Mast cells induced in vitro by interleukin 3 from native murine thymus cells.

To shed further light on the induction and characterization of thymus-derived mast cells, we cultured a variety of cell populations from murine thymus tissues (Balb/c) in the presence or absence of interleukin 3 (IL-3). The whole cell population and the non-adherent T cell-depleted population developed mast cells. The morphological studies revealed granulated cells; the granules were stained with toluidine blue, alcian blue (pH 3.0), and metachromatic dyes. Electron microscopy revealed altered mast cell granules. These cells contained relatively low amounts of histamine (approximately 1700 ng/10(6) cells), were IL-3 (but not IL-2)-dependent, and did not possess T-cell, B-cell, or macrophage markers. No phagocytosis was observed. The cells also had 20 alpha-hydroxysteroid dehydrogenase, and both IL-3 and IgE (145,600/cell) high affinity receptors. The frequency analysis showed 17 precursor cells per 10(6) thymic cells. The results indicate that the thymus indeed contains progenitors of mast cells responsive to IL-3, and that the mast cells are derived from non-T, non-phagocytic, and non-adherent cells of the thymus. Their T-lymphocyte product (IL-3) dependency, ultrastructural appearance, granular stainability, and low content of histamine may support the view that the mast cells originating from the thymus probably belong to a mucosal mast cell lineage.

Induction of granulocyte histaminase release by particle-bound complement C3 cleavage products (C3b, C3bi) and IgG.

The interaction of opsonized particles with human granulocytes promotes a number of important biologic functions, including phagocytosis, superoxide generation, and release of a variety of enzymes, including histaminase. We have previously determined that histaminase release occurs via a C3-dependent process. Although fluid-phase C3b dimers can mediate release, the relative effects of particle-bound C3b and C3bi and of IgG have not been examined. In this report we demonstrate that particle-bound C3 deposited on activators of the alternative C pathway effected histaminase release in the absence of IgG. Particle-bound C3bi and C3b were both effective as mediators of histaminase release. The extent of release varied as a function of the activating surface on which C3 was deposited (zymosan C3b was considerably more potent than C3b bound to rabbit erythrocytes, which was slightly more potent than C3b bound to neuraminidase-treated sheep erythrocytes). In contrast, C3b or C3bi deposited on nonactivating surfaces (such as sheep erythrocytes) at inputs of up to 2,000,000 molecules per granulocyte failed to induce histaminase release unless IgG was also present. The ability of C3b bound to particles that serve as activators of the alternative pathway to induce histaminase release is apparently not the result of decreased susceptibility of C3b to proteolysis or to an increased binding affinity to the C3b receptor, but may relate to the interaction of other surface structures on activating particles with the PMN membrane.

Role of human factor I and C3b receptor in the cleavage of surface-bound C3bi molecules.

Control of functions mediated by the third component of complement (C3) depends on the rate of generation and degradation of biologically active C3 fragments. To evaluate the mechanisms of degradation of active C3 fragments, the role of the control protein C3b/C4b inactivator (factor I) was investigated under conditions approximating those found in vivo, i.e. in the presence of plasma. The breakdown of human erythrocyte-bound C3bi molecules in serum or plasma was mediated only by factor I, since factor I-deficient or -depleted plasma was inactive until reconstituted with highly purified factor I. The rate of cleavage of C3bi bound to human erythrocytes by purified factor I was not affected by the presence or absence of beta 1H (factor H). The released breakdown product of C3bi has been shown to be C3c antigenically and on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Two different monospecific antibodies to the human C3b receptor totally abrogated factor I-mediated cleavage of cell-bound C3bi, suggesting that the C3b receptor (but not factor H) is required as an obligate cofactor. The rate of this C3b receptor-dependent, factor I-mediated cleavage of bound C3bi is strongly regulated by the surface to which C3bi is bound. Whereas C3bi bound to particulate nonactivators of the alternative complement pathway such as human erythrocytes is rapidly degraded by this mechanism, the rate of cleavage of C3bi bound to activators is significantly slower. These data suggest a physiologic role of C3b receptors in the degradation of biologically active C3 fragments deposited on host tissues. They also suggest that C3bi molecules on restricted surfaces are relatively stable and can thereby interact with complement C3 receptors in vivo.

The first component of complement. I. Purification and properties of native C1.

The first component of complement has been purified by using affinity chromatography on Sepharose-bound IgG. Unlike earlier procedures that yield the activated form of C1, in this method C1 is maintained in the native form by the protease inhibitor p-nitrophenyl, p'-guanidinobenzoate (NPGB). The procedure requires only two steps and yields pure C1 as judged both by SDS-PAGE analysis and by effective molecule calculations. The yields have varied from 30 to 50% in over 50 preparations. The functional properties of the purified native C1 correspond to those of C1 in serum. The dose-response activity profile is nonlinear, but becomes linear when C1 IS ALLOWED TO SELF-ACTIVATE. From SDS-PAGE analysis of the self-activated C1, all the C1r and C1s subcomponents are converted to the activated split products, indicating that all C1 molecules are biologically active. The recovery of C1 activity is dependent on the use of a heterologous source for the IgG on the affinity absorbant. The conditions of binding and elution from the Sepharose-IgG column are critical, indicating that immunoglobulin-bound C1 is rapidly inactivated under physiologic conditions by serum inactivators. The activation of the purified C1 in fluid phase has been explored both in the presence and absence of C1-inhibitor.

Native and activated properdin: interconvertibility and identity of amino- and carboxy-terminal sequences.

The development of a two-step purification procedure of native properdin with good yield has allowed the physical and chemical comparison of native and activated properdin. The two forms of properdin have identical electrophoretic mobility, subunit size, and amino- as well as carboxyl-terminal amino acid sequences. The two forms of properdin can be interconverted by using mild denaturing agents, indicating that the change in biologic activity is conformational. Circular dichroism analysis of properdin reveals a significant variability in the tertiary structure. However, the differences are a result of the method of purification and do not correspond to the biologic activity of the protein, because the spectra of the interconverted forms of properdin do not change. This indicates that the conformational transition that causes biologic activity changes is small, relative to the conformational variations produced by other conditions that do not alter the biologic activity.

Potentiation of antiboty-dependent cell-mediated cytotoxicity by target cell-bound C3b.

Antibody-dependent cytolytic effector lymphocytes are known to possess, in part, receptors for activated C3. Employing a model system consisting of 51Cr-labeled chicken erythrocytes and purified human peripheral lymphocytes, we investigated the effect of target cell bound C3b on antibody-dependent cellular cytotoxicity (ADCC). At concentrations of anti-target cell antibody too low to cause effective ADCC, target cell bound C3b cooperated with antibody to produce marked target cell lysis. In the presence of a 1/6.25 X 10(6) dilution of anti-chicken erythrocyte rabbit IgG, cell lysis increased from 20% to 65% by the attachment of 18,000 C3b molecules per cell. C3b-dependent enhancement of ADCC was dose dependent. It was augmented by attachment of activated properdin (P) to the C3b-bearing target cells.

Alternative pathway of complement: nonenzymatic, reversible transition of precursor to active properdin.

A method for the purification of properdin in precursor state (pre-P) has been elaborated. Precursor properdin, in contrast to activated properdin (P), does not initiate the formation of a C3 convertase when added to properdin-depleted serum, rather it requires the presence of an activating substance such as zymosan for expression of its activity. Comparing the activities of pre-P and P it was found that some P preparations contained significant amounts of pre-P because they were fully active only in the presence of zymosan. This observation indicated that P can partly revert to pre-P. Comparison by immunoelectrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed no charge or size differences between pre-P and P. Radiolabeled pre-P, when analyzed after its participation in the reactions of the pathway, displayed an unchanged subunit m.w. of 50,000. Taken together, these results strongly suggest that the transition of pre-P to P is due to a partly reversible change in the conformation of the protein rather than the result of proteolytic cleavage.

Fractionation of of proteolytic enzymes by affinity chromatography on sepharose aminocaproyl proflavin.

The serine proteinases trypsin, chymotrypsin, elastase, and acrosin bind to the proflavin resin, the sulfhydryl proteinases ficin, bromelain, and papain are retarded by the resin, whereas most proteins and enzymes tested are not bound. Elution of the bound activities is accomplished NaCl or by variation from the pH optimum of the enzyme. Commercially available enzymes that are bound or retarded are easily further purified by the column. The acrosin activity of sperm acrosomal extracts is separated into bound and unbound activities. Acrosin is purified 120-fold from sperm acrosomal extracts in a single step, yielding a specific activity of 96.

Alternative pathway of complement: recruitment of precursor properdin by the labile C3/C5 convertase and the potentiation of the pathway.

In this study the physiological role of properdin and the differential subunit composition of the solid phase enzymes of the pathway have been explored. Cell-bound C3 and C5 convertase differ in their C3b requirement. Apparently one molecule of C3b is sufficient to allow formation of C3 convertase (C3b,B), whereas two or more are required for generation of C5 convertase (C3bn,B). This conclusion was drawn from results indicating the critical role of the spacial distribution of C3b molecules on the cell surface in enzyme formation. While the C3/C5 convertase is fully capable of acting on C5 and thereby initiating the assembly of the cytolytic membrane attack complex, it is exceedingly labile and vulnerable to destruction by the C3b inactivator. It is the apparent role of properdin to confer a degree of stability upon the labile enzyme and to protect its C3 convertase function against enzymatic destruction. To achieve these effects, precursor properdin (pre-P) is recruited in a binding-activation reaction by the labile C3/C5 convertase. Multiple C3b molecules appear to be needed for the formation of properdin-activating principle. Three modes of regulation have been described, which involve spontaneous dissociation enzymatic degradation by C3b inactivator and disassembly by beta1H. The functional differences of pre-P and activated properdin (P) were delineated, pre-P displaying a weak affinity for C3b and P the capacity of strong interaction, P generating a soluble C3 convertase in serum and pre-P being unable to do so. Because of the profound differences between native pre-P and the laboratory product P, the question was raised as to whether soluble P represents an unphysiological form of the protein. On the basis of this and other studies, the conclusion was reached that in vitro properdin recruitment constitutes the terminal event of the properdin pathway, and that properdin augments the function of C3/C5 convertase without changing its substrate specificity.

A molecular concept of the properdin pathway.

The sequential events of the properdin system were analyzed. Properdin-depleted serum allows the formation of a Factor B- and D-dependent C3 convertase. This enzyme, called the properdin-receptor-forming enzyme, was shown to utilize a novel serum component, the initiating factor. The protein is a beta-globulin in precursor form and is distinct from immunoglobulins. The function of the enzyme is to deposit C3b on the surface of activator particles. Apparently doublets of C3b are required for the formation of the properdin-activating principle. It consists of a complex containing surface-bound C3b and activated Factor B. properdin precursor is activated by binding to this complex without detectable change in molecular weight. The transition of properdin precursor to activated properdin is probably caused by a conformational change. The complex, consisting of bound C3b, properdin, and activated Factor B, represents the enzyme that acts on C5, thereby initiating self-assembly of the membrane attack system. Native C3 is not needed for the function of the enzyme. It is disassembled by soluble C3 or C3b and its formation is under the control of the properdin-receptor-destroying enzyme, which may be identical with the C3b inactivator.

The serine protease nature of the C3 and C5 convertases of the classical and alternative complement pathways.

Activated Factor B, incorporated into the cobra venom factor (CVF)-dependent C3/C5 convertase, was inactivated by diisopropylfluorophosphate (DFP). Inactivation was time- and dose-dependent and was enhanced by the presence of substrate. Treatment of the zymogen of Factor B with DFP effected significant inactivation. Incorporation of [3H]diisopropylphosphate into the zymogen and into the activated form of Factor B was demonstrated after [3H]DFP treatment and subsequent electrophoresis of the proteins on polyacrylamide gels containing sodium dodecyl sulfate. Inactivation of activated C2 incorporated into the classical C5 convertase was observed on DFP treatment of the enzyme. DFP also reduced the activity of the C2 zymogen. The description of their serine proteinase nature further emphasizes the close structural and functional relationship of C2 and Factor B.

Properdin- and nephritic factor-dependent C3 convertases: requirement of native C3 for enzyme formation and the function of bound C3b as properdin receptor.

Two complex enzymes were assembled that both converted C3 to C3b, one consisting of activated properdin (P), native C3, proactivator (PA) and proactivator convertase (PAase), and the other of nephritic factor (NF) and the same three cofactors. By maintaining a critical concentration of PAase, the P-C3 convertase and the NF-C3 convertase were shown to function efficiently without formation of the C3b-feedback enzyme. The former two enzymes are distinct from the C3b-dependent C3 convertase in that they utilize native C3 instead of C3b and PA in an apparently uncleaved form. The P- and NF-C3 convertase express maximal activity within approximately 10 min at 37 degrees C and decay with a half-life of 35 min at 37 degrees C, which is in contradistinction to the reported lability of the C3b-feedback enzyme. P- and NF-C3 convertases are inhibited by their product C3b, which may constitute a heretofore unknown control of the alternative pathway. A direct physical interaction of P with native C3 and C3b was demonstrated by agglutination of C3b-bearing erythrocytes and by agglutination inhibition. Bound C3b thus constitutes the only known receptor of P and may fulfill an important localizing function for P and the P-C3 convertase in vivo. Although P and NF form functionally similar enzymes, they act independently of each other and are apparently immunochemically unrelated proteins.