Antibody-coated Surface Research Articles

Ligation of Integrin α3β1 by Laminin 5 at the Wound Edge Activates Rho-dependent Adhesion of Leading Keratinocytes on Collagen

Wounding of the epidermis signals the transition of keratinocytes from quiescent anchorage on endogenous basement membrane laminin 5 to migration on exposed dermal collagen. In this study, we attempt to characterize activation signals that transform quiescent keratinocytes into migratory leading cells at the wound edge. Previously, we reported that adhesion and spreading on collagen via integrin alpha(2)beta(1) by cultured human foreskin keratinocytes (HFKs) requires RhoGTP, a regulator of actin stress fibers. In contrast, adhesion and spreading on laminin 5 requires integrins alpha(3)beta(1) and alpha(6)beta(4) and is dependent on phosphoinositide 3-hydroxykinase (Nguyen, B. P., Gil, S. G., and Carter, W. G. (2000) J. Biol. Chem. 275, 31896-31907). Here, we report that quiescent HFKs do not adhere to collagen but adhere and spread on laminin 5. By using collagen adhesion as one criterion for conversion to a "leading wound cell," we found that activation of collagen adhesion requires elevation of RhoGTP. Adhesion of quiescent HFKs to laminin 5 via integrin alpha(3)beta(1) and alpha(6)beta(4) is sufficient to increase levels of RhoGTP required for adhesion and spreading on collagen. Consistently, adhesion of quiescent HFKs to laminin 5, but not collagen, also promotes expression of the precursor form of laminin 5, a characteristic of leading keratinocytes in the epidermal outgrowth. We suggest that wounding of quiescent epidermis initiates adhesion and spreading of keratinocytes at the wound edge on endogenous basement membrane laminin 5 via alpha(3)beta(1) and alpha(6)beta(4) in a Rho-independent mechanism. Spreading on endogenous laminin 5 via alpha(3)beta(1) is necessary but not sufficient to elevate expression of precursor laminin 5 and RhoGTP, allowing for subsequent collagen adhesion via alpha(2)beta(1), all characteristics of leading keratinocytes in the epidermal outgrowth.

An optical tweezers-based immunosensor for detection of femtomoles-per-liter concentrations of antigens.

We used optical tweezers--optical trapping with focused laser beams--to pull microspheres coated with antigens off of an antibody-coated surface. Using this technique, we could quantify the force required to separate antigen to antibody bonds. At very low surface density of antigen, we were able to detect the single antigen to antibody binding. The force required to break the antigen-antibody bonds and pull the microsphere off the surface was shown to increase monotonically with increasing surface density of antigens. Using the force determination as a transducer, we were able to detect concentrations of free antigens in solution as small as 10(-15) mol/L in a competitive binding assay.

Adhesion of Lymphoid Cells to CD44-Specific Substrata: The Consequences of Attachment Depend on the Ligand

Interactions between cell-surface adhesion receptors and immobilized specific substrata can exert profound effects on cell morphology. Using phase-contrast microscopy, we show that CD44-expressing mouse lymphoid cells display a spread morphology when adhering to CD44-specific monoclonal antibody (mAb) immobilized on plastic. This spread morphology is different from that of these same cells when adhering to immobilized hyaluronan, the natural ligand of CD44. Morphometric measurements, in combination with intracellular actin staining and fluorescence microscopy, revealed that the adhesion of lymphoid cells to hyaluronan required essentially no cytoskeletal reorganization and resulted in no fundamental change in morphology. On the other hand, cells adhering to immobilized CD44-specific mAb rearranged their actin structure and established multiple membrane contact sites (spread). Cell spreading on antibody, but not attachment to hyaluronan, was inhibited by cytoskeleton-disrupting agents. Transfection of CD44-negative lymphoid cells with full-length and tailless CD44 enabled these cells to bind to both immobilized hyaluronan and mAb. However, the transfectant lacking the cytoplasmic tail of CD44 spread only transiently on the antibody-coated surface. Our results suggest that CD44 may mediate lymphocyte attachment to its carbohydrate ligand hyaluronan by mechanisms broadly similar to those used by selectins. When immobilized CD44-specific antibody is the ligand, however, CD44 may regulate the activity of the cytoskeleton by mechanisms broadly similar to those used by integrins. In the latter case, the cytoplasmic domain of CD44 contributes to cell spreading.

Activation of complement receptor 3 on human monocytes by cross‐linking of very‐late antigen‐5 is mediated via protein tyrosine kinases

Bordetella pertussis interacts with very-late antigen-5 (VLA-5) receptors on the human monocyte resulting in cross-linking of these receptors followed by activation of complement receptor 3 (CR3) and firm adhesion of B. pertussis to these monocytes. In the present study we investigated whether protein tyrosine kinases are involved in the activation of CR3 on monocytes, which was assessed by the binding of C3bi-coated erythrocytes (EC3bi). Pre-incubation of monocytes with tyrphostin-A47, a specific protein tyrosine kinase inhibitor, before adherence of the cells to an anti-VLA-5 monoclonal antibody-coated surface, or addition of tyrphostin-A47 within 10 min of the adherence to such surface, reduced the binding of EC3bi to monocytes significantly. Pre-incubation of monocytes with tyrphostin-A47 reduced the binding of B. pertussis to such monocytes as well. Inhibitors of protein kinase A and/or C had no effect on EC3bi binding to monocytes. Cross-linking of VLA-5 on monocytes resulted in tyrosine phosphorylation of several proteins. Together, these results indicate that protein tyrosine kinases are involved in the VLA-5-induced activation of CR3 on human monocytes.

Adhesion mediated by bonds in series.

Cell adhesion in physiological situations and biotechnological applications is often mediated through serial protein/protein linkages. The adhesion strength of cell/substrate contacts through receptor/ligand bonds in series is explored with a simple mathematical model and quantified with an experimental adhesion assay. A deterministic, mass-action model is developed to describe the attachment and detachment of cell/substrate contacts through single and serial bonds. The experimental system is comprised of protein-coated beads, soluble antibody linkers, and an antibody-coated glass surface. Using the Radial-Flow Detachment Assay, the adhesion strengths of bead/substrate contacts through single and serial linkages are measured. Taken together, this work shows that the specific adhesion strength of the cell/substrate contacts comprised of two receptor/ligand bonds in series is less than the specific adhesion strength of the cell/substrate contacts comprised of either of the bonds separately. In addition, the force to rupture contacts comprised of bonds in series varies with the concentration of the solution linker. The model predicts that the locus of linkage fracture location has only a mild dependence on the ratio of relative bond affinities.

Experimental Study of the Rate of Bond Formation Betwwen Individual Receptor-Coated Spheres and Ligand-Bearing Surfaces

The efficiency of cell adhesion is highly dependent on the rate of association between adhesion molecules when membranes are at bonding distance. Whereas kinetic parameters of interactions involving at least one soluble molecular species have been extensively studied, the definition and experimental determination of corresponding parameters when both receptors and ligands are bound to surfaces are much more difficult to achieve. In the present work, we explore the feasibility of measuring the rate of association between antibody-coated spheres and antigen-derivatized surfaces in presence of an hydrodynamic shear force lower than the strength of a single bond. An image analysis procedure allows continuous recording of particle position with about 0.05 μm accuracy and a time resolution of 5 milliseconds. We present an original procedure allowing direct determination of the wall shear rate by processing the images of moving spheres. Further, simultaneous determination of the Brownian fluctuations perpendicular to the bulk fluid motion and the mean translational velocity of particles allows in principle a numerical determination of the sphere-to-substrate distance within a range of about 10 to 1000 nm. It is concluded that : i) particle motion is in rough agreement with current hydrodynamic theories based on creeping flow approximation. ii) In our experimental system, adhesion seems to be diffusion-limited, therefore, only a lower boundary for the kinetic constant of molecular association can be obtained. iii) Further improvement of our method will require the production of molecularly smooth receptor-coated surfaces.

Dynamic adhesion of CD8-positive cells to antibody-coated surfaces: the initial step is independent of microfilaments and intracellular domains of cell-binding molecules.

Cell adhesion is a multistep, metabolically active process usually requiring several minutes or even hours to complete. This results in the formation of strong bonds that cannot be ruptured by mechanical forces encountered by living cells in their natural environment. However, the first seconds after contact formation are much more sensitive to external conditions and may be the critical step of adhesion. This step is very difficult to monitor without disturbing the observed system. We addressed this problem by studying the interaction between anti-CD8-coated or control surfaces and murine lymphoid cell lines bearing wild-type CD8 molecules, or genetically engineered molecules bearing extracellular CD8 domains and transmembranar and intracytoplasmic domains of class I histocompatibility molecules, or with extensive deletion of intracytoplasmic domains. We used a new method that consisted of monitoring the motion of cells driven along adhesive surfaces by a hydrodynamic force weaker than the reported strength of single ligand-receptor bonds, but sufficient to make free cells move with an easily detectable velocity of several micrometers per second. Cells exhibited short-term (< or = 0.5 s) adhesions to the surface with a frequency of about one event per 30-s period of contact. These events did not require specific antigen-antibody bonds. However, when anti-CD8 were present, strong adhesion was achieved within < 1 s, since most arrests were longer than a standard observation period of 1 min. This bond strengthening was not affected by cytochalasin, and it did not require intact intracellular domains on binding molecules. It is concluded that the initial step in strong adhesion may be viewed as a passive, diffusion-driven formation of a new specific bonds.

Anti‐CD4 modifies the kinetics but not the magnitude of anti‐CD3‐induced interleukin 2 production in peripheral T cells

We have studied the effect of T cell receptor (TcR) cross-linking and TcR-CD4 cross-linking on the induction of interleukin (IL) 2 and IL 2 receptor mRNA expression in peripheral T cells. We confirm our previous results using bioassays, suggesting that CD4 is involved in T cell activation by up-regulating the expression of IL 2 receptors. Furthermore, we show that this also concerns other functional T cell genes such as IL 4 and interferon-gamma. In contrast, similar levels of IL 1 mRNA are found in T cells activated with anti-CD3 with and without anti-CD4 antibody, suggesting that the induction of the IL 2 gene is regulated by TcR cross-linking alone and not influences by CD4. However, anti-CD4 appears to accelerate the induction of IL 2 mRNA in the T cell populations, presumably by a more rapid adhesion of the cells to the antibody-coated surface.

Use of thiol-terminal silanes and heterobifunctional crosslinkers for immobilization of antibodies on silica surfaces

A procedure for covalent immobilization of functional proteins on silica substrates was developed using thiol-terminal silanes and heterobifunctional crosslinkers. Using this procedure, a high density of functional antibodies was bound to glass cover slips and silica fibers. The amount of anti-IgG antibody immobilized was determined to be in the range of 0.66 to 0.96 ng/mm 2 using radiolabeled antibody. The relative amount of IgG antigen bound by the immobilized antibody (0.37 to 0.55 mol antigen/mol antibody) was three to five times greater than other investigators have reported. In addition, the amount of protein nonspecifically adsorbed to the antibody-coated surface was further reduced by the addition of blocking agents so that nonspecific adsorption of protein antigens represented only 2–6% of the total antigen binding. With this low background, IgG antigen binding could be measured at levels as low as 150 fmol when an antigen concentration of 3 pmol/ml was applied. The process for antibody immonilization is straightforward, easy to perform, and adaptable for modifying mass quantities of biosensor components.

Mechanism of inhibition of immunoglobulin G-mediated phagocytosis by monoclonal antibodies that recognize the Mac-1 antigen.

We have investigated the effects of the monoclonal antibodies against the cell surface molecule Mac-1 on C3bi-mediated rosetting and IgG-mediated rosetting and phagocytosis by human peripheral blood monocytes. Highly purified M1/70 F(ab')2, used in the fluid phase, inhibited both monocyte functions. Half-maximal C3bi rosette inhibition occurred at a concentration of 2 nM F(ab')2 M1/70. An equivalent decrease in IgG-mediated rosetting required 10 nM M1/70 F(ab')2, and 50% inhibition of IgG-mediated phagocytosis required 7 nM antibody. Mo-1 F(ab')2 inhibited EC3bi binding with an ID50 of 0.3 nM, whereas 50% decrease in IgG-mediated rosetting required 70 nM of this antibody. OKM1 did not inhibit rosettes of sheep erythrocytes opsonized with IgG antibody (EA) at all. F(ab')2 M1/70 did not affect the binding of monomeric human IgG to monocytes, but did substantially decrease the binding of IgG aggregates. Half-maximal inhibition of aggregated IgG binding at 0 degrees C occurred at 8 nM F(ab')2 M1/70, very close to the concentration that caused equivalent inhibition of IgG-mediated phagocytosis. Aggregated IgG inhibited the binding of radiolabeled M1/70 to monocytes by approximately 40%, suggesting that some, but not all Mac-1 molecules were associated with IgG receptors under these conditions. When cells were allowed to adhere to surfaces coated with M1/70 or Mo-1 F(ab')2, C3bi-mediated rosetting was inhibited, but IgG mediated-phagocytosis was unaffected. Moreover, the dose response of inhibition of phagocytosis by fluid-phase F(ab')2, of anti-Mac-1 monoclonals was similar on monocytes adherent to albumin-coated and antibody-coated surfaces. Kinetic experiments showed that even prolonged incubation of monocytes on M1/70 coated surfaces did not lead to inhibition of EA binding nor did these incubations alter the dose response for inhibition of EA binding by fluid-phase M1/70 F(ab')2. This suggested that not all molecules recognized by M1/70 are freely mobile in the plasma membrane. Indeed, only approximately 60% of 125I-M1/70-biding sites were lost even after 4 h when monocytes were adherent to M1/70-coated surfaces. We conclude that some anti-Mac-1 antibodies can inhibit EA binding because of their epitope specificity, independent of any direct interaction with monocyte Fc receptors. This interference with IgG-Fc receptor-mediated binding and ingestion apparently occurs because of antibody binding to a subpopulation of Mac-1 molecules which are associated with IgG Fc receptors and remain on the apical membrane of monocytes adherent to anti-Mac-1-coated surfaces. We suggest that there may be two functionally distinct molecules on human monocytes recognized by M1/70 and Mo-1 that can be distinguished by their mobility in the plane of the monocyte membrane. The more mobile form of Mac-1 is involved in C3bi rosettes, and does not affect IgG-mediated phagocytosis. The other antigen recognized by M1/70 does not diffuse within the plane of the membrane; ligation of the latter molecule by antibody is associated with inhibition of IgG-mediated phagocytosis.

Dendritic cells in human peripheral blood: effective enrichment from the nonadherent cells

Dendritic cells (DC) from human peripheral blood were enriched using a method including adherence on plastic, depletion of phagocytic cells, flotation on density gradient column and panning on antibody-coated surfaces. The course of cell separation was evaluated by characterizing the morphological, antigenic and functional features of the different cell fractions. Using the population depleted of strongly adherent cells as the source, we were able to achieve a cell fraction containing 40–65% of DC (the main contaminants being monocytes and natural killer cells). Functionally these cells were highly stimulatory in autologous mixed leukocyte reaction. On the other hand, cells which primarily adhered well and were detached after overnight culture contained less than 5% of DC (the main contaminants being monocytes) after the same purification protocol. The calculated yields of DC were 1–2 × 10 6 and less than 0.5 × 10 6 from the nonadherent and adherent populations, respectively. Thus we concluded that the adhesiveness of DC from human blood is so weak that they can more efficiently and in a more reproducible way be enriched from the primarily nonadherent cell fraction.

7] Methods for the study of receptor-mediated phagocytosis

This chapter describes methods designed to measure the function of individual types of receptors in attachment and engulfment of ligand-coated particles. One method involves the use of ligand-or antibody-coated surfaces to selectively remove a single receptor type from the apical surface of the phagocyte. Such surfaces allow determining if an individual receptor is necessary for a given phagocytic event. The other principal method involves the use of erythrocytes that display a single type of ligand on their surface. These particles can usually bind to a single type of receptor on the surface of the phagocyte and permit to determine if a given ligand and its receptor are sufficient to cause phagocytosis. Many procedures for the purification of mononuclear phagocytes exploit the capacity of the cells to adhere to glass or plastic surfaces. The resulting monolayers are difficult to bring into suspension, and are thus unsuitable for experiments that require suspensions of phagocytes.

Mechanisms for adherence of eosinophils to an antibody‐coated surface

Eosinophils may act by degranulation after attachment to a surface. As the mechanisms of adherence are not understood, we have investigated the dependency on Fc(IgG) receptors and other mechanisms by studying the adherence of human eosinophils to albumin-Sepharose beads coated with either specific rabbit IgG antibody, F(ab')2 antibody fragments or serum under different conditions. Adherence to Sephadex beads and albumin-coated microtitration plates was also investigated. 50% of the eosinophils adhered spontaneously to all 3 different surfaces not coated with the antibody, whereas only 25% of neutrophils and less than 10% of mononuclear cells adhered. A small but significant increase in adherence to albumin-Sepharose or albumin-coated plastic occurred after addition of the IgG-antibody, but not after addition of F(ab')2 fragments, indicating that the Fc region was responsible for some increase in adherence. Incubation of eosinophils with IgG-Fc fragments prevented the additional antibody-mediated adherence. As Fc receptor-negative eosinophils adhered almost as well as Fc receptor-positive cells, it appears that the Fc receptors are of minor importance and instead, a nonspecific adherence mechanism, possibly unique for the eosinophil, seems to be the most important in eosinophil adherence to antibody-coated surfaces.

The mechanism of Fc-mediated interaction of eosinophils with immobilized immune complexes. II. Identification of two membrane proteins, modified by the interaction.

Human peripheral blood eosinophils attach to and flatten down onto antibody-coated surfaces and subsequently degranulate. An antibody-coated surface was prepared by treating a layer of agar, containing tetanus toxoid antigen and eosinophil chemotactic factor (ECF), with human anti-tetanus immunoglobin. Changes in eosinophil surface proteins during attachment to the antibody-coated agar layer were detected by lactoperoxidase catalysed iodination. Purified eosinophils were pre-treated with unlabelled iodide, lactoperoxidase and hydrogen peroxide to block pre-existing accessible tyrosine residues on the cell surface. They were then allowed to interact with the agar layer, and subsequently treated with lactoperoxidase and 125I-labelled iodide to label newly accessible surface proteins. Separation of the radioactive proteins by sodium dodecyl sulphate/polyacrylamide gel electrophoresis revealed that, while incubation of the cells in suspension restored the major proteins to the cell surface, interaction with the antibody-coated agar layer caused the appearance of additional proteins of apparent molecular weight 55K, 30K, 28K and 18K. The 55K, 28K and 18K proteins were greatly reduced when antibody was absent, but the 55K protein was distinguishable from immunoglobulin G (IgG) heavy chain, since it could be detected in low amounts even in the absence of antibody. It was found in purified plasma membranes and it could be separated from IgG heavy chain by iso-electric focusing. The possibility is discussed that this protein is either linked to the receptor for the Fc portion of IgG, or that it is itself the receptor. The 18K protein required both antibody and ECF for maximum expression, but was seen in limited amounts with ECF alone. Possibly it is concerned with an ECF-mediated recognition of IgG. Unlike the 55K protein, it binds concanavalin A. Plasma membranes were prepared from eosinophils by lysis in borate, followed by purification on a glass-bead column. Both the 55K and the 18K proteins were found to be major components of the eosinophil membrane.

Mechanism of fc-mediated interaction of eosinophils with immobilized immune complexes: I. effects of inhibitors and activators of eosinophil function

A protein of apparent molecular weight 55000, designated protein 3, becomes newly detectable on the eosinophil surface as a specific consequence of interaction with antigen-antibody complexes immobilized in agar layers. The effect of various agents upon this interaction has been determined by monitoring the appearance of this protein by lactoperoxidase-catalysed iodination. Other parameters that have been measured include: the attachment of eosinophils to the agar layers and their subsequent degranulation, as measured by the release of granule peroxidase, and the degree of spreading of the eosinophils, as assessed by electron microscopy. Attachment of eosinophils to antibody-coated layers is inhibited by heat-aggregated immunoglobulin G (IgG), suggesting that this attachment is mediated via eosinophil Fc receptors. In addition, agents, such as the eosinophil chemotactic factor Ala-Gly-Ser-Glu, that enhance the expression of Fc receptors also enhance the appearance of protein 3, while agents, such as hydrocortisone, that inhibit the expression of Fc receptors reduce its appearance. It is concluded that the appearance of protein 3 parallels the expression of Fc receptors. Attempts to block the Fc region of the bound antibody with staphylococcal protein A were not successful. These experiments indicated that the Fc region of bound IgG has different binding sites for protein A and for the Fc receptor. The correlation between the appearance of protein 3 and subsequent degranulation of the eosinophils was confirmed by the use of agents, such as cytochalasin D and levamisole, that enhance both the appearance of protein 3 and degranulation. Conversely, hydrocortisone reduces the appearance of protein 3 and inhibits degranulation. Protein 3 does not appear when eosinophils adhere to agar layers coated with concanavalin A instead of antibody and the eosinophils do not degranulate. Addition of the calcium ionophore A23187, while causing the release of granule peroxidase, does not elicit the appearance of protein 3. These observations provided additional evidence that the appearance of protein 3 is a specific consequence of the interaction of eosinophils with antibody-coated surfaces. The fact that protein 3 appears at the eosinophil surface as a direct consequence of the interaction with antibody suggests that this protein is closely associated with the eosinophil Fc receptor. The enhancement of the appearance of protein 3 in the presence of cytochalasin D indicates that the movement and reorientation of both this protein and the Fc receptor are constrained by association with cytoplasmic microfilaments.

Eosinophil membrane changes during interaction with antibody-coated non-phagocytosable surfaces.

Abstract Eosinophils attach themselves closely to large non-phagocytosable, antibody-coated targets, such as helminths, and release their granule contents directly on to the surface of the target. A model system has been developed in which human peripheral blood eosinophils flatten down and degranulate on to antibody-coated agar layers, which are too large to be phagocytosed. The agar layer contains tetanus toxoid antigen and the eosinophil chemotactic factor ala-glyser-glu (10 −6 M), and is coated with human antitetanus immunoglobulin. Changes in the organization of the eosinophil membrane proteins were detected by lactoperoxidase catalysed iodination. A protein of molecular weight 55,000 appeared at the cell surface as an early and specific consequence of interaction with antibody coated non-phagocytosable targets. The appearance of this protein preceded degranulation. A second protein of molecular weight 58,000, was seen when no chemotactic factor was present. In the complete system this protein may be blocked by the chemotactic factor. The role of these proteins in the attachment of eosinophils to antibody-coated targets is discussed.

Eosinophil membrane changes during interaction with antibody-coated non-phagocytosable surfaces

Eosinophils attach themselves closely to large non-phagocytosable, antibody-coated targets, such as helminths, and release their granule contents directly on to the surface of the target. A model system has been developed in which human peripheral blood eosinophils flatten down and degranulate on to antibody-coated agar layers, which are too large to be phagocytosed. The agar layer contains tetanus toxoid antigen and the eosinophil chemotactic factor ala-glyser-glu (10 −6M), and is coated with human antitetanus immunoglobulin. Changes in the organization of the eosinophil membrane proteins were detected by lactoperoxidase catalysed iodination. A protein of molecular weight 55,000 appeared at the cell surface as an early and specific consequence of interaction with antibody coated non-phagocytosable targets. The appearance of this protein preceded degranulation. A second protein of molecular weight 58,000, was seen when no chemotactic factor was present. In the complete system this protein may be blocked by the chemotactic factor. The role of these proteins in the attachment of eosinophils to antibody-coated targets is discussed.

The Mechanism of Activation of the First Component of Complement by a Univalent Hapten-IgG Antibody Complex

Abstract We have previously shown that the univalent hapten, 2,4-dinitrophenyl (DNP)-polylysine when bound to anti-DNP-IgG antibody, yields a 7S complex which is a powerful activator of the classical pathway (Goers et al., 1975, J. Biol. Chem., 250:4918). In this paper we compare this univalent hapten-antibody complex with unassociated antibody, reduced and alkylated antibody, and IgG to which polylysine has been covalently attached. Of these, only the univalent hapten-antibody complex fixes complement. However, both antibody and reduced and alkylated antibody bind equally well to the C1q subunit of the first component, whereas only the univalent hapten-antibody complex shows enhanced binding to the intact first component, C1. Moreover, the antibody-hapten complex or hapten alone will bind to activate C1r,C1s to give C1̄r,C1̄s. The C1s, by itself, is not activated by hapten. Moreover, reassociated C1, composed of C1q,C1r,C1s, is activated by the hapten-antibody complex but not by hapten alone. These results allow us to postulate the existence of a polylysine-sensitive activation site on C1r,C1s which is concealed when C1r, C1s is associated with C1q. Furthermore, we suggest that the binding of hapten-antibody complex to C1 induces a conformational change which causes this site to become exposed. Finally, since all of the substances tested bind equally well to C1q, but only the hapten-antibody complex activates C1, we suggest that peripheral subunits on C1q serve only as anchors to bind C1 to antibody-coated cell surfaces or immune complexes and are not directly involved in the C1 activation process. Activation may occur, we speculate, when the antibody-hapten complex combines with the central stalk of C1q,C1r,C1s.