Cytokine-mediated up-regulation of CD55 and CD59 protects human hepatoma cells from complement attack.

Hepatic parenchymal cells respond in many different ways to acute-phase cytokines. Some responses may protect against damage by liver-derived inflammatory mediators. Previous investigations have shown that cytokines cause increased secretion by hepatoma cells of soluble complement regulatory proteins, perhaps providing protection from complement attack. More important to cell protection are the membrane complement regulators. Here we examine, using flow cytometry and Northern blotting, the effects of different cytokines, singly or in combination, on expression of membrane-bound complement regulators by a hepatoma cell line. The combination of tumour necrosis factor-alpha, IL-1beta, and IL-6 caused increased expression of CD55 (three-fold) and CD59 (two-fold) and decreased expression of CD46 at day 3 post-exposure. Interferon-gamma reduced expression of CD59 and strongly antagonized the up-regulatory effects on CD59 mediated by the other cytokines. Complement attack on antibody-sensitized hepatoma cells following a 3-day incubation with the optimum combination of acute-phase cytokines revealed increased resistance to complement-mediated lysis and decreased C3b deposition. During the acute-phase response there is an increased hepatic synthesis of the majority of complement effector proteins. Simultaneous up-regulation of expression of CD55 and CD59 may serve to protect hepatocytes from high local concentrations of complement generated during the acute-phase response.

Expression of the beta-chain of the complement regulator C4b-binding protein in human ovary.

Human C4b-binding protein (C4BP) is an important regulator of the complement system that also binds and inactivates the anticoagulant vitamin K-dependent protein S. These two activities are performed by two distinct polypeptides of 70 kDa and 45 kDa known as alpha and beta chains, respectively. C4BP is present in plasma in various isoforms with different alpha/beta composition. We report here that C4BPbeta, but not C4BPalpha, is expressed in adult human ovary. Expression of C4BPbeta was detected in all ovarian biopsies analyzed (n = 15), independently of age and phase of the menstrual cycle. In situ hybridization and immunostaining analyses on cryostat sections demonstrated expression of C4BPbeta in both regressing corpus luteum and corpus albicans, but not in the follicles, the corpus luteum, the ovary stroma or the vascular cells. In addition, we noted that the expression pattern of the C4BPbeta mRNA resembles that described for the connective tissue that invades the degenerating corpus luteum and causes a progressive fibrosis that gradually converts it into a scar, the corpus albicans. RT-PCR and immunostaining analyses of primary cultures derived from human ovaries demonstrated the presence of fibroblast-like cells that express C4BPbeta. As a whole, these data suggest a role for the C4BPbeta in human ovary during the healing and scar resorption processes that leads to the formation of the corpus albicans and its replacement by ovarian stroma.

Modulation of C4b-binding protein isoforms during the acute phase response caused by orthopedic surgery.

Orthopedic surgery is described as an event with a high risk of thromboembolic diseases. This is probably a consequence of a synergistic combination of different risk factors in the patients subjected to this type of surgery, including age, immobilization, anesthesia and different hypercoagulable states. After surgery patients develop an acute-phase response that leads to changes in several plasma proteins. One of these proteins is the complement regulator C4b-binding protein (C4BP). We have recently shown that in some acute-phase patients C4BP is incorrectly controlled (with elevation of the C4BP beta-containing isoforms), leading to a potential hypercoagulable state by decreasing the plasma levels of free (active) protein S. Here we have studied whether patients subjected to orthopedic surgery have an appropriate modulation of the C4BP isoforms during their postoperative acute-phase responses. We have analyzed the evolution of the C4BP isoforms in serial samples from 11 patients who have undergone knee (or hip) prosthesis surgery (mean age 70 years), or scoliosis surgery (mean age 18 years). Our data suggest a similar evolution of C4BP isoforms in all these patients, with an almost exclusive increase of C4BP isoforms lacking C4BP beta polypeptides and steady levels of free protein S.

Isoforms of human C4b-binding protein. I. Molecular basis for the C4BP isoform pattern and its variations in human plasma.

Human C4b-binding protein (C4BP) is an important regulator of the complement system that also binds and inactivates the anticoagulant vitamin K-dependent protein S. These two activities are performed by two distinct polypeptides of 70 kDa and 45 kDa known as alpha- and beta-chains, respectively. C4BP is present in plasma in various isoforms with different alpha beta composition. Here we report multiple discrete variations of the relative levels of the C4BP isoforms among normal individuals and provide evidence that they are determined by genetic factors that segregate with the regulator of complement activation gene cluster. We also report the characterization of the C4BP molecules secreted by HepG2 and Hep3B cells, as well as transfection experiments in COS cells, to illustrate that the relative levels of expression of the C4BPA and C4BPB genes play a major role in determining the proportion in which the different C4BP isoforms are synthesized. Altogether, the data indicate that the human C4BP isoform pattern is genetically determined, but can be modified by factors with a differential effect on the expression of the C4BPA and C4BPB genes. These observations provide a new way to explore the possible association between elevated levels of C4BP and an increased risk to thromboembolic disorders.

Isoforms of human C4b-binding protein. II. Differential modulation of the C4BPA and C4BPB genes by acute phase cytokines.

Human C4b-binding protein (C4BP) controls activation of the complement system and inactivates the anticoagulant vitamin K-dependent protein S using two distinct polypeptides known as C4BP alpha and C4BP beta, respectively. C4BP presents three isoforms, alpha 7 beta 1, alpha 7 beta 0, and alpha 6 beta 1, the proportion of which depends on the relative levels of C4BP alpha and C4BP beta. To better understand the regulation of C4BP during the acute phase response we analyzed the C4BP isoforms in 23 serial samples of acute phase patients and characterized the effect of various acute phase cytokines on the expression of the C4BPA and C4BPB genes using Hep3B cells. We show that the elevation of C4BP during acute phase response leads to changes in the proportion of the C4BP isoforms. However, there are striking differences among acute phase individuals. Some of them present a pattern of induction that primarily affects the alpha 7 beta 0 isoform, whereas others present the opposite situation, increasing the C4BP beta-containing isoforms. In vitro studies demonstrate that IL-6, IL-1 beta, and INF-gamma increase the levels of both C4BP alpha- and C4BP beta-mRNAs, whereas TNF-alpha down-regulates these mRNAs. INF-gamma shows, in addition, a differential effect on the C4BP alpha- and C4BP beta-mRNAs. Differential modulation of the C4BPA and C4BPB genes has been postulated as an efficient mechanism to maintain steady concentrations of C4BP beta when C4BP is induced. A synergistic 10-fold induction of C4BP alpha-mRNA, but a marginal increase of C4BP beta-mRNA, was observed when INF-gamma was used together with TNF-alpha, suggesting that association of these cytokines is critical to avoid elevation of C4BP beta during the acute phase induction of C4BP.