Aberrant expression of complement regulatory proteins, membrane cofactor protein and decay accelerating factor, in the involved epidermis of patients with vitiligo.

BACKGROUND: Vitiligo is a pigmentary disorder of the skin characterized by the complete absence of melanocytes from the lesion. Complement-activating antimelanocyte antibodies have been implicated in vitiligo pathogenesis. As membrane regulators of complement activation, membrane cofactor protein, decay accelerating factor and CD59 protect cells from elimination by autologous complement, their absence or downregulation on melanocytes may be associated with autoantibody and complement-mediated melanocyte destruction in vitiligo. OBJECTIVES: We studied the expression of these regulatory proteins in non-lesional, perilesional and lesional vitiligo skin compared with those of control specimens. METHODS: We used immunohistochemistry to study the expression of the regulatory proteins, and flow cytometric analysis of cultured melanocytes to investigate possible constitutive changes in the expression levels of these molecules. We also investigated whether melanocytes can influence keratinocyte susceptibility to autologous complement by regulating keratinocytic decay accelerating factor and membrane cofactor protein expression levels. RESULTS: Immunohistochemical data showed that expression of membrane cofactor protein and decay accelerating factor in whole epidermis was lower in lesional and perilesional skin in comparison with non-lesional skin. The reduced in situ expression appeared to be specific to vitiligo. However, coculture experiments indicated that melanocytes do not influence keratinocyte susceptibility to autologous complement. Further, flow cytometric analysis of cultured melanocytes convincingly demonstrated that non-lesional vitiligo and control melanocytes have comparable decay accelerating factor, membrane cofactor protein and CD59 expression levels. CONCLUSIONS: It is therefore concluded that there is no constitutive melanocyte defect per se that could be related to the in vivo expression of these molecules in vitiligo. Nevertheless, the present data suggest that both keratinocytes and melanocytes in the involved vitiliginous whole epidermis express lower levels of decay accelerating factor and membrane cofactor protein compared with controls that could render them more vulnerable to autologous complement attack.

Therapeutic inhibition of the complement system. Y2K update.

Activation of complement is an essential part of the mechanism of pathogenesis of a large number of human diseases; its inhibition by pharmacological means is likely to suppress disease processes in complement mediated diseases. From this point of view low molecular weight synthetic inhibitors of complement are being developed and high molecular weight natural inhibitors of human origin present in plasma or embedded in cell membrane are being purified or produced in their recombinant forms. This review is concerned with high molecular weight inhibitors, some of which are already in clinical use but may be efficacious in many other diseases in which they have not yet been tried. C1-esterase inhibitor (C1-INH) concentrate prepared from human plasma is being successfully used for the treatment of hereditary angioneurotic edema. Recently, C1-INH has been found to be consumed in severe inflammation and has been shown to exert beneficial effects in several inflammatory conditions such as human sepsis, post-operative myocardial dysfunction due to reperfusion injury, severe capillary leakage syndrome after bone marrow transplantation, reperfusion injury after lung transplantation, burn, and cytotoxicity caused by IL-2 therapy in cancer. Factor I has been used for the treatment of factor I deficiency. Recombinant soluble forms of membrane cofactor protein (MCP), and decay accelerating factor (DAF) have not yet been tried in humans but have been shown to be effective in immune complex mediate inflammation in animals. Organs of pigs transgenic for one or more of human membrane regulators of complement namely membrane cofactor protein (MCP), decay accelerating factor (DAF) or CD59, are being produced for transplantation into humans. They have been shown to be resistant to hyperacute rejection in non-human primates; acute vascular rejection is still a problem in their clinical use. It is hoped that these observations together with future developments will make xeno-transplantation in clinical practice a reality. Several recombinant variants of complement receptor 1 (CR1) have been produced. The most effective of these appears to be sCR1-SLe x, sCR1 part of which inhibits complement and carbohydrate Sle x moiety inhibits selectin mediated interactions of neutrophils and lymphocytes with endothelium. Although clinical trials of sCR1 in humans is eagerly awaited, several of the recombinant versions of sCR1 have been shown to suppress ischemia/reperfusion injury, thermal trauma, and immune complex mediated inflammation. They have also been shown to be effective in experimental models of systemic sclerosis, arthritis, myasthenia gravis, Guillain Barré syndrome and glomerulonephritis. Intravenous immunoglobulin, three of the most prominent properties of which are neutralization of autoantibody activity, suppression of autoantibody production and inhibition of complement activity, is being used in several diseases. These include autoimmune thrombocyopenic purpura, Kawasaki disease and several neurological diseases such as myasthenia gravis and Guillain Barre syndrome. In many uncontrolled small scale studies intravenous immunoglobulin has been shown to be effective in many immunological including dermatological diseases; controlled clinical trials in a large number of patients with these diseases is needed to establish the efficacy. It is hoped that in future therapeutic inhibition of complement will be one of the major approaches to combat many human diseases.

Synthesis of complement components C3 and factor B in human keratinocytes is differentially regulated by cytokines.

The complement system plays an important part in host defense and inflammation. Locally synthesized complement may perform these functions at tissue and organ level. In skin the keratinocyte is the major cell type, it is known to produce two soluble complement components, C3 and factor B. In this study we investigated the regulation of synthesis of these components in foreskin keratinocytes by cytokines. Human keratinocytes were cultured in the presence of supernatant of activated peripheral blood mononuclear cells, interleukin-1alpha, interleukin-2, interleukin-6, transforming growth factor-beta1, tumor necrosis factor-alpha, or interferon-gamma. C3 and factor B proteins were measured in culture supernatant by enzyme-linked immunosorbent assay and C3 and factor B transcripts in harvested cells by reverse transcriptase-polymerase chain reaction. Cultured keratinocytes constitutively produced C3 and factor B. Supernatant of activated mononuclear cells upregulated C3 and factor B production by 27- and 15-fold, respectively. interleukin-1alpha, interferon-gamma, and tumor necrosis factor-alpha upregulated C3 synthesis by 7-, 8-, and 22-fold, and interleukin-1alpha, interleukin-6, and interferon-gamma upregulated factor B synthesis by 3-, 3-, and 34-fold, respectively. Tumor necrosis factor-alpha induced production of C3 and interferon-gamma induced production of factor B were inhibited by cycloheximide. Cytokine induced upregulation of C3 and factor B proteins was always associated with the upregulation of levels of C3 and factor B mRNA. This indicated that, as expected, cytokine-induced enhancement in C3 and factor B levels was due to an increase in synthesis rather than their possible release from intracellular stores. In conclusion, synthesis of C3 and factor B in keratinocytes is regulated by some cytokines, known to be produced by inflammatory cells and keratinocytes.

Transforming growth factor-beta isoforms regulate the surface expression of membrane cofactor protein (CD46) and CD59 on human keratinocytes [corrected].

We studied the regulation of the expression of complement regulatory proteins, membrane cofactor protein (MCP), decay accelerating factor (DAF) and CD59, on human keratinocytes by supernatant of activated mononuclear cells and by some individual cytokines present therein. Cultured keratinocytes expressed MCP, DAF and CD59. Supernatant of activated mononuclear cells and recombinant forms of transforming growth factor (TGF)-beta variants (beta1, beta2 and beta3) up-regulated MCP and CD59 but not DAF. Recombinant IL-1alpha, IL-2, IL-6, TNF-alpha and IFN-gamma had no influence. TGF-beta present in the supernatant was likely responsible for up-regulation of MCP and CD59. A monoclonal anti-TGF-beta antibody, which neutralized TGF-beta1, -beta2 and -beta3, did not inhibit the up-regulation of MCP and CD59 by the supernatant. These results indicated that TGF-beta and an additional factor(s) present in the supernatant may be responsible for up-regulating the expression of MCP and CD59 on keratinocytes; both may be acting non-synergistically.

Effects of UVB on the synthesis of complement proteins by keratinocytes.

UVB exposure of the skin results in increased production of several cytokines by keratinocytes and infiltration of inflammatory cells. We hypothesized that UVB may increase the expression of complement (C) components and C-regulatory proteins by keratinocytes. In vivo, UVB may upregulate these proteins by direct effects or via cytokines released by keratinocytes or infiltrating inflammatory cells. In vitro, UVB may upregulate these proteins only directly, because of dilution of released cytokines in the medium. To test this, we exposed cultured human keratinocytes to UVB (0-64 J per m2) and monitored C3 and Factor B release in the medium by enzyme-linked immunosorbent assay, and surface expression of decay accelerating factor, membrane cofactor protein, and CD59 by flow cytometry. Keratinocytes produced small amounts of C3 and Factor B, which remained unaffected by UVB. UVB (32 J per m2) caused a transient upregulation of all three C-regulatory proteins. Decay accelerating factor expression was maximal at 48 h (1.81 +/- 0.06-fold increase in mean fluorescence intensity over nonexposed cells), membrane cofactor protein at 72 h (2.13 +/- 0.09-fold increase in mean fluorescence intensity), and CD59 at 120 h (1.96 +/- 0.09-fold increase in mean fluorescence intensity), returning to baseline values within 96, 192, and 192 h, respectively. Exposure to 64 J per m2 resulted in significant cell death; cells surviving this dose up to 48 h expressed a higher level of all the three proteins than those surviving 32 J per m2. In conclusion, UVB upregulated membrane cofactor protein, decay accelerating factor, and CD59 on keratinocytes without affecting the constitutive release of C3 and Factor B. Thus, UVB can increase the resistance of keratinocytes against their own C known to be produced excessively in response to cytokines of inflammatory cells that infiltrate the skin following UVB exposure.

Molecular heterogeneity of second and fourth components of complement and their genes in systemic sclerosis and association of HLA alleles A1, B8 and DR3 with limited and DR5 with diffuse systemic sclerosis.

Deficiency of the complement component C4 at the functional, protein and gene level and deficiency of complement component C2 at the functional level were investigated and HLA analysis was performed on patients with limited and diffuse systemic sclerosis (SSc). One of the patients with limited SSc (n = 15) had subnormal C4, 1 subnormal C2 and 1 subnormal C4 and C2 activities; the latter patient had HLA alleles A11;B35;Dw1 associated with type II C2 deficiency and therefore most likely had a defect at the C2 locus. One of the patients with diffuse SSC (n = 12) had subnormal C4 and 1 subnormal C4 and C2 activities. C2 deficiencies in patients other than the one with the haplotype associated with C2 deficiency appeared not to be determined by the gene at the C2 locus. The incidence of partial C2 deficiency in a normal Caucasian population is reported to be 16 in 10,000, and that of partial C4 deficiency also appears to be very low. The percentages of C4A*Q0 and C4B*Q0 alleles in normal controls (n = 45) were within the reported range. Seven patients with limited SSc (n = 14) had one or two C4A*Q0 alleles and 2 with diffuse SSc (n = 13) had one C4A*Q0 allele. Thus, the incidence of C4A*Q0 was higher than normal in limited SSc and within the normal range in diffuse SSc. The two-sided Fisher's exact test applied on these data revealed that the association of C4A*Q0 with limited SSc did not reach a significant level (p = 0.10). Two of the 3 patients with limited SSc, who had two C4A*Q0 alleles, carried a heterozygous C4A-21-hydroxylase A (OHA) gene segment deletion as detected by Southern blotting. There was no correlation between the subnormal activity of C4 and the occurrence of one or two C4A*Q0 (and C4A-21-OHA segment deletion). HLA alleles A1, B8 and DR3 (p = 0.002) were associated with limited SSc (n = 23) and DR5(w11) (p = 0.018) with diffuse SSc (n = 17).

Relative contributions of decay accelerating factor (DAF), membrane cofactor protein (MCP) and CD59 in the protection of melanocytes from homologous complement.

Complement regulatory molecules, membrane cofactor protein (MCP), decay accelerating factor (DAF) and CD59, protect body cells from autologous complement. They have wide tissue distribution but nothing is known about the expression of these molecules on human melanocytes. Since melanocytes are lysed in the lesional skin of patients with a depigmentary disorder vitiligo, it is important to compare the protection offered by complement regulatory molecules to melanocytes present in normal and vitiligo epidermis, against autologous complement. From this point of view, we investigated the differential expression of MCP, DAF and CD59 on normal cultured human melanocytes and assessed their individual contribution in the protection of these cells against complement-mediated damage. Flow cytometric analysis showed that MCP and DAF but not CD59 were expressed on cultured melanocytes. When heat inactivated sera of patients with vitiligo were used as a source of anti-melanocyte antibody to sensitize melanocytes, and guinea pig serum (GpS) or normal human serum (NHS) as a source of complement, GpS was found to be more effective in causing the lysis of melanocytes than NHS. When melanocytes were sensitized with autoantibody as well as F(ab')2 fragment of either anti-MCP or anti-DAF and subsequently incubated with NHS or GpS, both antibody fragments increased the killing of melanocytes by NHS as well as by GpS. F(ab')2 fragment of anti-DAF was much more effective in causing enhancement of lysis than that of anti-MCP. Thus, cultured normal human melanocytes express functionally active MCP and DAF but not CD59. Contribution of DAF in protecting melanocytes against complement attack was much more than that of MCP.

Prolonged increase of cis-urocanic acid levels in human skin and urine after single total-body ultraviolet exposures.

Cis-urocanic acid (cis-UCA), a mediator of immunosuppression, is formed from trans-UCA upon UV-exposure of the skin. This study describes a liquid chromatographic method for the simultaneous quantification of cis- and trans-UCA in skin, urine and plasma of nonirradiated volunteers. It also describes cis- and trans-UCA kinetics in UV-irradiated volunteers. New procedures to remove interfering substances from urine and plasma are reported. Normal levels of cis-UCA in skin, urine and plasma of nonirradiated volunteers were 0.5 nmol/cm2, 0.03 mumol/mmol creatinine (median 0.00) and undetectable and those of trans-UCA were 17.1 nmol/cm2, 1.36 mumol/ mmol creatinine and 0.5 microM, respectively. Upon single total body UVB (290-320 nm) exposures of 250 J/m2, epidermal cis-UCA levels immediately reached a maximum and returned to basic levels 3 weeks later. The cis-UCA levels in urine reached a maximum in 5-12 h postirradiation and reached baseline values in 8-12 days. Additionally, a single total body UVA (320-400 nm) irradiation of 200 kJ/m2 yielded a similar pattern. The kinetics of cis-UCA in plasma could not be followed due to low concentrations; however, that of skin and urine was informative in relation to solar exposures and phototherapy.

Pharmacological manipulation of the complement system in human diseases.

Complement is one of the powerful effector systems involved in the body's defense. When present in a dormant state it can, in concert with other components of immune system, protect the individual from foreign pathogens. However, inappropriately activated complement can cause disease. Several disease states such as immune complex and autoimmune diseases and deficiencies of some complement regulators are associated with inappropriate activation of complement. In some diseases complement is activated for a long or indefinite period while in others for a comparatively short time; in some it is activated systemically, in others locally; in some whole cascade is activated, in others only a few components are activated; in some classical pathway is activated, in others alternative pathway. In some diseases activation of complement takes place on cell and tissue surfaces. In many complement activating diseases biological activities of complement fragments become detrimental resulting in tissue injury and disease. Inhibition of complement by specific inhibitors is likely to arrest complement mediated disease processes. From this point of view, some laboratories are developing low molecular weight synthetic inhibitors whereas others are focusing on the development of high molecular weight plasma or cell surface complement inhibitors in their natural or recombinant forms for therapeutic purposes. A review concerning development of low molecular weight inhibitors with the eventual aim of manipulating complement system in human diseases was recently published (1,2). This review is concerned with high molecular weight natural or recombinant complement inhibitory molecules in human plasma or cell membranes, some of which are already in clinical use.

Incomplete functional deficiencies of the fourth (C4) and second (C2) components of complement in a patient with linear frontoparietal scleroderma and his family. Deficiencies determined by a gene not linked to human leukocyte antigen system.

BACKGROUND: In a previous study, a patient suffering from linear frontoparietal scleroderma and some of his family members were found to have an incomplete functional deficiency of the second component (C2) of complement (C). In this study, the proband and the rest of his family members were investigated for functional deficiencies of C2 and the fourth component of C (C4). A search for null alleles of C2 (C2*Q0) and C4 (C4*Q0) was made to find out whether their occurrence is responsible for incomplete functional deficiencies. HLA analysis was performed to find out whether deficiencies are linked to HLA alleles known to be associated with C4*Q0 and C2*Q0. Possible large deletions at C4 and 21-hydroxylase (21-OH) gene loci were also investigated in some family members. OBSERVATIONS: The proband had a combined functional deficiency of C4 and C2. Some of his family members had a partial functional deficiency of C4, some of C2 and some of C4 and C2; none had null alleles of C2 (C2*Q0), factor B (B*Q0) or C4B (C4B*Q0). C4*Q0 or functional C4 deficiency in this family was not associated with HLA-A1;B8;DR3 alleles. C2 deficiency was also not associated with HLA antigens known to be associated with type I and II C2 deficiencies. No gene deletion or unusual polymorphism at C4A and 21-OHA loci could be seen by restriction fragment length polymorphism (RFLP) studies. CONCLUSIONS: Combined and isolated partial functional deficiencies of C4 and C2 observed in the proband and many of his family members were not caused by C activation or null alleles. They were not linked to HLA system and were reminiscent of those observed previously in a family in which C4 deficiency was determined by a gene not linked to the HLA system.

Morphoea lesions are associated with aberrant expression of membrane cofactor protein and decay accelerating factor in vascular endothelium.

One of the main features of systemic and localized forms of scleroderma is vascular damage, the mechanism of which is not yet understood. Recently, we have shown undetectable or decreased expression of complement regulatory molecules, membrane cofactor protein (MCP) and decay-accelerating factor (DAF), in cutaneous endothelium of patients with systemic sclerosis (SSc). In some patients, CD59 expression in endothelium was also altered. As these molecules protect endothelial cells from damage by autologous complement, their decreased expression could be part of the mechanism of vascular damage in SSc. In the present study, we investigated the expression of MCP, DAF and CD59 in the endothelium of lesional and non-lesional skin of patients with localized morphoea. Normal skin and lesional skin from patients with systemic lupus erythematosus, and three inflammatory diseases, were included as relevant controls. The results showed that the extent of expression of the three molecules in non-lesional skin of patients with morphoea, on all the skin cells and structures, was identical to that of normal skin. In lesional skin, however, the expression of MCP and DAF in endothelium was either undetectable or only present to a very slight degree. CD59 expression in endothelium in lesional skin was normal. No such aberrant expression was observed in the lesions of any of the control diseases. These results indicate a decreased ability of endothelial cells in lesional areas to protect themselves from autologous complement, and this could contribute to vascular damage in morphoea lesions.

Aberrant expression of membrane cofactor protein and decay-accelerating factor in the endothelium of patients with systemic sclerosis. A possible mechanism of vascular damage.

BACKGROUND: One of the main features of systemic sclerosis (SSc) is vascular damage, the mechanism of which is not understood. The aim of this study was to investigate if complement (C) regulatory molecules, membrane cofactor protein (MCP), decay-accelerating factor (DAF), and CD59, which normally protect endothelial cells from damage by autologous C, are absent or down-regulated in vascular endothelium of patients with SSc. A deficiency or persistent down-regulation of the above molecules is likely to render vascular endothelium of these patients susceptible to damage by physiologically or pathologically activated C. From this point of view, expression of MCP, DAF, and CD59 was tested on endothelium of skin of normal subjects and patients with diffuse and limited forms of SSc. EXPERIMENTAL DESIGN: Punch biopsies of normal skin (N = 11) and lesional and non-lesional skin of patients with diffuse (N = 5) and limited (N = 5) forms of SSc were obtained and serial sections prepared. Immunoperoxidase staining of these sections was carried out using four monoclonal antibodies (MoAbs) directed to different epitopes of DAF, four to different epitopes of MCP, one to CD59 and one to von Willebrand factor. von Willebrand factor served as a marker of endothelial cells. Besides normal skin, lesional skin of systemic lupus erythematosus and several inflammatory and proliferative diseases, described below, served as controls. RESULTS: The endothelium of normal skin strongly expressed all the three proteins. However, the endothelium of lesional and nonlesional skin of all the 10 patients with diffuse or limited forms of the disease tested, expressed either decreased or undetectable amounts of MCP and DAF. CD59 expression was normal in some patients and lower than normal in others. Aberrant expression of MCP, DAF, or CD59 was not found in other control inflammatory, connective tissue and proliferative diseases studied. CONCLUSIONS: In view of the common function of MCP and DAF to protect self cells from autologous C, their decrease or virtual absence from the endothelium of patients with SSc strongly suggests that this deficiency may contribute to vascular damage, resulting in intima proliferation and, finally, fibrosis.

Glycosylphosphatidylinositol (GPI)-anchored membrane proteins are constitutively down-regulated in psoriatic skin.

Hyperproliferation of keratinocytes (KCs) in psoriasis has been found to be associated with excessive activation of a phospholipase C (PLC)/protein kinase C (PKC) signal transduction system. The molecular species of PLCs which are activated in psoriasis have not been thoroughly investigated. It was envisaged that if glycosylphosphatidylinositol (GPI)-specific PLC was activated in the membrane of psoriatic epidermal cells, it would render these cells devoid of those proteins which are anchored to the cell membrane through their GPI moiety. In order to test this possibility, four GPI proteins (CD16, CD55, CD58, and CD59) were determined immunohistochemically in normal and psoriatic skin. In normal skin, CD55 and CD59 were strongly expressed on epithelium and vascular structures, whereas CD16 and CD58 were strongly expressed only on epithelium. The expression of all four GPI proteins was decreased in non-lesional psoriatic skin and virtually abolished in lesional psoriatic skin. A control transmembrane protein, CD46, was strongly expressed in normal and non-lesional psoriatic skin, and its expression was not significantly decreased in psoriatic lesions. The absence or reduction of GPI proteins was not seen in the lesions of several other inflammatory and proliferative diseases studied.

HLA associations in vitiligo patients in the Dutch population.

This study characterizes the HLA class I and class II antigens in a group of patients with vitiligo and a control group, both of Dutch descent. Earlier reports had shown a significant positive association with DR4 and a significant negative association with DR3. We found that, after correction for the broad antigens studied, only Cw7 and DR6 were significantly associated with vitiligo. The significant positive association of DR6 with vitiligo is interesting since vitiligo has an autoimmune component in its pathogenesis and DR6 may be a marker for high immune responsiveness.

Molecular heterogeneity of the fourth component of complement (C4) and its genes in vitiligo.

In view of evidence suggesting vitiligo is an autoimmune disease, we investigated whether vitiligo is associated with inherited deficiencies of the fourth (C4) and second (C2) component of complement and with certain human leukocyte antigens (HLA). Analysis of functional activities of C4 and C2 in sera of patients with vitiligo (n = 42) showed that 17% of them had a heterozygous C4 deficiency and 5% had a heterozygous C2 deficiency. In the normal control group (n = 30), 3% had a heterozygous C4 deficiency and none had a C2 deficiency. C4 typing by Western blot analysis showed the frequency of the C4A*Q0 allele in the vitiligo patient group to be close to normal. However, the frequency of one C4B*Q0 allele was three times higher, and that of two C4B*Q0 alleles five times higher in the vitiligo patient group than the reported frequencies in normal control groups. Southern blot analysis of Taq1 digests of DNA using C4 and 21-hydroxylase probes showed that two patients with two C4B*Q0 alleles had a deletion of a 21-OHA-C4B segment. In the other patients, having one or two C4B*Q0 alleles, these null alleles probably occurred due to a loss of C4 gene expression. HLA analysis did not show any allelic association of C4A*Q0 or C4B*Q0 with any HLA antigen in vitiligo, but confirmed the previous findings of a negative association with HLA-DR3 and a positive association with HLA-DR4. These results suggest that abnormalities of the C4B gene and the above-mentioned associations with HLA antigens may be some of the risk factors in vitiligo.