA)-prestimulated Jurkat or human PBMC on non-activated Jurkat cells, indicating that APO2L is released by these cells and contributes to AICD. A combination of neutralizing anti-APO2L and anti-Fas mAb blocks around 60 % of the toxicity associated with supernatants from PHA-activated human PBMC. These results show that FasL and APO2L account for the majority of cytotoxic activity released during AICD, and suggest that additional uncharacterized factors may also contribute to this process.