We studied the serologic properties of monoclonal autoantibodies that were produced by hybridomas derived from lymphocytes of patients with systemic lupus erythematosus. The hybridomas were made by fusion of a human lymphoblastoid cell line, GM 4672 (derived from a patient with multiple myeloma), with peripheral-blood or splenic lymphocytes from six patients with lupus. Thirty monoclonal autoantibodies, selected for their ability to react with denatured DNA, were analyzed. Eighteen of them reacted with three or more additional polynucleotides, including native DNA, left-handed double-helical DNA (Z-DNA), poly(l), and poly(dT). Ten reacted both with nucleic acids and the phospholipid cardiolipin. The multiple binding reactions of the monoclonal autoantibodies may be explained by the presence of appropriately spaced phosphodiester groups in both the polynucleotides and the phospholipid. The sharing of antigenic groups by polymers of different natures may contribute to the apparent diversity of serologic reactions in systemic lupus erythematosus. These findings suggest that DNA itself need not be the immunogenic stimulus for autoantibody formation in this disease.

These experiments tested the hypothesis that unmutated germline genes from normal mice can encode autoantibodies. We found that the unmutated VHIdCR gene segment, which encodes a large proportion of antiarsonate antibodies in A/J mice, also encodes antibodies with the ability to bind to DNA and cytoskeletal proteins. After Ars immunization, at a time when the VHIdCR gene segment mutates and antibody affinity for the hapten increases, reactivity with the autoantigens was lost. Six antibodies obtained after immunization with Ars bound both the Ars and DNA. Results of competitive inhibition assays suggested that the same variable region site in the antibodies bound to both Ars and DNA. The properties of the individual germline-encoded antibodies, which include reactivity to both DNA and cytoskeletal proteins, suggest that autoantibodies characteristic of SLE might be a subset of antibodies encoded by unmutated germline V genes.

A B S T R A C T Recent evidence has suggested that a particulate 02-forming system is responsible for the respiratory burst in activated neutrophils. The respiratory burst is normally a transient event, lasting only 30-60 min. To investigate the mechanism by which the btrst is terminated, we examined the 02-forming activity of neutrophil particles as a function of time in the presence and absence of agents known to affect the function of intact cells. Measurements of the°2-forming capacity of the particles against time of exposure of neutrophils to opsonized zymosan, a potent stimulating agent, revealed a rapid fall in activity when exposure was continued beyond 3 min. Exposure to zymosan under conditions in which the myeloperoxidase system was inactive (i.e., in the presence of myeloperoxidase inhibitors, or in the absence of oxygen) resulted in a substantial increase in the initial 02-forming activity of particles from the zymosan-treated cells, but did not prevent the sharp fall in activity seen when zymosan exposure exceeded 10 min. The fall in activity was, however, prevented when activation took place in the presence of cytochalasin B (1.5 ,ugml), an agent thought to act largely by paralyzing the neutrophil through an interaction with its microfilament network.We conclude from these findings that the termination of the respiratory burst results at least in part from the inactivation of the particulate 02-forming system. This inactivation involves at least two processes which probably act simultaneously. One is the destruction of the system through the action of myeloperoxidase. The other appears to require active cell motility and is independent of oxygen. The current view holds that the 02-forming system of the neutrophil is located in the plasma membrane. It may be that the second process involves the internalization and degradation of this membrane-bound system.

Neutrophil chemotaxis, phagocytosis, and oxygen-dependent microbicidal activity are initiated by interactions of stimuli with the plasma membrane . However, difficulties in neutrophil plasma membrane isolation have precluded studies on the precise structure or function of this cellular component. In this paper, a method is described for the isolation of representative human neutrophil plasma membrane vesicles, using nitrogen cavitation for cell disruption and a combination of differential centrifugation and equilibrium ultracentrifugation in Dextran gradients for membrane fractionation . Multiple biochemical markers and galactose oxidase-tritiated sodium borohydride surface labeling were employed to follow the yield, purity, and distribution of plasma membranes, nuclei, lysosomes, endoplasmic reticulum, mitochondria, and cytosol . According to these markers, neutrophil plasma membranes were exposed to minimal lysosomal hydrolytic enzymes and could be isolated free of other subcellular organelles . In contrast, disruption of neutrophils by mechanical homogenization resulted in >20% lysosomal rupture and significant plasma membrane proteolysis . Electron microscopy demonstrated that plasma membranes isolated after nitrogen cavitation appeared to be sealed vesicles with striking homogeneity .Participation of polymorphonuclear leukocytes (PMN) in host defense against infection is dependent on the ability of this cell to respond to its chemical and physical environment . Although the list of "factors" that stimulate or inhibit PMN function is enormous and ever increasing, the cell's repertoire of responses is essentially limited to three general processes: (a) locomotion in a random or directed fashion (chemotaxis), (b) phagocytosis and killing of ingested material, and (c) secretion of lysosomal constituents . A fourth process, namely protein synthesis, may contribute to the above responses as well as lead to the appearance of new proteins (i .e ., leukocytec pyrogen [1] or serum amyloid A protein [2]) . Each of these major PMN responses is thought to be initiated by an interaction of the appropriate activating factor with the neutrophil plasma membrane . Despite the central role of the plasma membrane in neutrophil function, little information is available on the structural organization and biochemical nature of this cellular component.

Neutrophils from a five-year-old boy with recurrent bacterial infections failed to spread on surfaces, leading to a severe defect in chemotaxis and a mild impairment in phagocytosis. Failure to spread was also seen in a fraction of the neutrophils from the patient's mother and sister, but cells from his father and brother were normal. Gel electrophoresis revealed that a protein with a molecular weight of 110,000 daltons (designated gp 110) present in the particulate fraction of normal neutrophils was absent from the patient's cells, and that its levels were below normal in cells from his mother and sister but normal in neutrophils from his father and brother. These findings suggest that gp 110 is necessary for the spreading of neutrophils onto surfaces, that the functional abnormality in the patient's cells is caused by its absence, and that deficiency of gp 110 is an X-linked congenital disease.