The interleukin-6-hepcidin-hemoglobin circuit in systemic lupus erythematosus flares.

Objective Hepcidin regulates iron availability and may be responsible for the anemia of chronic disease because it is induced by interleukin-6. This study investigated the IL-6-hepcidin-hemoglobin axis in patients with lupus. Methods IL-6 and hepcidin were measured in serial serum samples collected before, during and after lupus flares by specific ELISAs. Results During renal and non-renal SLE flare cycles IL-6 did not predict hepcidin and hepcidin did not predict hemoglobin. When lupus nephritis patients were in remission, IL-6 and hepcidin were correlated, but hepcidin and hemoglobin were not. Conclusion Hepcidin does not contribute significantly to anemia during active lupus.

Evidence that abnormally large seasonal declines in vitamin D status may trigger SLE flare in non-African Americans.

Cross-sectional studies have shown that low vitamin D (25-hydroxyvitamin D (25(OH)D)) is associated with increased systemic lupus erythematosus (SLE) activity. This study is the first to assess the temporal relationship between 25(OH)D levels and onset of SLE flare. This assessment was made possible because of the specimen bank and database of the Ohio SLE Study (OSS), a longitudinal study of frequently relapsing SLE that involved regular bimonthly patient follow-up. We identified for this study 82 flares from 46 patients that were separated by at least 8 months from previous flares. Serum 25(OH)D levels were measured at 4 and 2 months before flare, and at the time of flare (a flare interval). We found that for flares occurring during low daylight months (LDM, Oct-Mar), 25(OH)D levels were decreased at the time of flare, but only in non-African American (non-AA) patients (32% decrease at flare, compared to 4 months prior, p < 0.001). To control for seasonal effects, we also measured 25(OH)D levels in the LDM "no-flare" intervals, which were intervals that matched to the same calendar months of the patients' LDM flare intervals, but that didn't end in flare (n = 24). For these matches, a significant decrease occurred in 25(OH)D levels during the flare intervals (18.1% decrease, p < 0.001), but not during the matching no-flare intervals (6.2% decrease, p = 0.411). For flares occurring during high daylight months (HDM), 25(OH)D levels changed only in non-AA patients, increasing slightly (5.6%, p = 0.010). Analysis of flare rates for the entire OSS cohort (n = 201 flares) revealed a tendency for higher flare rates during LDM compared to HDM, but again only in non-AA patients (p = 0.060). Flare rates were lower during HDM for non-AA patients compared to AA patients (p = 0.028). In conclusion, in non-AA SLE patients, unusually large declines in 25(OH)D during LDM may be mechanistically related to SLE flare, whereas relatively high 25(OH)D levels during HDM may protect against flare.

Vitamin D deficiency as marker for disease activity and damage in systemic lupus erythematosus: a comparison with anti-dsDNA and anti-C1q.

OBJECTIVES: To study the sensitivity and specificity of vitamin D deficiency for predicting disease activity and damage of systemic lupus erythematosus (SLE) in comparison with anti-dsDNA and anti-C1q. METHODS: Consecutive patients who fulfilled four or more ACR criteria for SLE were studied. Levels of 25-hydroxyvitamin D3, anti-C1q, anti-dsDNA and complement levels were measured. Relationship among these markers, concurrent disease activity and damage scores of SLE was studied by Spearman's rank correlation method. RESULTS: In total, 290 SLE patients were studied (95% women; mean age 38.9 ± 13.1 years; SLE duration 7.7 ± 6.7 years). Clinical or serological lupus activity (SLEDAI ≥ 1) was present in 225 (78%) patients. Vitamin D deficiency (< 15 ng/ml) was detected in 78 (27%) patients. Levels of 25-hydroxyvitamin D3 correlated inversely with the clinical SLE disease activity score (Rho = -0.26; p < 0.001). A negative correlation was also observed between 25-hydroxyvitamin D3 and anti-dsDNA levels (Rho = -0.13; p = 0.02), or anti-C1q (Rho = -0.14; p = 0.02). However, there was no significant relationship between levels of 25-hydroxyvitamin D3 and complement C3 (Rho = 0.09; p = 0.12) or C4 (Rho = 0.09; p = 0.13). Both 25-hydroxyvitamin D3 deficiency and anti-C1q were more specific but less sensitive than anti-dsDNA for concurrent clinical renal and non-renal SLE activity. Levels of 25-hydroxyvitamin D3, anti-dsDNA or anti-C1q did not correlate significantly with the SLE damage scores. CONCLUSIONS: 25-hydroxyvitamin D3 correlated inversely and significantly with clinical SLE activity, anti-C1q and anti-dsDNA titers, but not with complement levels or damage scores. Deficiency of 25-hydroxyvitamin D3 was as specific as anti-C1q, but less sensitive than anti-dsDNA, for detecting concurrent renal and non-renal clinical activity of SLE.

The complex nature of serum C3 and C4 as biomarkers of lupus renal flare.

To assess the relationship between serum C3 or C4 levels and lupus renal flare, C3 and C4 levels were measured bimonthly in 71 lupus nephritis patients for a mean of 35 months, during which time 70 renal flares were identified. Comparing baseline, pre-flare, and at-flare values indicated that neither C3 nor C4 levels decreased pre-flare, but both decreased on average significantly at flare. However, sensitivity/specificity for C3 (75%/71%) and C4 (48%/71%) were low. To account for other influencing factors, multiple regression was performed that included bimonthly values of C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR), and genotype data on C3 (S/F), CRP (1846G > A), and the complement regulator factor H (Y402H). This analysis revealed that reduced levels of C4, but not C3, were independently associated with the two-month pre-flare period. Conversely, reduced levels of C3, but not C4, were independently associated with the flare visit. Significant pro-flare interactions included low C3 levels with the factor H 402HH-encoding genotype, and low CRP levels with the C3 F allele. Together these data suggest that C4 activation is critical for initiating renal flare while C3 activation is involved in the actual tissue damage, and that these effects are influenced by genetic variability in complement activation and regulation.

Phenotypes, genotypes and disease susceptibility associated with gene copy number variations: complement C4 CNVs in European American healthy subjects and those with systemic lupus erythematosus.

A new paradigm in human genetics is high frequencies of inter-individual variations in copy numbers of specific genomic DNA segments. Such common copy number variation (CNV) loci often contain genes engaged in host-environment interaction including those involved in immune effector functions. DNA sequences within a CNV locus often share a high degree of identity but beneficial or deleterious polymorphic variants are present among different individuals. Thus, common gene CNVs can contribute, both qualitatively and quantitatively, to a spectrum of phenotypic variants. In this review we describe the phenotypic and genotypic diversities of complement C4 created by copy number variations of RCCX modules (RP-C4-CYP21-TNX) and size dichotomy of C4 genes. A direct outcome of C4 CNV is the generation of two classes of polymorphic proteins, C4A and C4B, with differential chemical reactivities towards peptide or carbohydrate antigens, and a range of C4 plasma protein concentrations (from 15 to 70 mg/dl) among healthy subjects. Deliberate molecular genetic studies enabled development of definitive techniques to determine exact patterns of RCCX modular variations, copy numbers of long and short C4A and C4B genes by Southern blot analyses or by real-time quantitative PCR. It is found that in healthy European Americans, the total C4 gene copy number per diploid genome ranges from 2 to 6: 60.8% of people with four copies of C4 genes, 27.2% with less than four copies, and 12% with more than four copies. Such a distribution is skewed towards the low copy number side in patients with systemic lupus erythematosus (SLE), a prototypic autoimmune disease with complex etiology. In SLE, the frequency of individuals with less than four copies of C4 is significantly increased (42.2%), while the frequency of those with more than four copies is decreased (6%). This decrease in total C4 gene copy number in SLE is due to increases in homozygous and heterozygous deficiencies of C4A but not C4B. Therefore, it is concluded that lower copy number of C4 is a risk factor for and higher gene copy number of C4 is a protective factor against SLE disease susceptibility.

Spot urine protein/creatinine ratios are unreliable estimates of 24 h proteinuria in most systemic lupus erythematosus nephritis flares.

The diagnosis of glomerulonephritis flares in systemic lupus erythematosus (SLE) is usually based on whether the magnitude of proteinuria has changed. Our study tests two methods to assess proteinuric change: protein/creatinine (P/C) ratios of intended 24-h urine collections or that of spot urine samples. Sixty-four patients with glomerulonephritis due to SLE followed in the Ohio SLE Study provided bimonthly paired spot and intended 24-h urine collections. Completeness of each collection was estimated as the ratio of the measured creatinine to the expected creatinine based upon Cockroft-Gault. Intended 24-h urine collections with measured/expected creatinine ratios between 0.5 and 0.9 (237 samples overall) showed ratios that were not significantly different from ratios of complete 24-h urine collections with ratios of 0.9-1.1 (159 samples). To compare spot and 24 h P/C ratios, we randomly selected pairs of samples with measured/expected ratios above 0.75. Consistent with previous studies, spot and 24-h urine P/C ratios showed good correlation over the range of values as well as reasonably strong concordance. Over the range of most SLE glomerulonephritis flares, however, correlation was present but concordance was poor. Our work suggests that the use of spot urine P/C ratios will yield more false-positive and -negative diagnoses of glomerulonephritis flares in patients with SLE than the ratio in 24-h urines.

Consumption of erythrocyte CR1 (CD35) is associated with protection against systemic lupus erythematosus renal flare.

Erythrocyte complement receptor type one (E-CR1) is thought to protect against immune complex (IC) disease through interactions that lead to E-CR1 consumption, and low E-CR1 levels are characteristic of systemic lupus erythematosus (SLE). The purpose of this study was to test the hypothesis that E-CR1 consumption can predict or mark SLE flare. Recurrently active SLE patients [n = 43; 28 with past or present major renal manifestations (SLER) and 15 without (SLENR)], were evaluated every 2 months by detailed protocol testing (mean follow-up 22 months), including direct measurements of E-CR1 levels using a radioimmunoassay. In all patients, detectable E-CR1 levels fluctuated widely through acute periods of consumption and regeneration, preventing the use of any single value as a baseline. However, when individual chronic baseline values were used, determined as the mean of all E-CR1 values 4 months or more from a flare, a clear trend was observed. In 16 of 16 instances of non-renal flare in SLER patients, E-CR1 levels decreased at flare (mean decrease 34%, P < 0.0001). In contrast, no consistent difference was observed for flare in SLENR patients or for renal flare in SLER patients. Changes in E-CR1 levels did not correlate with plasma CR1 levels. In conclusion, single occurrences of E-CR1 consumption did not generally predict or mark SLE flare. However, compared to the average E-CR1 levels measured during no-flare intervals, E-CR1 consumption in SLER patients at flare was strongly associated with freedom from signs of renal involvement. We postulate that E-CR1 consumption reflects E-CR1 function that includes protecting against SLE nephritis.

Identification of the Kna/Knb polymorphism and a method for Knops genotyping.

BACKGROUND: DNA mutations resulting in the McCoy and Swain-Langley polymorphisms have been identified on complement receptor 1 (CR1)-a ligand for rosetting of Plasmodium falciparum-infected RBCs. The molecular identification of the Kna/Knb polymorphism was sought to develop a genotyping method for use in the study of the Knops blood group and malaria. STUDY DESIGN AND METHODS: CR1 deletion constructs were used in inhibition studies of anti-Kna. PCR amplification of Exon 29 was followed by DNA sequencing. A PCR-RFLP was developed with NdeI, BsmI, and MfeI for the detection of Kna/Knb, McCa/McCb, and Sl1/Sl2, respectively. Knops phenotypes were determined with standard serologic techniques. RESULTS: A total of 310 Malian persons were phenotyped for Kna with 200 (64%) Kn(a+) and 110 (36%) Kn(a-). Many of the Kn(a-) exhibited the Knops-null phenotype, that is, Helgeson. The Kna/b DNA polymorphism was identified as a V1561M mutation with allele frequencies of Kna (V1561) 0.9 and Knb (M1561) 0.1. CONCLUSION: The high frequency (18%) of Knb in West African persons suggests that it is not solely a Caucasian trait. Furthermore, because of the high incidence of heterozygosity as well as amorphs, accurate Knops typing of donors of African descent is best accomplished by a combination of molecular and serologic techniques.

Of mice and men: the relevance of the mouse to the study of human SLE.

A number of mouse models have been utilized to study the pathophysiology of immune complex (IC) disease, and the hallmark IC disease systemic lupus erythematosus (SLE). Many of these studies have provided exciting new insights into IC-mediated inflammation and autoimmunity. However, numerous differences exist between mice and humans that suggest that mouse studies are not always applicable to human disease. These differences can be found in the biological systems that interact with circulating IC, in the specifics of disease presentation, and in the general physiology of the two species. Furthermore, although the mechanisms of SLE-like autoimmune disease in the mouse are being defined through analyses of the murine models of SLE, it remains to be proven that these mechanisms are relevant to human SLE. Thus, generalizing the results of the mouse studies to human SLE and other human IC diseases must be done with caution.

CR1 and CR1-like: the primate immune adherence receptors.

Immune adherence describes the phenomenon in which complement-opsonized substrates, such as immune complexes (IC), viruses, or bacteria, are bound by primate erythrocytes via erythrocyte complement receptors. In vivo studies have shown that this binding allows the erythrocyte to act as an inert shuttle, targeting IC to the monocyte phagocytic system and away from vulnerable tissue. Thus, immune adherence appears to play an integral role in the primate in promoting the safe clearance of circulating IC and preventing IC-mediated pathologies. The complement receptors that mediate immune adherence comprise two unique but closely related gene products, either the type one complement receptor (CRI) in humans or CRI-like in non-human primates. This review focuses on the structure, function, and physiological role of the primate immune adherence receptors.

Molecular identification of Knops blood group polymorphisms found in long homologous region D of complement receptor 1.

Complement receptor 1 (CR1) has been implicated in rosetting of uninfected red blood cells to Plasmodium falciparum-infected cells, and rosette formation is associated with severe malaria. The Knops blood group (KN) is located on CR1 and some of these antigens, ie, McCoy (McC) and Swain-Langley (Sl(a)), show marked frequency differences between Caucasians and Africans. Thus, defining the molecular basis of these antigens may provide new insight into the mechanisms of P falciparum malaria. Monoclonal antibody epitope mapping and serologic inhibition studies using CR1 deletion constructs localized McC and Sl(a) to long homologous repeat D of CR1. Direct DNA sequencing of selected donors identified several single nucleotide polymorphisms in exon 29 coding for complement control protein modules 24 and 25. Two of these appeared to be blood group specific: McC associated with K1590E and Sl(a) with R1601G. These associations were confirmed by inhibition studies using allele-specific mutants. A sequence-specific oligonucleotide probe hybridization assay was developed to genotype several African populations and perform family inheritance studies. Concordance between the 1590 mutation and McC was 94%; that between Sl(a) and 1601 was 88%. All but 2 samples exhibiting discrepancies between the genotype and phenotype were found to be due to low red cell CR1 copy numbers, low or absent expression of some alleles, or heterozygosity combined with low normal levels of CR1. These data further explain the variability observed in previous serologic studies of CR1 and show that DNA and protein-based genetic studies will be needed to clarify the role of the KN antigens in malaria.

The chimpanzee and cynomolgus monkey erythrocyte immune adherence receptors are encoded by CR1-like genes.

The human erythrocyte immune adherence (IA) receptor is the Mr 220,000 type one complement receptor, or CR1. Nonhuman primate IA receptors are comprised of a family of smaller erythrocyte complement receptors (E-CRs) of unknown origin. Recently, the Mr 65,000 baboon E-CR was identified as a glycophosphatidylinositol (GPI)-linked protein encoded by a partially duplicated CR1 gene termed CR1-like. The purpose of this study was to determine the genetic origin of the Mr 75,000 chimpanzee E-CR. Two previously identified cDNAs, an alternative splice product of CR1 termed CR1a and a chimpanzee form of CR1-like, were synthesized and amplified from chimpanzee bone marrow RNA, and transiently expressed in COS-7 cells. By SDS-PAGE, the CR1a protein had a relative mobility slightly greater than chimpanzee E-CR, whereas that of the CR1-like protein was slightly less. Affinity chromatography demonstrated that little chimpanzee CR1a bound to human C3i linked to activated thiol-Sepharose (C3i-ATS), while over 50% of both chimpanzee CR1-like and chimpanzee E-CR bound to C3i-ATS. Treatment with phosphatidylinositol-specific phospholipase C (PIPLC) to assess GPI linkage released E-CR from chimpanzee erythrocytes, and E-CR from cynomolgus monkey erythrocytes. Based on size, ligand-binding specificity, and PIPLC sensitivity, we conclude that the chimpanzee E-CR is encoded by the CR1-like gene. Furthermore, based on PIPLC sensitivity, the cynomolgus monkey E-CR is also likely encoded by a CR1-like sequence. Thus, CR1-like, which is a genetic element of unknown significance in humans, is the gene that encodes the erythrocyte IA receptor of many nonhuman primates.

Effects of immune complex formation and complement activation on circulating platelets in the primate.

Primate platelets are different from rodent and rabbit platelets in that they do not express receptors for C3a or C5a or immune adherence receptors. This study assessed the effects of immune complex (IC)-induced complement activation on primate platelets in the circulation. Cynomolgus monkeys (CYN, N = 4) immunized to bovine gamma globulin (BGG) were infused with BGG over 5 min to induce acute intravascular IC formation and complement activation. The studies were carried out under normal complement conditions (N = 12), partial complement inhibition (CAB-2 treated, N = 3), or total complement inhibition (CVF treated, N = 1). Under normal complement conditions, BGG infusion increased C3a levels from undetectable to an average of 11.9 +/- 2.6 micrograms/ml. At this time, decreases occurring in both circulating neutrophils (85 +/- 6%) and monocytes (78 +/- 6%) were significantly greater than decreases in circulating platelets (13 +/- 3%, p < 0.001). Partial complement inhibition had an equivocal effect on the BGG-induced changes in circulating leukocytes, while total complement inhibition abrogated these changes. In contrast, platelet changes were unaffected by complement inhibition. We conclude that, compared to circulating leukocytes, circulating platelets are insensitive to intravascular complement activation induced by IC in the nonhuman primate. These results contrast with previous studies in rodents which demonstrate strong effects of IC-induced intravascular complement activation on both circulating neutrophils and platelets.

Prevention of acute murine cardiac allograft rejection: anti-CD4 or anti-vascular cell adhesion molecule one monoclonal antibodies block acute rejection but permit persistent graft-reactive alloimmunity and chronic tissue remodelling.

We treated C57BL/6 mouse recipients of DBA/2 cardiac allografts with anti-CD4 monoclonal antibodies (mAb) or anti-vascular cell adhesion molecule 1 mAb to promote long-term allograft survival and subjected both the recipient animals and the long-surviving allografts to a battery of histologic and immunologic tests. The results were similar regardless of the mAb used for antirejection therapy. At all tested times after transplantation, the allografts displayed histologic evidence of ongoing microvascular endothelial activation and interstitial leukocytic infiltration. Reverse transcription polymerase chain reaction analyses revealed continuous intragraft expression of messenger RNA for interleukin 1, interleukin 2, interleukin 4, interleukin 6, tumor necrosis factor, interferon gamma, and transforming growth factor beta. All grafts had histologic evidence of ongoing vascular and parenchymal tissue remodeling, including interstitial fibrosis and vascular neointimal hyperplasia. The graft recipients retained limiting dilution analysis--detectable, donor-reactive cytolytic T lymphocyte, and helper T lymphocyte in their spleens and produced high liters of donor-reactive alloantibodies. Variable amounts of allogeneic microchimerism were detectable in some, but not all of the long-surviving graft recipients. In general, these observations indicate that (1) a similar immune status is achieved in long-surviving allografts and their recipients when either anti-CD4 mAb or anti-vascular cell adhesion molecule-1 mAb was used for antirejection therapy, in spite of the major differences in lineage and distribution of cells targeted by these two mAbs, (2) this immune status is characterized by continuous, long-term inflammatory and immune processes very similar to those observed during acute allograft rejection, and (3) in spite of these processes the allografts continue to function, although they invariably develop a chronic rejection-like histopathologic condition that may ultimately limit graft function. In this regard, the recipients of long-surviving allografts do not seem to be tolerant of their graft alloantigens.

Effect of enalapril therapy on glomerular accumulation of immune complexes and mesangial matrix in experimental glomerulonephritis in the nonhuman primate.

The present study is a prospective, controlled, blinded trial of enalapril therapy in experimental immune complex (IC)-mediated glomerulonephritis (GN) in the nonhuman primate (cynomolgus monkey [CYN]). Two groups of CYNs were studied: those with established GN (study A) and those in which GN was being induced (study B). In study A, 12 CYNs had GN established by 8 or 10 weeks of daily intravenous infusion of bovine gamma-globulin (BGG). These CYNs were then assigned to either 4 weeks of daily oral enalapril therapy (n = 6) or daily oral placebo therapy (n = 6). The daily BGG infusions were continued during the 4 weeks of enalapril or placebo therapy. At the start of the enalapril/placebo protocol, the two groups were similar with respect to proteinuria and level of precipitating antibody to BGG, which determined the daily BGG dose. Renal biopsy was performed in each CYN at the start and end of the 4-week period of enalapril/placebo protocol. In study B, 15 normal CYNs were immunized to BGG over a period of 4 weeks. The CYNs were then assigned to daily oral enalapril therapy (n = 8) or placebo therapy (n = 7) based on level of precipitating antibody to BGG. At this point, daily intravenous BGG was begun along with daily enalapril or placebo for 8 weeks. Renal biopsy was performed in each CYN before and at the end of this 8-week period. In study A, enalapril therapy was associated with a significant decrease in mesangial matrix volume (mean change, -27.7%; P = 0.031) and a trend toward decreased mesangial matrix deposits (mean change, -34.1%; P = 0.188). By contrast, in CYNs receiving placebo therapy, mesangial matrix volume increased compared with the enalapril group (P = 0.002) and mesangial deposits were unchanged. In study B, both the enalapril and placebo groups showed significant increases in mesangial matrix volume, mesangial deposits, mesangial cell volume, and capillary wall deposits during the 8 weeks of daily BGG infusion. However, none of the differences between the groups achieved statistical significance. Changes in mesangial cell volume and capillary wall deposits were also evaluated in study A and study B, but were not found to be different between the enalapril and placebo groups. In both study A and study B, blood pressure was lower in the enalapril groups. In conclusion, in the initial phase of IC-GN induction (0 to 8 weeks), enalapril therapy does not significantly influence the glomerular accumulation of mesangial matrix or immune deposits. However, in established IC-GN (after 8 weeks of GN induction), enalapril therapy significantly decreases the further accumulation of mesangial matrix and may decrease the further accumulation of mesangial deposits. Whether this benefit of enalapril therapy was related to lower blood pressure or to other effects of angiotensin-converting enzyme (ACE) inhibition was not determined in this study.

The baboon erythrocyte complement receptor is a glycophosphatidylinositol-linked protein encoded by a homologue of the human CR1-like genetic element.

The human erythrocyte CA receptor (E-CR) is the type 1 complement receptor (CR1), the most common form of which is a 220,000 Mr integral membrane glycoprotein composed of 30 short consensus repeats (SCRs). The E-CR of many nonhuman primates is a smaller receptor of unknown genetic origin. Recently, we identified a chimp cDNA, termed CR1b, which represented transcription of a homologue of the human genetic element, CR1-like. The purpose of this study was to identify CR1b in the baboon and, if present, determine whether it encodes the 65,000 Mr baboon E-CR. Baboon bone marrow cDNA was amplified by PCR using primers specific for the signal peptide-encoding region of human CR1 and the 3' region of chimp CR1b. This amplification yielded a CR1b sequence predicted to encode seven SCRs followed by a hydrophobic region, with an N terminus homologous to the N terminus of baboon E-CR. Expression of baboon CR1b yielded a membrane protein that reacted with an anti-CR1 mAb, was identical in size to baboon E-CR, and, like baboon E-CR, could bind baboon C3 linked to activated thiol-Sepharose (C3i-ATS), but not human C3i-ATS. Phosphatidylinositol-specific phospholipase C (PIPLC) released CR1b from Chinese hamster ovary cells and E-CR from baboon erythrocytes, demonstrating that both of these proteins are glycophosphatidylinositol linked to the membrane. Thus, the data indicate that baboon CR1b, a homologue of the human CR1-like genetic element, encodes a glycophosphatidylinositol-linked protein that is the baboon E-CR.

Effect of chronic human recombinant erythropoietin therapy on antibody responses to immunization in chronic hemodialysis patients.

There are multiple lines of evidence suggesting that human recombinant erythropoietin (rEPO) could influence immune responses by direct effects of rEPO on T or B cells. The present study tested this hypothesis by measuring antibody responses after immunization to tetanus toxoid (TT, a T cell dependent antigen) or pneumococcal capsular polysaccharide antigen (PA, a T cell independent antigen). The patients chosen for this prospective study were chronic hemodialysis patients receiving chronic rEPO therapy, and a comparable group of chronic hemodialysis patients not receiving rEPO therapy. We found that the patients immunized with PA and receiving rEPO therapy (N = 15) had IgG anti-PA responses comparable to that of those not receiving rEPO therapy (N = 15). In contrast, in the patients immunized with TT, those receiving rEPO (N = 15) developed significantly higher IgG anti-TT levels than those not receiving rEPO (N = 14) (time-group interaction P = 0.005). The peak difference between these groups was at two weeks, where the rEPO-treated patients developed a 4.1-fold mean increase in IgG anti-TT level and those not receiving rEPO developed only a 1.4-fold mean increase in IgG anti-TT level (P < 0.01). The difference in immune response to TT in the rEPO compared to the non-rEPO-treated patients could not be explained by differences between the groups in any of the parameters measured at baseline or during the post-immunization period. In conclusion, rEPO therapy increased immune response to TT but not PA, which suggests that rEPO enhances immune response to T cell dependent antigens.

Erythrocyte complement receptors.

Primate erythrocytes express complement receptors (E-CR), which can extrinsically bind C3b and Cb4. This interaction allows primate erythrocytes to bind complement opsonized particles and immune complexes, a phenomenon historically referred to as immune adherence. The binding of C3b and C4b by E-CR also leads to inhibition of complement activation. The human E-Cr is the complement activation. The human E-CR is the complement receptor type 1, or CR1, which is codominantly expressed as four polymorphic allotypes, ranging in size from 190,000 to 280,000 M(r). Non-human primate E-CR are similar to CR1 in function and antigenicity and are likely homologous to CR1 in structure; however, they are one third to one half the size of CR1. The physiological role of E-CR, determined from studies in monkeys and humans, is to allow erythrocytes to perform as inert shuttles for circulating immune complexes (IC), safely directing IC to the organs of the monocyte phagocytic system, thus preventing indiscriminate IC deposition in vulnerable tissue. In IC-mediated diseases, such as systemic lupus erythematosus (SLE), detectable erythrocyte CR1 levels are reduced, an abnormality that in part is acquired during disease activity. The loss of erythrocyte CR1 may be an important pathogenic factor in the development and severity of SLE.

Rate of antigen entry into the circulation in experimental versus naturally occurring immune complex glomerulonephritis.

This study tested the hypothesis that the rate of antigen entry into the circulation in systemic lupus erythematosus (SLE), a naturally occurring immune complex glomerulonephritis (IC-GN), is slow compared with that of traditional experimental models of IC-GN, in which the antigen is delivered rapidly as a daily iv bolus. This hypothesis was tested by comparing rates of decline of the third component of complement (C3) and of circulating neutrophils (PMN) in SLE patients with active disease with those of cynomolgus monkeys (CYN) undergoing induction of experimental IC-GN by means of a daily bolus infusion of antigen. It has previously been shown that, as antigen enters the circulation and forms circulating IC, C3 levels and circulating PMN decline acutely. Thus, acute changes in these parameters can be surrogates for the rate of antigen entry into the circulation. In the CYN undergoing induction of IC-GN (N = 11), infusion of the antigen (bovine gamma-globulin) over 10 min resulted in acute declines in C3 levels (25 +/- 6.6%; P = 0.0018 and PMN counts (59 +/- 9%; P < 0.0002). In addition, the CYN experienced the onset of acute respiratory distress and hypotension. By contrast, in patients with active SLE (N = 9), C3 and white blood cell counts measured at 24-h intervals did not change significantly, and episodes of acute hypotension or respiratory distress were not observed. In the CYN, the onset of visible vasculitic lesions in the omentum were also documented within minutes of the infusion of bovine gamma-globulin. The rapidity of onset of these vascular lesions suggests that the tempo at which lesions develop in experimental models of IC disease is faster than that of naturally occurring IC diseases. It was concluded that, in naturally occurring IC diseases, antigen probably enters the circulation slowly over prolonged periods of time, rather than as large boluses over short periods of time, as in traditional experimental models of IC-GN. Thus, models of IC-GN involving a daily bolus infusion of antigen may not be clinically relevant, particularly when IC clearing mechanisms are tested, because the efficiency of these mechanisms may be markedly influenced by the rate at which IC form in the circulation.

Effects of complement activation products on the synthesis of decay accelerating factor and membrane cofactor protein by human mesangial cells.

We previously demonstrated that activation of terminal complement components (C8 and/or C9) increases the synthesis and expression of decay accelerating factor (DAF) on human glomerular cells. DAF is a cell membrane-associated complement regulatory protein that inhibits complement activation on cell surfaces. In the present studies we evaluated, first, the mechanisms by which complement activation stimulates DAF synthesis, and second the effect of complement activation on the synthesis. and expression of membrane cofactor protein (MCP), another complement regulatory protein, by human mesangial cells (HMC) in culture. Complement activation by immune complexes resulted in increased DAF mRNA levels by at least two mechanisms: deposition of activated C3 on HMC and generation of soluble complement activation products, specifically C5a. The increase in DAF mRNA levels induced by activated C3 or C5a was short lived (less than 4 hr). In contrast, the up-regulation of DAF mRNA levels induced by activation of the complete complement cascade persisted for at least eight hours. The effect of complement activation on DAF mRNA levels was not affected by cycloheximide, a protein synthesis inhibitor. However, cycloheximide alone resulted in a significant up-regulation of DAF mRNA levels on HMC. In contrast to those findings complement activation did not cause an up-regulation of MCP mRNA, nor an increase in the synthesis of this protein. However, by FACS, complement produced a small but significant increase of MCP protein levels on HMC. In conclusion, both MCP and DAF are present on HMC. Several activated complement components are capable of increasing DAF mRNA levels, but DAF protein levels increase only after activation of the whole complement cascade.(ABSTRACT TRUNCATED AT 250 WORDS)