Human Alternative Pathway Research Articles

Guinea pig erythrocytes after their contact with influenza virus, activate the human alternative pathway through virus-induced desialation of the cells

Guinea pig erythrocytes after their contact with influenza virus, activate the human alternative pathway through virus-induced desialation of the cells

Antibody restores human alternative complement pathway activation by mouse erythrocytes rendered functionally deficient by pretreatment with pronase.

Activation of the human alternative pathway of complement (C) by surfaces requires the initial deposition of C3b by fluid-phase C3 convertase and sustained C3 cleavage by C3 convertases fixed to the surface. Nonactivating particles have previously been characterized by an inability to sustain the function of C3 convertases on their surfaces because these sites were susceptible to the regulatory action of the control proteins. Pronase converts the mouse erythrocyte (E) from an activator to a nonactivator by markedly decreasing the ability of the cell to affix C3b generated by a fluid-phase C3 convertase; this conversion is unrelated to the action of the control proteins on bound C3b. The capacity of antibody and its F(ab')2 or Fab' fragments to restore the alternative pathway-activating function of pronase-treated mouse E indicates that the contribution of antibody is mediated by its combining site without a requirement for bridging or for the Fc portion. The fab'-dependent activation of the alternative pathway of C relates to the deposition of C3b on the particle surface, which is a first and continuing step in alternative pathway activation. The antibody effect is not directed to the action of the regulatory proteins. Thus, particle-dependent activation of the alternative C pathway has been shown for the first time to be abolished and restored by cell surface-directed mechanisms that function independently of the regulatory proteins.

Antibody-independent activation of the alternative complement pathway by measles virus-infected cells.

When HeLa cells acutely infected with measles virus were incubated in a mixture containing only the six proteins of the alternative pathway of complement activation (C3, factors B and D, beta 1H, C3b inactivator, and native properdin) without antibody, there was activation of the alternative pathway as shown by progressive uptake of 125I-labeled C3b onto the cell surface. This C3b uptake was blocked by EDTA and was not shown by uninfected cells. The rate of 125I-labeled C3 uptake by infected cells was the same in the absence and presence of properdin; however, when antiviral IgG was bound to the cell surface, the rate of C3 uptake was increased in the presence of properdin. Significant 125I-labeled C3 uptake was first detectable when cells were studied at 12 hr after infection, when cells expressed viral polypeptides on their surface. There was also progressive uptake of 125I-labeled C3 onto measles virus-infected cells incubated in human serum depleted of both IgG and C4. Hence, the human alternative pathway of complement activation can be initiated on the surface of measles virus-infected cells independent of IgG antibody. However, lysis of the infected cells only occurs when antiviral antibody is present.

Surface-associated heparin inhibits zymosan-induced activation of the human alternative complement pathway by augmenting the regulatory action of the control proteins on particle-bound C3b.

Discrimination by the human alternative pathway between activating and nonactivating particles occurs after deposition of C3b by the continuous low-grade interaction of the alternative pathway components in biologic fluids and is dependent on the modulation by surface constituents of the interaction of bound C3b with the control proteins, beta 1H, and C3b inactivator (C3bINA). When heparin glycosaminoglycan was coupled to activating particles, such as zymosan or Sepharose, by cyanogen bromide activation, their capacity to activate the human alternative pathway was inhibited. The loss of alternative pathway-activating capacity was directly correlated to the number of heparin molecules bound/zymosan particle, whether the ratio was varied by increasing the amounts of heparin in the initial coupling reactions or by treating a fully inhibited particle with incremental concentrations of heparinase. Analysis by linear regression of the inhibitory effect of each procedure (r = 0.97, r = 0.98, respectively) for adjusting the number of heparin molecules/particle revealed that the dose-response relationships were identical and that complete inhibition occurred with greater than 12 X 10(8) molecules of heparin/zymosan particle. The coupling of heparin to zymosan did not impair the uptake of C3b from the fluid-phase interaction of C3, B, and D, and did not alter the capacity of bound C3b to associate with B so as to permit its inactivation by D. Although the regulatory proteins present in normal serum chelated with EDTA or presented as a combination of purified C3bINA and beta 1H were relatively inefficient in inactivating C3b function on an activating particle of the alternative pathway such as zymosan or zymosan-cyanogen bromide, the control proteins rapidly inactivated C3b on a nonactivating particle wuch as a sheep erythrocyte or zymosan with coupled heparin. The increased numbers of C3b sites susceptible to inactivation by C3bINA in the presence of beta 1H were significantly correlated to the number of molecules of heparin/particle. By linear regression analysis of the correlation (r = 0.99) the number of heparin molecules/particle required to promote total inactivation of bound C3b by purified control proteins was 13.8 X 10(6). This molecular analysis suggests that the action of heparin coupled to an activating particle of the alternative pathway is to promote the interaction between particle-bound C3b and the regulatory proteins, thereby preventing particle-associated amplified C3 cleavage. It is noteworthy that both surface constituents known to maintain a particle as a nonactivator of the alternative pathway, sialic acid and N-sulfated mucopolysaccharide, act by facilitating the inactivation by regulatory proteins of the function of particle-bound C3b.