Involvement of cyclic AMP and nitric oxide in immunoglobulin E-dependent activation of Fc epsilon RII/CD23+ normal human keratinocytes.

Epidermal keratinocytes (EK) are exposed to multiple inflammatory stimuli and paracrine factors secreted by various dermal cells (lymphocytes, mast cells, macrophages, fibroblasts) during wounding, cutaneous allergy, and infections. We have previously demonstrated that after stimulation with interleukin 4 or interferon-gamma, human EK express the low-affinity receptor for IgE (Fc epsilon RII/CD23) on their surface. In the present study, we showed that the ligation of CD23 by IgE/anti-IgE immune complexes or specific monoclonal antibody induces a dose-dependent release of interleukin 6 and tumor necrosis factor-alpha from EK. CD23-ligation activates the nitric oxide-dependent pathway, as demonstrated by the high levels of nitrites released in cell supernatants, and the accumulation of intracellular cyclic nucleotides in EK. These second messengers are required for IgE-dependent stimulation of cytokine production by these cells, inasmuch as this is completely abolished by the use of cAMP or nitric oxide synthase antagonists. Human epithelial keratinocytes may thus participate in IgE-mediated immune responses, through their ability to express functional CD23 antigen.

The effect of intravenous administration of a chimeric anti-IgE antibody on serum IgE levels in atopic subjects: efficacy, safety, and pharmacokinetics.

CGP 51901 is a non-anaphylactogenic mouse/human chimeric anti-human IgE antibody that binds to free IgE and surface IgE of IgE-expressing B cells but not to IgE bound to high affinity IgE receptors (Fc epsilonR1) on mast cells and basophils or low affinity IgE receptors (Fc epsilonR2) on other cells. A phase 1 double-blind, placebo-controlled, single dose study with doses of 3, 10, 30, and 100 mg of CGP 51901 was conducted in 33 pollen-sensitive subjects who had raised levels of serum IgE and received either intravenous CGP 51901 or placebo. The administration of CGP 51901 was well tolerated and resulted in a decrease of serum free IgE levels in a dose-dependent manner, with suppression after 100 mg of CGP 51901 reaching > 96%. Time of recovery to 50% of baseline IgE correlated with the dose of administered antibody and ranged from a mean of 1.3 d for the 3 mg to 39 d for the 100 mg dose. Total IgE, comprised of free and complexed IgE, increased as stored and newly synthesized IgE bound to CGP 51901. Complexed IgE was eliminated at a rate comparable with the terminal half-life of free CGP 51901 (11-13 d at all doses). Only one subject showed a weak antibody response against CGP 51901. We conclude that the use of anti-human IgE antibody is safe and effective in reducing serum IgE levels in atopic individuals and provides a potential therapeutic approach to the treatment of atopic diseases.

Structural model of the collagen-like region of C1q comprising the kink region and the fibre-like packing of the six triple helices.

A detailed three-dimensional model of the collagenous part of C1q was derived by model building and computer-aided energy refinement calculations. The proposed structure is based on the collagen-like (-Gly-Xaa-Yaa-) repeating sequence of 78 to 81 residues in the N-terminal regions of the constituent A, B and C chains, on the mode of disulphide linkage between the 18 chains of C1q, and on its electron microscopically derived gross structure. It is demonstrated that the interruptions of the repeating sequence about half-way along the length of the collagenous regions (Gly36-Ile37-Arg38-Thr39 in the A chain and Ala36-Ile37-Hy138 in the C chain) do not lead to a disruption of the triple helical conformation but rather to a bend of about 60 degrees in an otherwise continuous triple helix. These features are consistent with a flexibility comparable with that of regular triple helices and with the observed low proteolytic susceptibility of the kink region. The azimuthal orientation of the kink is defined approximately by ArgA38 being located in the cap of the knee. Because of this extra residue between two glycine residues, a bad contact that would arise between the methyl group of AlaC36 and the peptide carbonyl of IleA37 in a straight triple helix is relaxed. The model features also a cluster of hydrophobic contacts between large hydrophobic side-chains in the interaction edges between the six collagen triple helices aligned with their about 10 nm long N-terminal regions in the fibril-like endpiece of C1q. The azimuthal orientations of the triple helices were derived by energy calculations of side-chain interactions previously applied to fibre-forming collagens. Independently, the same orientations and interaction edges were derived from the azimuthal orientation of the kink and the electron microscopically observed orientations of the triple helical arms that emerge from the endpiece, and which carry the C-terminal globular binding domains. The structural model has a number of implications for the assembly of the first component of complement from C1q and the zymogen complex C1r2C1s2 and possible mechanisms of its activation.

Dissociation of C1 and concentration dependence of its activation kinetics.

The activation of the zymogen C1s to the enzyme C1s in the human C1 complex [C1q(c1rC1s)2] was studied as a function of the concentrations of (C1rC1s)2 and C1q which were saturated with oligomers of rabbit IgG. A large concentration dependence of the sigmoidal kinetics was observed in the 2-180 nM concentration range. This was explained by association-dissociation equilibria between the antibody-saturated C1q and various forms of the (C1rC1s)2 complex (unactivated to activated). The establishment of these equilibria (binding constant 2 x 10(7) M-1) was assumed to be fast as compared to the rates of the activation steps (rate constants 10(-3) and 10(-2) sec-1 at 30 degrees C). The fast re-equilibration of the C1 complex explains the finding that small amounts of antibody-saturated C1q catalysed the activation of large amounts of C1s. The interpretation of the kinetic results was supported by a direct demonstration of the dissociation of C1 into C1q and (C1rC1s)2 by analytical and density gradient centrifugation. No difference was found between the rates of activation and the dissociation properties of reconstituted C1 and C1 isolated from serum.

A molecular mechanism for the activation of the first component of complement by immune complexes.

The proposed activation mechanism is based upon several key concepts, including the "S"-structure for the folding of the C1r2C1s2 tetramer among the C1q arms [Poon, et al., J. molec. Biol. 168, 563-577 (1983)]; the locations of the catalytic domains on the tetramer and the resulting functional relevance of the "S"-structure [Colomb et al., Phil. Trans. R. Soc. B306, 282-292 (1984)]; the structure of C1-inhibitor [Odermatt et al., FEBS Lett. 131, 283-289 (1981)]; and the control of C1 activation by C1-inhibitor [Ziccardi, J. Immun. 128, 2505-2508 (1982)]. The proposed activation mechanism has four main features: steric exclusion of C1-inhibitor from C1 when it binds to an immune complex; signal generation through multivalent binding of the C1q heads to an irregularly-arranged cluster of antibody Fc regions, and signal transmission through the movement of the stiff C1q arms about their semi-flexible joints, causing distortion of the symmetrical cone of C1q arms; induction of rapid activation by a shift in equilibrium favoring the autocatalytic conformation of C1r2C1s2; and release of the activated C1s from the C1q arms, so that the ends of the tetramer are free for interaction with C4 and C2 and C1-inhibitor, and the C1q subcomponent becomes more flexible, allowing access of C1-inhibitor to C1r.

Expression of CD23 by human bone marrow stromal cells.

CD23 is a surface antigen expressed by a variety of human hematopoietic cells and shown to display multiple biological functions. In present work, we assayed CD23 expression by human bone marrow (BM) or by stromal cells derived from this tissue. While freshly isolated BM-cells showed low CD23 expression, a subset of long term BM-culture (LTBMC)-derived stromal cells expressed CD23 mRNA at high levels in their steady state and secreted soluble CD23 in their culture supernatants. To assay the role of CD23 in LTBMC, these cultures were initiated in the presence of neutralizing anti-CD23 mAb. A dramatic decrease in total numbers of hematopoietic cells and CFU-GM recovery was observed in these cultures as compared to controls. These data suggest a role of CD23 expression in stroma cell functions and further confirm the ability of this antigen to regulate human hematopoietic cell development.

Regulation of human IgE response in hu-PBL-SCID mice.

The human IgE response was investigated in hu-PBL-SCID mice created by ip injection of human PBL into C.B.17 scid/scid (scid) mice. With 30-100 x 10(6) PBL/mouse, 80 to 90% of the animals responded with human IgE serum levels of 3-1000 ng/ml after 2 weeks. PBL from all donors analyzed (total number > 20) responded with IgE production. The half-lives of human IgE, IgM, and IgG in scid mice were determined to test the possibility of a passive transfer of the immunoglobulins in contrast to de novo synthesis. The values found were 88, 128, and 126 hr, respectively. In general, immunoglobulin production of all isotypes continuously increased over a period of 7-9 weeks after PBL injection, indicating de novo synthesis had taken place. The kinetics of the IgE response exhibited two phases: An initial burst of IgE production occurred between Days 12 and 22. This burst reached levels of 25-70 ng/ml IgE. After a rapid decline to about 50% of the peak value there was a sustained, slow, increase of IgE production for several weeks, excluding a passive transfer for IgE. About half of the donors lacked the initial burst of IgE production and only exhibited a slowly rising IgE production that is indistinguishable from the slow phase of the former donor population. The levels reached in this second phase of IgE production were 20-40 ng/ml after 6-7 weeks. This kinetics may reflect the presence of two different B cell populations, of which only one is present in all donors. The initial IgE burst was only partially dependent on the presence of human IL-4, reflected by a partial inhibition of this response by a neutralizing monoclonal anti-IL-4 antibody. The IgE response in scid mice seems to consist therefore of an IL-4-independent and an IL-4-dependent part, indicating the response to be partially driven by preswitched B cells. Injection of exogenous recombinant human IL-4 (rhIL-4) was not suitable due to the short half-life of rhIL-4 in scid mice of 12 min. Attempts to supply a constant source of rhIL-4 by injection of IL-4-producing Chinese hamster ovary cells failed because of toxic effects produced by these cells. The human IgE production in the scid mice was suppressed by interferon-alpha (BD) to 60-80% compared to that of untreated mice. The suppression was not isotype specific, however, because production of IgG and IgM was inhibited to similar extents.(ABSTRACT TRUNCATED AT 400 WORDS)

Activation of C1 by monoclonal antibodies directed against C1q.

Eleven monoclonal antibodies directed against the subcomponent C1q of the first component of human complement, C1, were prepared and tested for binding to intact C1q and to the collagenous portion, the C1q stalks. All of the monoclonals bound well to the intact C1q. Eight out of the eleven exhibited strong binding to the collagenous stalks, while three bound very weakly, if at all, to the stalks and, thus, were presumed to bind to the pepsin-sensitive region which includes the C1q heads. For one of the latter monoclonals, this was confirmed by electron microscopy. Five of the monoclonals were purified by C1q affinity chromatography. When tested with C1 reassembled from its subunits, two of these purified monoclonal antibodies markedly enhanced the rate of spontaneous activation.

Assembly of subcomponents C1r and C1s of first component of complement: electron microscopic and ultracentrifugal studies.

Monomeric C1s (Mr, 85,000; s20,w, 4.3S), a subcomponent of first component of complement (C1), the dimer (Mr, 170,000; s20,w, 6.7 S) of C1r, another subcomponent, and the tetrameric complex (C1r,C1s)2 (Mr, 340,000; s20,w, 8.7 S) are elongated molecules. Hydrodynamic equivalents of cylindrical shape have a diameter of 3.3 nm and lengths of 20 nm for C1s, 36 nm for (C1r)2, and 64 nm for (C1r,C1s)2. In electron micrographs the C1r,C1s complex appears as a chain composed of six to eight globular domains with a contour length of 51 nm. A structure is proposed in which (C1r)2 forms a core to which C1s protomers are associated at both ends. The C1 complex (s20,w, 16.3 S) reconstituted from C1q, C1r, and C1s dissociates under the conditions used for electron microscopy. Some features of the C1 complex are revealed in the dissociation products.

Activation of the first component of human complement, C1, by monoclonal antibodies directed against different domains of subcomponent C1q.

Two monoclonal antibodies directed against C1q, and their (Fab)2 and Fab fragments, were used to study the mechanism of C1 activation. Monoclonal antibody 2A10, an IgG2a, was digested by pepsin to yield fully immunoreactive (Fab')2. Monoclonal antibody 1H11, an IgG1, was digested by papain to yield fully immunoreactive, bivalent (Fab)2. Previously 1H11 had been shown to bind to the C1q "heads," whereas 2A10 bound to stalks. Activation of C1 was followed by the cleavage of 125I-C1s in the presence of C1 inhibitor (C1-Inh) at 37 degrees C. Spontaneous activation was minimal at inhibitor concentrations above 0.4 micron (1.3 X physiologic inhibitor concentration); all results were corrected for the spontaneous activation background. Heat-aggregated IgG activated completely in this system and was taken as 100% activation. Monoclonal antibody 2A10 caused precipitation of C1 and slow activation; neither the (Fab')2 nor the Fab' derived from 2A10-caused activation. Probably, aggregates of intact 2A10 and C1 were serving as immune complexes to activate other molecules of C1. In contrast, both 1H11 and its (Fab)2 activated completely and stoichiometrically; that is, maximal activation was achieved at a ratio of one C1q head to one antibody combining site. The monovalent Fab derived from 1H11 bound well to C1q, but no activation of C1 was observed. Thus, bivalent binding of this head-binding monoclonal is required for C1 activation, but not the presence of the antibody Fc portion. Neither 1H11 nor its (Fab)2 fragments caused C1 precipitation; however, the 1H11 did form complexes composed of two C1q cross-linked by multiple 1H11, which were visualized by electron microscopy. The presence of these dimeric complexes correlated well with activation. A model for C1 activation is proposed in which two C1q subcomponents are held together by multiple (Fab)2 bridging C1q heads. The model is roughly analogous to touching opposing pairs of fingers and thumb tips, the two hands representing the two C1q, forming a cage. C1-Inh, which probably binds to C1r through the open end of the C1 cone, is too long asymmetric to be included within the cage. Thus, according to this model, the dimers of C1 are released from the inhibitory action of C1-Inh, and activation proceeds spontaneously and rapidly at 37 degrees C.

Interferon-alpha suppresses the capacity of T cells to help antibody production by human B cells.

This study was designed to analyze the effect of interferon-alpha (IFN-alpha) on the potential of T cells to help B-cell differentiation in vitro. Human splenic T cells preactivated via the T-cell receptor (TCR)/CD3 complex, as well as murine EL4 thymoma T cells preactivated with phorbol esters, stimulated human B cells via a species cross-reactive physical interaction to differentiate into antibody-producing cells. If the human or murine T cells were activated in the presence of IFN-alpha, normal proliferation and interleukin-2 (IL-2) production occurred, but the cells did not acquire any B-cell helper potential. Therefore, IFN-alpha modulates the B-cell stimulatory potential of T cells by interfering with the T-cell activation process. In contrast, IFN-alpha neither acted on B cells directly nor on already activated T cells, because it did not suppress B-cell differentiation induced by T cells preactivated in the absence of IFN-alpha. IFN-alpha did not induce the production of inhibitory T-cell factor(s), since T cells preactivated in the presence of IFN-alpha did not inhibit the interaction of B cells with T cells optimally preactivated in the absence of IFN-alpha. Taken together the data indicate that IFN-alpha suppresses the potential of T cells to stimulate B-cell differentiation by interfering with the T-cell activation process, but acts neither on B cells directly nor on already activated T cells.

Role of CD23 in astrocytes inflammatory reaction during HIV-1 related encephalitis.

Soluble factors released by intra-cerebral activated cells are implicated in neuronal alterations during central nervous system inflammatory diseases. In this study, the role of the CD23 pathway in astrocyte activation and its participation in human immunodeficiency virus-1 (HIV-1)-induced neuropathology were evaluated. In human primary astrocytes, CD23 protein membrane expression was dose-dependently upregulated by gp120. It was also upregulated by gamma-interferon (gamma-IFN) and modulated by interleukin-1-beta (IL-1beta) whereas microglial cells in these stimulation conditions did not express CD23. Cell surface stimulation of CD23 expressed by astrocytes induced production of nitric oxide (NO) and IL-1beta which was inhibited by a specific inducible NO-synthase (iNOS) inhibitor (aminoguanidine), indicating the implication of this receptor in the astrocyte inflammatory reaction. On brain tissues from five out of five patients with HIV-1-related encephalitis, CD23 was expressed by astrocytes and by some microglial cells, whereas it was not detectable on brain tissue from five of five HIV-1-infected patients without central nervous system (CNS) disease or from two of two control subjects. In addition, CD23 antigen was co-localized with iNOS and nitrotyrosine on brain tissue from patients with HIV1-related encephalitis, suggesting that CD23 participates in iNOS activation of astrocytes in vivo. In conclusion, CD23 ligation is an alternative pathway in the induction of inflammatory product synthesis by astrocytes and participates in CNS inflammation.

Kinetics of C1 activation by a monoclonal anti-C1q antibody and its (Fab)2 fragments.

Monoclonal antibody 1H11, which binds to the "head" portion of C1q, has been shown to be a strong, stoichiometric activator of C1, the first component of human complement, maximal activation being achieved at a ratio of one antibody-combining site per one C1q head; moreover, this activation occurs even in the presence of C1-inhibitor, as reported previously. In the present paper, the kinetics of activation are shown to be biphasic; that is, a portion of the C1 is activated very rapidly, and the remainder slowly. These two processes can be separated by the order of mixing of preincubated components; thus, only the rapid activation rate is observed if C1q and the monoclonal antibody are preincubated together and are added subsequently to a mixture of C1r2C1S2 and C1-inhibitor. Only the slow activation rate is observed when C1q, C1r2C1S2, and C1-inhibitor are preincubated and are added subsequently to monoclonal antibody 1H11. Similar results are obtained by using either the intact 1H11 antibody or else the (Fab)2 obtained from it by proteolytic digestion and purification. The rapid phase is independent of the concentration of 1H11 over the range employed; the slow phase depends on 1H11 concentration. Plausible activation schemes are presented to explain the two distinct activation rate processes, and kinetic models are developed which provide a reasonable simulation of the experimental data.

Human Fc epsilon R II and IgE-binding factors.

The low-affinity receptor for IgE (Fc epsilon R II) is mainly expressed on B lymphocytes, although it may also be found on some monocytes, eosinophils, and platelets. The presence of Fc epsilon R II on T cells is still controversial, but our results demonstrate that it is expressed on some HTLV-I-transformed T lymphocytes, and they strongly suggest that it may be found on a small proportion of normal T cells. Fc epsilon R II is a 45-KD glycoprotein containing one N-linked carbohydrate of complex type, O-linked carbohydrates, and sialic acid residues. Fc II is cleaved into soluble fragments with molecular weights of 37, 33, 25, and 12 KD, the first three retain the ability of binding to IgE, i.e., they are IgE-binding factors (IgE-BFs). The enzymes involved in their proteolytic cleavage are cell bound. The cDNA coding for Fc epsilon R II was cloned and functionally expressed. The predicted sequence has no homology with that of murine IgE-BFs which are of T cell origin. However, there is a striking homology with several animal lectins, and since the IgE-binding site is located in the homology region, it is possible that it binds to IgE via the carbohydrates expressed on the Fc region of this immunoglobulin. The expression of Fc epsilon R II on B cells and the release of IgE-BFs are upregulated by interleukin 4 and suppressed by gamma and alpha interferons.(ABSTRACT TRUNCATED AT 250 WORDS)

New concepts of IgE regulation.

B cell switch to IgE expression is mediated by IL-4 and is regarded as a T helper cell-related phenomenon. In this overview we describe that IgE switch can also be induced by mast cell/basophil like cells (from splenic non-B, non-T cells), activated by IgE receptor cross-linking and/or IL-3 which results in IL-4 production by these cells. Furthermore, activated mast cells produce their own growth factors, IL-3 and GM-CSF. Thus, activation of mast cells can provoke an ongoing local allergic reaction as long as antigen confrontation is maintained, a process which is sustained by further IgE production as well as renewal of mast cells. It is furthermore demonstrated that in certain established immune situations the IgE response may become independent of IL-4, namely in the spontaneous in vitro IgE expression of cells from atopic individuals as well as in an in vitro antigen-induced secondary IgE response of spleen cells derived from previously immunized mice. Thus, IgE-switched B cells may persist in vivo and may represent a pool of potentially IgE-producing cells. Finally, a selective inhibition of the IgE response is described in vitro and in vivo by the use of so-called non-anaphylactic monoclonal anti-IgE antibodies. Such antibodies bind to surface IgE+ B cells, but not to IgE-sensitized mast cells, and thereby inhibit IgE responses. Non-anaphylactic antibodies blocked the binding of allergen-specific IgE to mast cells by competing with the Fc epsilon on these cells. As a consequence they do not induce but rather prevent allergen-induced mediator release by mast cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of Fc epsilon RII/CD23 and L-arginine-dependent pathway in IgE-mediated stimulation of human monocyte functions.

Elevated IgE levels are commonly observed during the inflammatory responses in allergy and a variety of infections. This Ig activates the release of multiple mediators from monocytes/macrophages. In the present work, we attempted to clarify the IgE-dependent events involved in the activation of monocyte functions. IgE-anti-IgE immune complexes induce the production of tumor necrosis factor-alpha, oxygen radicals, IL-6 and thromboxane B2 from normal human purified monocytes. Expression and cross-linkage of Fc epsilon RII/CD23 were essential for these IgE-mediated effects. Cytokine production following CD23 ligation depended on nitric oxide transduction pathway, as it was inhibited by NG-monomethyl-L-arginine, a competitive inhibitor of the conversion of L-arginine to L-citroline by nitric oxide synthase. Furthermore, addition of the nitric oxide chemical donator, Sin-1, enhanced IgE-induced monokine release. CD23-ligation also induced the production of nitrites by these cells. This work linked CD23 to the L-arginine-dependent transduction pathway and shows their involvement in IgE-mediated stimulation of human monocytes.

Cloning and expression of the cDNA coding for a human lymphocyte IgE receptor.

Low-affinity receptors (Fc epsilon R) and secreted factors (IgE-BF) which bind to immunoglobulins of the IgE isotype play a key role in the regulation of human IgE synthesis. We report here the cloning of a cDNA coding for the Fc epsilon R of the human B-lymphoblast cell line RPMI 8866. The nucleotide sequence of this cDNA predicts a polypeptide with 321 amino acids and a mol. wt of 36,281 daltons. A functional Fc epsilon R capable of binding IgE was expressed in Chinese hamster ovary cells after stable transformation with the cDNA which had been cloned into a mammalian expression vector. Amino acid sequence analysis of IgE-BF purified from RPMI 8866 cells revealed an amino-terminal sequence of 19 residues which coincides with the predicted amino acid sequence of the Fc epsilon R, starting at residues 148 and 150. A computer search with the translated amino acid sequence of the Fc epsilon R revealed a domain of 120 amino acids having striking homology to the human asialoglycoprotein receptors.

Cytokine effects of CD23 are mediated by an epitope distinct from the IgE binding site.

Human CD23 and its soluble forms (sCD23) display various biological activities, in addition to their IgE binding function (IgE/BF). The IgE binding domain was recently mapped to residues between Cys163 and Cys282 but its involvement in IgE-independent, CD23 functions remains unknown. In order to clarify this point, a series of N-terminal, C-terminal and internal deletion mutants of CD23 or sCD23 were expressed in CHO cells and tested for their ability (i) to bind to IgE, (ii) to induce colony formation by human myeloid precursor cells, (iii) to promote mature T cell marker expression by early prothymocytes, and (iv) to regulate IgE synthesis. The present study indicates that cytokine activities require the presence of Cys288, while this amino acid is not necessary for IgE/BF. Blocking experiments using various conformation-sensitive monoclonal antibodies further suggest that active epitope(s) of CD23 in cytokine assays is(are) distinct from those involved in IgE/BF.