Arthropathy in hereditary haemochromatosis segregates with elevated erythrocyte mean corpuscular volume.

Objective: To evaluate the relationship between erythrocyte parameters and the presence or absence of arthritis in HFE C282Y homozygous hereditary haemochromatosis (HH) subjects compared to control groups of non-HH subjects with arthritis.Method: Erythrocyte and arthritis parameters [mean corpuscular volume (MCV) and mean cell haemoglobin (MCH)] were obtained from consecutive HH subjects (n = 119) who were referred for initial evaluation and management. For comparison, MCV and MCH values were collected from randomly selected non-HH subjects with rheumatoid arthritis (n = 100) and osteoarthritis (n = 100), consisting of equal numbers of men and women. Two other comparison groups comprised 16 men and women who were heterozygous for C282Y with arthritis, and 38 non-HH subjects with type 2 polyarticular osteoarthritis (T2POA).Results: MCV values were significantly higher in HH subjects with arthritis (95 ± 0.56 fL) than in HH subjects without arthritis (92.75 ± 0.50 fL, p = 0.037). HH subjects with or without arthritis demonstrated a higher mean MCV than the control groups of non-HH osteoarthritis (90.12 ± 0.46 fL, p < 0.001) and non-HH rheumatoid arthritis (90.94 ± 0.57 fL, p < 0.001). HH subjects with arthritis also demonstrated a higher MCV than heterozygous C282Y subjects with arthritis (93.18 ± 1.55 fL, p = 0.025) and non-HH subjects with a similar pattern of arthritis, notably T2POA (91.13 ± 0.50 fL, p < 0.01). An MCV of ≥ 97.85 fL provided a likelihood ratio of 2.2 for development of arthritis in HH subjects.Conclusion: This study demonstrated a relationship between elevated MCV and arthritis in incident cases of HH.

Factors effecting the measurement of tumor-associated MUC1 mucins in serum.

MUC1 epithelial mucins are produced by both normal and malignant epithelial cells. Serum proteins reactive with monoclonal antibodies against MUC1 mucins were studied using several techniques. Separation of proteins by native PAGE showed that anti-MUC1 core protein antibodies reacted with a high M(r) mucin but also with a 70-kD protein (p70) in normal women and women with ovarian cancer. After purification by gel filtration, p70 was not reactive in a double-determinant ELISA (CASA) and only weakly reactive in an inhibition assay. Serum CASA levels increased during pregnancy but p70 disappeared. Neuraminidase treatment of serum resulted in a greater increase in CASA in normal women and in ovarian cancer patients with low initial CASA than in ovarian cancer patients with high CASA and pregnant women (p < 0.05). These findings suggest that pregnancy and ovarian cancer-derived mucins are less heavily sialylated than mucin derived from normal tissues. An inhibition assay was developed which was more sensitive, but provided no diagnostic advantage over CASA. MUC1 is present in the serum of all women, reactivity in assays utilizing core protein antibodies is probably dependent not only on molar concentration but on the degree of exposure of peptide epitopes.

The use of mucin-specific monoclonal antibodies and lectins in the detection of tumor-associated serum markers in gynecological cancer.

The use of the mucin-specific lectin from Sambucus sieboldiana (SSAM) in the detection of tumor-associated serum antigens produced by patients with ovarian, cervical, and uterine cancer was investigated. Two-site assays were developed which used either SSAM or the MUC1 core protein-specific monoclonal antibody (mab) BC2 as capture, and biotinylated SSAM to detect bound mucin (SSAM and BC2SSAM assays respectively). These new assays were compared to the CA125 assay, and another assay for MUC1 (CASA), which utilizes the core protein reactive mabs BC2 and BC3. some asymptomatic women and patients with benign disease showed very high levels in the SSAM assay, while this was not the case in the other assays. When cutoff levels were set to exclude healthy women and patients with benign disease, the levels of detection in patients with ovarian cancer were 51% with CASA (> 6.7 units/ml), 71% with CA125 (> 250 units/ml), and 38% with BC2SSAM (> 8.6 units/ml). The levels of detection in cervical and uterine cancer patients were 28% and 25% with CASA, 0% and 8% with CA125, and 28% and 25% with BC2SSAM respectively. Of particular interest was the very different spectrum of reactivity observed with the CASA and BC2SSAM assays which use the same capture mab, indicating that each assay detects different glycoforms of the MUC1 mucin. Indeed, when used in combination, the CASA and BC2SSAM assays gave 62% of ovarian cancer patients, and 50% of cervical or uterine cancer patients with elevated marker levels. The additional use of BC2SSAM gave no advantage over the combined use of the CASA and CA125 assays in ovarian cancer, with 80% of patients detected, but the CASA/BC2SSAM combination was particularly useful in the cervical and uterine cancers due to the low level of detection with CA125. In fact, the additional use of CA125 gave no advantage over the CASA/BC2SSAM combination in these patients. Furthermore, the BC2SSAM assay may also be useful in monitoring patients with high preoperative BC2SSAM levels (> 10 units/ml), since this assay predicted recurrence in 5/5 cases, and was negative in all cases with no evidence of disease. Furthermore, the performance of this assay in monitoring these patients was equal or superior to CA125 and CASA.

Serum mucin antigens CASA and MSA in tumors of the breast, ovary, lung, pancreas, bladder, colon, and prostate. A blind trial with 420 patients.

BACKGROUND: The tumor markers CASA (cancer-associated serum antigen) and MSA (mammary serum antigen) have previously been shown to be useful in the clinical management of ovarian and breast carcinoma, respectively, but have not been assessed in other types of cancer. These assays were compared with carcinoembryonic antigen (CEA) and prostate-specific antigen (PSA) in a blind trial using sera from the Mayo Clinic-National Cancer Institute (NCI) Diagnostic Serum Bank. METHODS: CASA and MSA were assessed retrospectively in a blind trial using 465 serum samples from the Mayo Clinic-NCI Diagnostic Serum Bank representing malignant and benign disease of the breast, ovary, lung, pancreas, bladder, colon, and prostate and age-matched and gender-matched healthy control donors. CASA, MSA, and PSA levels were determined using commercially available kits, and CEA values and clinical details were later provided by the Mayo Clinic. RESULTS: CASA and MSA showed good reproducibility in 45 duplicate samples. CASA values were significantly elevated in the serum of patients with malignant tumors of the breast (44%), ovary (58%), lung (56%), prostate (48%), and bladder (54%), but not in those with benign conditions of these organs or pancreatic or colon cancer. MSA levels were only elevated significantly in cancers of the breast (52%) and ovary (58%). CASA showed significantly better sensitivity than either CEA (20%) or MSA (25%) in the detection of lung cancer, whereas CEA showed significantly superior detection of colon cancers (78%). CASA was not as sensitive as PSA in prostate cancer (48% versus 96%), but gave superior specificity in nonmalignant conditions of the prostate (93% versus 70%), although this was not statistically significant. CONCLUSIONS: The commercial CASA and MSA assays are reliable and reproducible tests for these tumor markers. In addition to ovarian cancer, CASA is also elevated significantly in many patients with breast, lung, prostate, and bladder cancer and has potential clinical use in patients with these tumors. The use of the MSA assay appears restricted to breast cancer.

Circulating tumour-associated mucin concentrations, determined by the CASA assay, in healthy women.

This investigation was undertaken to establish a reference range for tumour-associated MUC1 mucins in the serum of healthy women of the ages at risk for adenocarcinoma of the ovary and breast. Blood samples and clinical information were obtained from 5,000 women attending a breast screening mammography clinic. Data from women diagnosed with breast carcinoma and those subsequently diagnosed with other cancers were omitted from the reference range. Mucin concentrations were measured using the CASA assay which detects the protein core of MUC1 encoded mucins. Multiple linear regression analysis showed no effect on CASA concentrations by non-malignant changes to the breast, menopausal status, presence/absence of the reproductive tract, parity or history of hormone use. However, CASA concentrations were significantly increased in smokers (P < 0.001) and progressively increased with age (P < 0.001). These data show that these factors must be given consideration when setting upper limits of normal using MUC1 protein core binding assays.

The management of ovarian carcinoma is improved by the use of cancer-associated serum antigen and CA 125 assays.

BACKGROUND: The new tumor-associated mucin assay, cancer-associated serum antigen (CASA), was assessed with the CA 125 assay for use in the management of patients with epithelial ovarian cancer. METHODS: CASA and CA 125 were assessed retrospectively for use in (1) monitoring 28 patients with Stage 3 or 4 ovarian carcinoma during therapy, (2) predicting the outcome of 41 second-look laparotomies (SLL), and (3) predicting the survival outcome by measuring these levels after surgery but before chemotherapy in 65 patients with Stage 3 disease. RESULTS: Of 20 patients with recurrence after an initial response, the presence of CASA levels detected recurrence in 65% before clinical detection; CA 125, 50%; and the combination of CASA and CA 125, 80%. Six patients whose disease was in long-term remission did not have elevations of either marker. When used to predict the results of SLL, the positive predictive values of CASA and CA 125 were 77% and 100%, respectively. The negative predictive values for CASA and CA 125 were 71% and 66%, respectively. CASA detected 50% of positive SLL where microscopic disease only was found; the CA 125 test did not. Multivariate analysis of survival rates using levels of CASA and CA 125, age, residual disease, tumor type and grade, or the presence or absence of cisplatin in the chemotherapeutic regimen found that postoperative CASA levels ranked above all prognostic factors except age. CASA levels may be more accurate than surgical reporting of residual disease or they may define a subset of patients with biologically more aggressive ovarian carcinoma. CONCLUSIONS: The CASA test is sensitive to ovarian carcinoma, and both CASA and CA 125 are more useful when used in conjunction.

Comparative study of mammography and mammary serum antigen estimation for breast cancer screening.

OBJECTIVE: To compare mammary serum antigen (MSA) levels with mammography as a screening test for breast cancer. To determine the value of MSA testing to decrease the need for women to undergo mammography. DESIGN: A blind prospective comparison of MSA levels and mammography to detect breast cancer. SETTING: Royal Women's Hospital Breast Cancer Screening Clinic. Women were mainly self-referred. RESULTS: MSA levels had a wide range in normal women and women with mammography-detected breast cancer. Mean MSA levels in women with breast cancer reflected tumour volume, but a wide range was again seen. At 60% specificity, the sensitivity of an elevated MSA level for breast cancer was 63% for invasive cancer and zero for in-situ disease. MSA levels were modestly but significantly elevated in smokers over non-smokers. CONCLUSION: The MSA level is an insufficiently sensitive or specific marker to have a role in screening for breast cancer.

Preoperative discrimination between ovarian carcinoma, non-ovarian gynecological malignancy and benign adnexal masses using serum levels of CA125 and the polymorphic epithelial mucin antigens CASA, OSA and MSA.

Serum levels of the tumor associated antigens CA125, CASA, OSA and MSA were determined preoperatively in a non-consecutive series of patients with: invasive epithelial ovarian cancer (OC, n = 87), ovarian tumors of low malignant potential (LMP, n = 9), benign adnexal masses (BAM, n = 48) and other peritoneal and pelvic malignancies (n = 48). In addition, serum levels of CASA, OSA, and MSA were determined in 3477 asymptomatic well women. Ninety-eight percent of the asymptomatic women had CASA levels < 6.0 U ml-1, OSA levels < 5.5 U ml-1 and MSA levels < 80.0 U ml-1. Serum CA125 levels were> 35 U ml-1 in 89% of OC, in 44% of LMP, and in 23% of BAM. Serum CASA levels were> 6.0 U ml-1 in 58% of OC, in 0% of LMP, and in 0% of BAM. Serum OSA levels were> 5.5 U ml-1 in 61% of OC in 0% of LMP and in 4% of BAM. Serum MSA levels were> 80.0 U ml-1 in 56% of OC, in 11% of LMP, and in 10% of BAM. When cut-off levels were set to exclude all patients with BAM, the best discrimination from OC using a single assay was achieved using CASA (58%). However, a combination of CASA and CA125 gave positive levels in 69% of OC at levels which precluded BAM. All markers were also elevated in some colon cancers, cervical cancers, uterine cancers and other peritoneal malignancies. A combination of CA125 and CASA levels, obtained preoperatively may assist the general gynecologist in avoiding potentially difficult oncologic surgery.

Quantitative analysis of adenine nucleotides during the prelytic phase of cell death mediated by C5b-9.

The nucleated cell death mediated by C5b-9 depends on the extent of C fixation and parameters that affect the ability of the cell to eliminate C5b-9. When C5b-9 formation exceeds elimination, cell death can be initiated. High Ca2+ in the medium accelerates Ehrlich ascites cell death induced by a large number of C5b-9, whereas osmotic prevention of cell swelling has little effect in protecting Ehrlich cells from killing by C5b-9. In the present study, we investigated the interrelationship between intracellular Ca2+, intra- and extracellular adenine nucleotides, and mitochondrial membrane potential, to understand the mechanism of acute cell death induced by C5b-9. When Ehrlich cells carrying C5b-8 were exposed to C9, rapid and profound ATP depletion in the cell was observed before cell death. Leakage of the adenine nucleotides ATP, ADP, and AMP also began during the prelytic phase. Studies using digital imaging fluorescence microscopy showed that loss of mitochondrial membrane potential was noted immediately after C9 addition but before nuclear staining with propidium iodide. These findings suggest that an increase in intracellular Ca2+ through C5b-9 channels and loss of mitochondrial membrane potential may initiate rapid cell death. The prelytic leakage of ATP precursors may also contribute to cell death by decreasing nucleotide pools, because recovery of ATP production was observed after a similar degree of ATP loss in cells exposed to sublethal doses of KCN, in which ADP and AMP leakage was not present.

Role of complement C9 and calcium in the generation of arachidonic acid and its metabolites from rat polymorphonuclear leukocytes.

We have previously shown that antibody-sensitized mouse peritoneal macrophages release arachidonic acid (C20:4) and its oxygenated derivatives when treated with complement, and that the major part of the release depended on the terminal complement complexes (TCC). To further delineate the process(es) responsible for this release we have extended our studies to rat peritoneal polymorphonuclear leukocytes (PMNs). Experiments were performed with antibody-sensitized rat PMNs labeled with [3H]C20:4 and carrying the TCC, C5b-7, C5b-8 or C5b-9. In contrast to the results of other studies, production of leukotriene B4 (LTB4), the major radiolabeled derivative, was strictly dependent on the presence of C9. However, low levels of C20:4 and prostaglandins (PGs) were produced prior to the C5b-9 stage. Kinetic studies demonstrated that release of LTB4 was rapid; the initial release occurred within 4-6 min and a second rise in release coincided with cell death. Virtually all the LTB4 produced was released as we found no evidence of retention of intracellular LTB4 at either the C5b-8 or C5b-9 stages. In the absence of extracellular calcium, the release of LTB4 was completely abolished and the release of C20:4 and PGs was drastically reduced. [3H]C20:4-labeled PMNs carrying C5b-9 did release substantial amounts of radiolabeled material in the presence of EGTA; however, the majority of this lipid was in the form of intact phospholipid and triglyceride. These results indicate that release of C20:4 and its oxygenated derivatives from rat PMNs is (1) dependent on the participation of C9 in the preexisting C5b-8 complex in the cell membrane, and (2) largely dependent on the presence of calcium.

Release of arachidonic acid and formation of oxygenated derivatives after complement attack on macrophages: role of channel formation.

Treatment of [3H]arachidonic acid ([3H]C20:4)-labeled, antibody-sensitized mouse resident peritoneal macrophages with rabbit serum complement, or C6-deficient rabbit serum + C6, caused hydrolytic release of incorporated [3H]C20:4 from phospholipids, followed by conversion to oxygenated derivatives. The C6 dose-response curve for release of C20:4 plus its metabolites was monotonic, which indicates dependence on channel formation, whereas the dose-response curve for lysis displayed multi-hit behavior. High-performance liquid chromatography demonstrated that the major radiolabeled products in the aqueous phase co-eluted with C20:4, 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), and prostaglandin E2. Kinetic studies of the release of 6-keto-PGF1 alpha, the major metabolite, displayed biphasic characteristics; a moderate amount of this prostaglandin was released before the onset of cell lysis. Experimental evidence obtained by freeze-thaw or by incubation of these cells with melittin or A23187 indicated that cell lysis does not necessarily result in the production of inflammatory mediators. Furthermore, when macrophages were treated with serum complement, it was apparent that the major part of the release was due to C5b-9 and not to the action of C5a. We conclude that release of C20:4 and its derivatives from complement-treated macrophages does not depend on cytolysis, but is a consequence of insertion and channel formation.

Complement lysis of U937, a nucleated mammalian cell line in the absence of C9: effect of C9 on C5b-8 mediated cell lysis.

Previous studies have demonstrated that in general, nucleated cells are more resistant to killing by serum complement than are erythrocytes. During studies aimed at defining the mechanisms of nucleated cell resistance, we found that the human histiocytic cell line U937 was easily lysed by homologous serum. U937 cells were also killed by serum depleted of C9, but not by serum depleted of C8, implying that the C5b-8 complex was sufficient to cause lysis of these cells. Enumeration of complexes on the cell surface demonstrated that approximately 40-fold more complexes were required to lyse U937 cells in the absence of C9 than in the presence of an excess of C9. Examination of the effects of small amounts of C9 on lysis of U937 cells by the C5b-8 complex demonstrated that at very low doses, C9 inhibited C5b-8 mediated lysis. The use of radiolabeled anti-C8 antibody showed that C5b-8 complexes were eliminated from the surface of U937 cells at 37 degrees C, and C9 at the dose causing inhibition of lysis accelerated the elimination of complexes. These results suggest that the increased lytic potential resulting from binding of small amounts of C9 to C5b-8 complexes is outweighed by enhanced elimination of complexes resulting in decreased cell death.

The relationship between channel size and the number of C9 molecules in the C5b-9 complex.

We have recently shown by dose-response analyses with resealed erythrocyte ghosts that the channel formed by complement is a monomer of C5b-9 of the composition C5b61C71C81C9n, in which n = 1 for channels permitting passage of sucrose (0.9 nm molecular diameter) and n = 2 for channels allowing transit of inulin (3 nm molecular diameter) (1). We have now continued these experiments and expanded them by including ribonuclease A (molecular diameter, 3.8 nm) as a marker to assess whether additional C9 molecules enlarge the functional C5b-9 channel. Our results show that formation of C5b-9 channels displays one-hit characteristics with respect to C5b6 when tested by transmembrane passage of inulin or ribonuclease A. By contrast, analysis of dose-response curves of C9 indicate that n = 2-3 for channels allowing transit of inulin and n = 4 for channels allowing transit of ribonuclease A. We have also performed sieving experiments with ghosts carrying C5b-7 and containing two small markers, inositol and sucrose. Dose-response curves for C8 were performed in the presence of excess C9 to ensure conversion of all C5b-8 to C5b-9 channels. The results indicate that small channels (approximately 0.8 nm effective diameter) are not formed at high C9 multiplicity, thus confirming the results obtained with the larger markers, i.e., increase of C9 input leads to formation of larger channels.

Penetration of C8 and C9 in the C5b-9 complex across the erythrocyte membrane into the cytoplasmic space.

We have developed a technique in which transglutaminase is used to measure the penetration of terminal complement proteins across the erythrocyte membrane into the cytoplasmic space. Penetration of a given terminal complement protein into the cytoplasmic space was assessed by labeling the protein of interest with radioactive iodine, forming the complement channel using the labeled protein, adding transglutaminase to only one side of the membrane, and allowing the enzyme to cross-link the susceptible proteins on that side of the membrane. Cross-linking was assessed by measuring the increase in molecular weight of the appropriate molecule on sodium dodecyl sulfate gels under reducing conditions. The results of these experiments indicate that C8 and C9 are rapidly cross-linked to high molecular weight from either the interior or the exterior of the membrane. In order to determine whether the cross-linking mediated by enzyme on the interior was occurring from within the ghosts and not via enzyme that had leaked into the extracellular medium, experiments were performed with dimethylcasein in the extracellular medium. In the presence of this protein, cross-linking of C8 and C9 from outside was negligible. Hence, if cross-linking occurs when transglutaminase is trapped inside the ghosts, it cannot be due to leakage of enzyme, but must be attributable to cross-linking from the inside. The results show that C9 definitely penetrated across the membrane into the intracellular space. With respect to C8, statistical evaluation indicates that C8 probably penetrated into the intracellular space.

Complement lysis of nucleated cells: effect of temperature and puromycin on the number of channels required for cytolysis.

We have previously shown that lysis of a nucleated mammalian cell requires several complement channels unlike lysis of erythrocytes and that this difference is due primarily to rapid elimination of channels from the plasma membrane. We have now investigated this problem further by studying the rate of channel elimination at low temp, the osmotic fragility of the cells, and the effectiveness of the membrane-associated ion pumps. When complement channels were formed for 3 min at 37 degrees C, followed by prolonged incubation at 2 degrees C, the C6 lytic dose-response curves indicated that a single channel was required for lysis of a cell, whereas multiple channels were required when the entire process was carried out at 30 degrees C. The shift from multi- to one-hit lytic behavior can be explained by the drastic reduction in the rate of channel elimination at low temp. C6 lytic dose-response curves with puromycin-treated cells were also found to display one-hit behavior, but, in this case, the rate of channel elimination was reduced only about 35-40% (which would not suffice to explain the one-hit lytic characteristics). However, cell death was more extensive for puromycin-treated cells than normal cells after incubation in buffers of low ionic strength, suggesting that an increase in osmotic fragility may be a contributing factor in the shift from multi- to one-hit behavior. Blocking of the membrane-associated Na+/K+-ATPases with ouabain did not affect the multi-channel requirement. Presumably, this means that the ion pumping rate does not significantly influence the number of channels required for lysis.

Elimination of complement channels from the plasma membranes of U937, a nucleated mammalian cell line: temperature dependence of the elimination rate.

We have studied the release of radiolabeled small markers from nucleated cells carrying complement channels in order to determine the life-span of these channels at various temperatures. U937 cells, a human histiocytic cell line, were labeled with 14C-aminoisobutyric acid or 86RbCl, and treated with sublytic doses of C to form transmembrane channels. The cells were then incubated at various temperatures, and the persistence of channels was evaluated by measuring the release of the intracellular markers through the remaining channels. The results indicate that the life-span of the C channels in the plasma membranes of these cells varies markedly with temperature. Thus, at 2 degrees C, the half-life of the channels was about 2 hr, whereas at 37 degrees C, the half-life was estimated to be approximately 1 min. The rapid elimination of the transmembrane channels from the plasma membranes of these nucleated cells contrasts sharply with the long persistence of C channels in the membranes of erythrocytes or erythrocyte ghosts. It is likely that the multi-hit requirement recently reported for lysis of nucleated mammalian cells by C is due, at least in part, to the rapid disappearance of channels.

Cytolysis of nucleated cells by complement: cell death displays multi-hit characteristics.

Lysis of nucleated cells by complement was studied to determine whether the lytic process by C5b-9 conforms to a one-hit mechanism as in the case of erythrocytes. Two nucleated cell lines, Molt 4 and U937, derived from human T lymphocytes and histiocytes, respectively, were employed as targets. The antibody-sensitized cells were used to develop the titration curves, measuring cell death as a function of limiting quantities of human C6 or C5,6 complex in the presence of an excess of other complement components. The cytolysis curves generated in both experiments were sigmoidal, in sharp contrast to the monotonic curves observed in lysis of erythrocytes treated similarly. The sigmoidal curves of cytolysis indicate a cooperative action of several molecules of C6 or acid-activated C5,6 complex, C(56)a. In contrast to the multi-hit characteristics of cytolysis, dose-response measurements of the release of 86Rb indicated that only one effective molecule of C6 per cell is required for assembly of a 86Rb-releasing channel. This divergence indicates that lysis requires formation of several channels or, alternatively, assembly of large channels that are formed by several molecules of C6. Because prior studies with erythrocyte ghosts have shown that only a single effective molecule of C6 is required for assembly of a transmembrane channel, regardless of size, we prefer to interpret the multi-hit characteristics of nucleated cell lysis as an indication of a multi-channel requirement, rather than channel enlargement.

Size distribution and stability of the trans-membrane channels formed by complement complex C5b-9.

We have performed double marker sieving experiments with molecules ranging from ca. 0.5-3 nm dia. in order to evaluate the size distribution of the channels formed by complement in resealed sheep erythrocyte ghosts. Evidence is presented that marker release through the channels reached equilibrium between the ghosts and the extracellular fluid in a period of 3 hr and that the channels are stable at 37 degrees C for this period of time. Under these experimental conditions we have observed a differential in the endpoint release of inositol and sucrose, which indicates that some of the ghosts carried channels measuring between 0.7 and 0.9 nm dia. No differential was observed between release of sucrose and raffinose (0.9 and 1.1 nm mol. dia., respectively). Comparisons between sucrose and inulin (0.9 and 3 nm mol. dia, respectively) showed a difference in marker release. Also, there was substantial release of inulin, indicating the presence of channels above 3 nm in dia. Hence, the present data indicate formation of channels in three size ranges, namely, 0.7-0.9 nm, 0.9-3 nm and greater than 3 nm.

Size of the transmembrane channels produced by complement proteins C5b-8.

It has been shown previously that erythrocytes can be lysed by complement proteins C5b-8, albeit at a much lower rate than C5b-9. We have now performed kinetic sieving experiments with resealed erythrocyte ghosts using sucrose (0.9 nm molecular diameter) and inulin (3.0 nm molecular diameter) as markers. We found that treatment of the ghosts with C5b-8 released sucrose, but not inulin. Addition of C9 to ghosts carrying C5b-8 dramatically increased the rate of sucrose flux and, in addition, caused release of inulin. Hence, unlike C5b-9 channels, those formed by C5b-8 measure less than 3 nm in diameter. Formation of C5b-8 channels was very slow compared with that of C5b-9 channels. Also, we found that about two-thirds of the C5b-8 ghosts did not have sucrose-releasing channels, but such channels were formed on reaction with C9.

Transmembrane channel formation by complement: functional analysis of the number of C5b6, C7, C8, and C9 molecules required for a single channel.

Earlier studies have shown that sequential treatment of resealed erythrocyte ghosts with C5b6, C7, C8, and C9 leads to insertion of hydrophobic peptides from these complement proteins into the membrane and assembly of transmembrane channels. The number of molecules of each of the proteins required for assembly of the membrane-associated channel structure was evaluated by measuring the quantitative relationship between the doses of the individual proteins and the release of two trapped markers, sucrose and inulin, from ghosts after channel formation. The incubation period was sufficient to attain equilibrium of marker distribution between the ghosts and the extracellular fluid. Two markers of different size (sucrose and inulin, 0.9 and 3 nm molecular diameter, respectively) were used in order to develop information on the molecular composition of small and large channels, respectively. We found that participation of C5b6, C7, and C8 in channel formation displayed one-hit characteristics, regardless of marker size. By contrast, the participation of C9 was one-hit with respect to the sucrose marker, whereas with respect to the inulin marker the C9 reaction was multi-hit. Our results are compatible with the view that these markers are released through a channel structure in the membrane that is a monomer of C5b--9 of the composition C5b61 C71C81C9n, in which n = 1 for channels permitting passage of sucrose and n = 2 for channels allowing transit of inulin.