News and Publications

IL-5 Enhancement of Eosinophil Responsiveness to C3a

P.J. Daffern, J.E. Ember, T.E. Hugli

Heterogeneity in the responsiveness of various populations of eosinophils in peripheral blood may be associated with differences in the proportions of normodense and hypodense eosinophils. Stimulation with IL-5 in vitro causes conversion to a hypodense phenotype and a concomitant change in the activation state of the treated eosinophils. We observed marked variability in the ability of C3a, but not C5a, to induce superoxide production in eosinophils purified from peripheral blood. Therefore, we hypothesized that IL-5 stimulation alters the responsiveness of eosinophils to C3a.

Two subsets of eosinophils were classified as low or high responders on the basis of their production of superoxide after stimulation with C3a. Low responders had diminished production of superoxide (<10% of maximal cell responses to phorbol myristate acetate), whereas high responders had a marked increase in production (>20% of maximal response to phorbol myristate acetate). Incubation with IL-5 for 2--24 hours before stimulation with C3a increased superoxide production by low-responder eosinophils to more than 20% of the maximal response but had a minimal effect on high-responder eosinophils.

Binding of C3a to the C3a receptor on eosinophils was examined immediately after isolation of the cells and after overnight exposure to IL-5. Assays of freshly isolated eosinophils indicated an initial K_d of 4.45 ± 6.57 nM and 63,000--85,000 binding sites per eosinophil. The results of binding assays could not be used to differentiate between low- and high-responder groups. In the presence of IL-5, the K_d increased to 20--50 nM with 1 million to 1.2 million binding sites per cell, and binding appeared to be nonsaturable. This dramatic change in receptor affinity and apparent increase in receptor number may be due to receptor upregulation or to increased uptake or internalization of receptor-ligand complexes. The results of experiments with antibodies to the C3a receptor did not indicate increased numbers of receptors on the IL-5--treated cells. On the other hand, when intracellular transport inhibitors, monensin or brefeldin A, were used in binding studies, ligand binding was reduced by 30--50%. These results indicate that dynamic changes in C3a processing, and not an increase in receptor numbers, are responsible for the IL-5--induced changes in the binding characteristics of eosinophils.

Effect of Transforming Growth Factor-ß on the Chemokine Profiles of Airway Epithelial Cells In Vitro

M.A. Jagels, T.E. Hugli

Regulation of the production of chemotactic cytokines (chemokines) in primary cultures of human airway epithelial cells was examined. In response to physiologic stimuli, these cells are capable of producing IL-8, a CXC chemokine with chemotactic activity toward neutrophils; RANTES, a CC chemokine that is chemotactic for lymphocytes and eosinophils; and granulocyte-macrophage colony-stimulating factor (GM-CSF), which has chemokinetic activity toward both neutrophils and eosinophils.

Production and release of each of these chemokines were stimulus specific: TNF- induced release of IL-8 and GM-CSF but not of RANTES; IFN- induced release of IL-8 and RANTES but not of GM-CSF; and transforming growth factor-ß (TGF-ß) led to production of GM-CSF and RANTES but not of IL-8. Stimulus-specific induction of mRNA for each chemokine was confirmed by ribonuclease protection assays. When combined with either IFN- or TNF-, TGF-ß suppressed production and release of IL-8. However, TGF-ß plus IFN- synergistically enhanced production of RANTES, and TGF-ß plus TNF- synergistically enhanced production of GM-CSF. TGF-ß therefore biases airway chemokine production away from neutrophil-specific signals (IL-8) and toward eosinophil- and lymphocyte-dominant signals (GM-CSF and RANTES).

These results suggest that airway epithelial cells are an important component of inflammatory responses through the production of chemotactic signals. Aberrant production or responsiveness to TGF-ß may therefore contribute to eosinophilic and lymphocytic airway inflammation, such as occurs in allergic and asthmatic conditions, through the effects of TGF-ß on the production of chemokines by epithelial cells.

Characterization of 2 Isoforms of the Guinea Pig C3a Receptor

Y. Fukuoka,* J.A. Ember, T.E. Hugli

* Tohoku University, Sendai, Japan

The anaphylatoxin C3a receptor (C3aR) is a G protein--coupled receptor with 7 transmembrane domains. Until recently, only human and mouse C3aR cDNA had been cloned. We have now successfully cloned cDNA for both rat and guinea pig C3aR. The C3aR contains a large extracellular loop between domains 4 and 5 that appears to be unique to this family of receptors. Sequence comparisons between the C5a receptor from 5 species and the C3aR from 4 species indicated an additional major difference between the 2 anaphylatoxin receptors. The N-terminal extracellular domain of C3aR is significantly shorter than that of the C5a receptor. It is well documented that a ligand binding site exists on the N-terminal extracellular domain of the C5a receptor. Although the C3a-binding sites on C3aR have not been determined, apparently the large extracellular loop, not the shorter N-terminal domain of C3aR, contributes to a C3a-binding site.

When guinea pig C3aR was cloned, a second form of C3aR was isolated, a form that has a lower molecular weight than that of C3aR from other species. Sequence analysis revealed that this second form of C3aR has a 34-residue deletion from the large extracellular loop. Reverse transcriptase--polymerase chain reaction indicated that all guinea pig tissues tested expressed both forms of the receptor equally. Each form of C3aR was transfected into L cells, and C3a-binding affinity and the cellular calcium response to C3a stimulation were measured. Both forms of C3aR had similar C3a affinities and cellular calcium responses. The biological consequences of having 2 isoforms of this receptor are not known, but the data provide evidence that a considerable part of the large extracellular loop of C3aR can be deleted without affecting the binding of C3a to the receptor.

Regulation of IL-6 Synthesis in Human Peripheral Blood Mononuclear Cells by C3a and C3a_desArg

W.H. Fischer,* M.A. Jagels, T.E. Hugli

* University of Würzburg, Würzburg Germany

The anaphylatoxin C3a reportedly has immunomodulatory effects on a number of different cell types. Therefore, we examined the effects of C3a and C3a_desArg on gene expression and protein secretion of IL-6 in human peripheral blood mononuclear cells, either alone or in combination with lipopolysaccharide or IL-1ß. C3a or C3a_desArg alone had no effect on either expression or secretion of IL-6. However, when the cells were stimulated with lipopolysaccharide or IL-1ß, both C3a and C3a_desArg enhanced IL-6 release in a dose-dependent manner.

Because C3a can induce production of prostaglandin E₂ by monocytes, and prostaglandin E₂ influences production of cytokines, the potential role of this prostaglandin in C3a-mediated enhancement of IL-6 production induced by lipopolysaccharide or IL-1ß was examined. Indomethacin blocked release of prostaglandin E₂ but had no influence on the observed effects of C3a. These findings suggest that the effects of C3a on IL-6 production by monocytes are independent of the formation of prostaglandin E₂. Northern blot analysis showed that both C3a and C3a_desArg enhanced lipopolysaccharide-induced mRNA levels for IL-6. Pretreatment of peripheral blood mononuclear cells with pertussis toxin blocked the observed functions of C3a and C3a_desArg, indicating that the actions of these 2 molecules are mediated by a G protein--coupled (i.e., receptor) pathway.

Furthermore, the effect of C3a and C3a_desArg on induction of NF-B and binding of activator protein 1 were evaluated. Both molecules enhanced lipopolysaccharide induction of NF-B and binding of activator protein 1. These results indicate the capacity of intact C3a and the physiologic (i.e., circulating) desArg form to exert immunomodulatory effects on peripheral blood mononuclear cells in vitro.

Processing of C5a by Human Neutrophils

G. Hetland,* P.H. Pfeifer,** T.E. Hugli

* National Institute of Public Health, Oslo, Norway
** BMA Biomedicals, Augst, Switzerland

Uptake of human C5a by neutrophils was monitored in vitro by using both radiolabeled and unlabeled C5a. The ligand was internalized by the cells in a dose-dependent manner, and maximal binding and uptake occurred after 5 minutes of incubation. Neutrophils were incubated with labeled C5a, and the cytosol and supernatant were analyzed. C5a degradation products were present primarily in the supernatant; most of the protein remained intact in the cytosol even after 60 minutes of incubation. Cytosol from neutrophils incubated for 20 minutes with unlabeled C5a contained antigenically intact C5a and retained the ability to induce a neutrophil response (a change in shape). The functional activity of C5a recovered from the cytosol was inhibited by antibodies to either C5a or the C5a receptor (CD88).

These data support our hypothesis that although it is internalized with its receptor, C3a remains antigenically intact and functionally active inside the cell and is primarily degraded extracellularly. The dogma that bioactive factors, such as C5a and many cytokines, that interact with G protein--coupled receptors are processed intracellularly may need to be reexamined.

PUBLICATIONS

Ames, R.S., Tornetta, M.A., Foley, J.J., Hugli, T.E., Sarau, H.M. Evidence that the receptor for C4a is distinct from the C3a receptor. Immunopharmacology 38(Special Issue):87, 1997.

Carney, D.F., Jagels, M.A., Hugli, T.E., Sands, H., Rubin, H. Effect of serine proteinase inhibitors on neutrophil function: Alpha-1-proteinase inhibitor, antichymotrypsin, and a recombinant hybrid mutant of antichymotrypsin (LEX32) modulate neutrophil adhesion interactions. J. Leukoc. Biol. 63:75, 1998.

Ember, J.A., Hugli, T.E. Complement factors and their receptors. Immunopharmacology 38(Special Issue):3, 1997.

Ember, J.A., Jagels, M.A., Hugli, T.E. Characterization of complement anaphylatoxins and their biological responses. In: The Human Complement System in Health and Disease. Volanakis, J.E., Frank, M.M. (Eds.). Marcel Dekker, New York, 1998, p. 241.

Fischer, W.H., Hugli, T.E. Regulation of B cell functions by C3a and C3a_desArg: Suppression of TNF-, IL-6, and the polyclonal immune response. J. Immunol. 159:4279, 1997.

Fukuoka, Y., Ember, J.A., Hugli, T.E. Cloning and characterization of rat C3a receptor: Differential expression of rat C3a and C5a receptors by LPS stimulation. Biochem. Biophys. Res. Commun. 242:663, 1998.

Fukuoka, Y., Ember, J.A., Yasui, A., Hugli, T.E. Cloning and characterization of the guinea pig C5a anaphylatoxin receptor: Interspecies diversity among the C5a receptors. Int. Immunol. 10:275, 1998.

Gray, G.D., Hasslen, S.H., Ember, J.A., Carney, D.F., Herron, M.J., Erlandson, S.L., Nelson, R.D. Receptors for the chemoattractants C5a and IL-8 are clustered on the surface of human neutrophils. J. Histochem. Cytochem. 45:1461, 1997.

Hetland, G., Pfeifer, P.H., Hugli, T.E. Processing of C5a by human polymorphonuclear leukocytes. J. Leukoc. Biol. 63:462, 1998.

Hsu, M.H., Ember, J.A., Wang, M., Prossnitz, E.R., Hugli, T.E., Ye, R.D. Cloning and functional characterization of the mouse C3a anaphylatoxin receptor gene. Immunogenetics 47:64, 1997.

Mainwaring, R.D., Lamberti, J.J., Hugli, T.E. Complement and cytokine activation following modified Fontan procedure. Ann. Thorac. Surg., in press.

Nakashima, K., Sakurada, T., Imayama, S., Masukawa, S., Ember, J.A., Hugli, T.E, Abe, M. A case of episodic angioedema associated with blood eosinophilia: Upregulated C5a receptor expression on eosinophils. Allergy 53:320, 1998.

Pfeifer, P.H., Hugli, T.E., Davie, E.W., Fujikawa, K. Complement activation in EDTA blood/plasma samples may be caused by coagulation proteases. In: Techniques in Protein Chemistry VIII. Crabb, J.W., Marshak, D.R. (Eds.). Academic Press, San Diego, 1997, p. 363.

Schmid, E., Piccolo, M.-T.S., Friedl, H.P., Warner, R.L., Mulligan, M.S., Hugli, T.E, Till, G.O., Ward, P.A. Requirements for C5a in dermal and lung vascular injury following thermal trauma to rat skin. Shock 8:119, 1997.