Ig Molecules Research Articles

Multiple genomic defects result in an alternative RNA splice creating a human gamma H chain disease protein.

Heavy chain diseases (HCD) are human lymphoproliferative disorders in which a clonal B cell population produces Ig molecules made of truncated H chains without associated L chain. We characterized the rearranged H chain gene and its mRNA from the leukemic cells of a patient (RIV) with gamma-HCD. The abnormal RIV serum Ig consisted of shortened, dimeric gamma 1-chains which had an amino terminus within the hinge region. RIV lymphoblasts possessed a foreshortened (1200 bp) gamma 1-mRNA which had sequences for only the leader, hinge, second, and third constant region domains (CH2 + CH3), but lacked variable (VH) and CH1 information. Sequence of the productive gamma 1 allele revealed it had undergone VH-JH and H chain class switch recombinations. However, normal RNA splice sites had been eliminated by a DNA insertion/deletion (VH acceptor site), mutations (JH donor site), or a large deletion (CH1 region). Inserted sequences were of non-Ig and apparently non-genomic origin. These DNA alterations resulted in aberrant mRNA processing in which the leader region was spliced directly to the hinge region, accounting for the HCD protein.

Non-immune immunoglobulin binding by "Haemophilus somnus".

In-vitro culture of Haemophilus somnus in liquid or solid media supplemented with bovine blood or serum resulted in non-immune binding of immunoglobulin (Ig) by the organism. This binding was independent of the antigen-combining site of the Ig molecule, since binding of an IgG preparation specific for the hapten dinitrophenol was unaffected by the presence of the homologous antigen. Quantitative comparison of the binding of Ig fragments Fab and Fc demonstrated that the non-immune binding occurred in the Fc region of bovine IgG. The isotypes of Ig that became bound to H. somnus included both bovine IgG subclasses (IgG1 and IgG2), which were bound equally, and bovine IgM.

Regulation of an idiotype+ B cell lymphoma. Effects of antigen and anti-idiotopic antibodies on proliferation and Ig secretion.

The B cell surface Ig molecule plays an important regulatory role in delivering inductive/tolerogenic signals to the cell. In this paper, the effect of Ag and anti-idiotopic antibodies on the in vitro proliferation and Ig secretion of a B cell tumor was studied. The tumor (BCL1), which had been transfected with the TEPC-15 VH and VL Ig genes, expresses surface Ig and secretes antibody that binds the hapten phosphorylcholine. We found that Ag (C polysaccharide and phosphorylcholine carrier Ag) and two different anti-idiotopic antibodies, in the absence of T cells, all inhibited the proliferation of the T15+ transfectant cell line. The anti-idiotopic antibodies, but not Ag, also inhibited the secretion of T15 Ig by this cell line, suggesting different functional roles for Ag vs anti-Id in the regulation of B cell inactivation. The inhibition of secretion and proliferation appears to be cell cycle phase related. In addition, mouse rIL-4 could override the inhibition of proliferation induced in these studies. These phenomena, demonstrating that binding of surface Ig can result in the transduction of negative growth signals to a B cell tumor, can be viewed as a manifestation of immunologic tolerance. These findings collectively demonstrate that Ag and anti-Id mediate different signals to B cells via interaction with the surface Ig. Because of the monoclonal nature of the T15 transfectant and the anti-idiotypic antibodies, this system can be used to investigate the underlying molecular reactions involved in the B cell response and induction of tolerance.

Monoclonal antibodies to axolotl immunoglobulins specific for different heavy chains isotypes expressed by independent lymphocyte subpopulations

An immunoblotting analysis of purified axolotl immunoglobulins (Ig) separated by SDS—PAGE reveals two heavy (H) chains isotypes: a 76 kDA chain recognized by the monoclonal antibody (mAb) 33.45.1 and a 66–68 kDa doublet recognized by the mAb 33.39.2. The 76 kDa chain is associated to high molecular weight (HMW) Ig molecules and the 66–68 kDa H chains are associated to low molecular weight (LMW) Ig of 172 kDa. Both H chains isotypes are linked to identical light (L) chains, labelled in immunoblotting by the mAb 33.101.2. Two different axolotl lymphocyte subpopulations are characterized by these two distinct H chains isotypes. One population of splenic lymphocytes (∼40%) is labeled by indirect immunofluorescence with mAb 33.45.1, specific for the 76 kDa H chain isotype. Another population (∼20%) is labeled by mAb 33.39.2 specific for the 66–68 kDa H chain isotype. Both populations of splenic lymphocytes are stained by mAb 33.101.2 specific for the axolotl L chains. Therefore, the presence of at least two independent Ig classes is now confirmed in a urodele amphibian species at the humoral and cellular levels.

Immunoglobulin diversification in embryonic chicken bursae and in individual bursal follicles.

Previous studies have shown that the same immunoglobulin (Ig) V lambda gene (V lambda 1) is rearranged in all chicken B cells, and that extensive sequence diversification of this gene occurs during B cell development in the bursa of Fabricius. We used two-dimensional gel electrophoresis to compare the heterogeneity of Ig lambda light chains produced by B cells at different stages of bursal development. Somatically diversified light chains were observed in Ig molecules produced by bursal cells as early as 15 days of embryonic incubation. The two principal species of light chain observed probably represent glycosylated and nonglycosylated forms of lambda chain encoded by alleles of a single lambda gene. Extensive diversification was observed during late embryogenesis. We also studied lambda light chain diversity in cyclophosphamide-treated birds repopulated with normal bursal cells. In these birds, individual bursal follicles are repopulated by single B cell precursors. Follicular cells derived from single B cell precursors were able to produce a spectrum of light chains almost as diverse as that of the total bursal cell population. We used two monoclonal anti-idiotype antibodies to study idiotype expression in individual normal or reconstituted follicles. About 30% of follicles contained 0.1% to 5% of lymphocytes which reacted with one or both of the antibodies. The results indicate that within individual bursal follicles bursa stem cells undergo Ig hyperdiversification.

Partial molecular genetic map of the rabbit VH chromosomal region.

Thirty VH-containing cosmid clones, isolated from rabbit germ-line DNA libraries, were restriction mapped and shown to contain approximately 100 VH genes in 765-kb of DNA. Twenty-two of the cosmid clones were grouped into seven distinct clusters. The VH genes were separated by an average of 8 kb, although some were separated by less than 3 kb. Comparison of the nucleotide sequences of two of these VH genes with the sequences of another 11 VH genes showed that they were all generally more than 80% homologous suggesting that rabbit VH genes are members of one highly homologous gene family. Most rabbit Ig molecules have the VH allotypic specificities a1, a2, or a3 and are designated VHa-positive. A small number (less than 30%) of Ig molecules lack these VHa allotypic specificities and are designated VHa-negative. The VH containing cosmid clones were hybridized with synthetic oligomer probes designed to be specific for genes encoding VHa-positive or VHa-negative molecules. At least 50% of the germ-line VH genes hybridized with the VHa-negative oligomer and thus presumably encode VHa-negative molecules; as few as 15% of the genes could be identified as encoding VHa-positive molecules based on hybridization with the VHa-positive oligomer. Approximately 35% of the VH genes did not hybridize with either oligomer and could not be classified as VHa-negative or VHa-positive. We propose that the predominance of serum VHa-positive molecules, in contrast to the predominance of VHa-negative encoding germ-line genes, may reflect preferential usage of a few germline VH genes. The implications of this idea toward explaining the allelic inheritance of VHa allotypes are discussed.

Transfected plasmacytoma cells do not transport the membrane form of IgM to the cell surface.

Expression vectors coding for membrane-bound IgM antibodies were introduced into myeloma and B lymphoma cells. Only the lymphoma but not the myeloma cells were able to express the antibodies on the cell surface, although in both cases, complete antibodies were assembled intracellularly. In myeloma cells, the Ig molecules did not reach the Golgi compartment. Thus, the intracellular transport of membrane-bound antibodies is controlled in the B cell lineages in a developmentally ordered fashion.

Suppressor T cells, immunoglobulin and Igh restriction.

Why do some T cell express idiotypes that are serologically similar to those of the B cells, since it is now well established that T cells do not use Ig genes for their antigen specific receptors? In this review article, we have summarized some the evidence for the influence of Igh linked genes on the Suppressor T cell repertoire, using anti-u treated mice as a model system. We investigated whether B cells and Ig molecules play a role in the generation of Ts repertoire. While our results clearly confirmed that B cells and Ig are important in the establishment of Ts repertoire, our experiments failed to resolve the fundamental question - 'what dictates the Igh restriction specificity of Ts?' Ts cells from anti-u treated mice did not lose all Igh restriction specificity. Instead, they expressed an altered restriction specificity. These results suggest that there are at least two independent mechanisms responsible for the generation of the suppressor T cell repertoire. A pre-selected germ line one, which is Ig independent, and a mature one, which is Ig dependent. The precise mechanisms responsible for the generation of germ line Ts repertoire remain to be determined.

Transcriptional Controlling Elements in the Immunoglobulin and T Cell Receptor Loci

Publisher Summary The proteins that fulfill the function of identifying foreign molecular structures for the humoral response are immunoglobulins (Igs) and for the cellular response are T cell receptors (TCRs). Because both Igs and TCRs carry out antigen recognition, it is not surprising that the structure of Ig and TCR proteins and the structure, organization, and rearrangement of the genes that encode them are quite similar. Ig molecules are secreted and recognize soluble antigens, while TCR molecules remain on the T cell surface in close association with other membrane proteins and recognize cellular antigens, many more Ig molecules are synthesized by terminally differentiated B cells than TCR molecules are synthesized by mature T cells. The chapter presents overviews of the various ways in which Ig and TCR genes are regulated during development. However, the detailed portions of the review in the chapter is limited to transcriptional regulation, and it focuses on cis-acting DNA elements and trans-acting cellular proteins, which are required to regulate transcription of Ig and TCR genes. Transcriptional enhancers, which were first discovered in animal viruses, are defined functionally as cis-acting DNA sequences, which activate transcription-initiation in an orientation- and distance-independent manner. In cases in which the difference in transcriptional activity has been quantitated, it seems that the preference is not absolute, because the low levels of transcription are observed in some non-B cell lines. The chapter discusses several models that may explain its B cell-specific activity in immunoglobulin genes.

Detection of private and recurrent idiotopes on natural anti-tubulin antibodies by monoclonal anti-idiotopic antibodies.

Two monoclonal anti-idiotopic antibodies (anti-Id) were raised in mice against a human monoclonal IgA,K displaying a monospecific anti-tubulin (anti-alpha- and anti-beta-tubulin) activity. One anti-Id (IgG,K) recognized a private idiotope, TID 3.2, present only on the IgA,K immunogen, close to or within the antigen-combining site. The other anti-Id (IgM,K) recognized a recurrent idiotope, TID 7.1, outside the paratope and present in normal human and BALB/c mouse serum, on 2 of 11 polyspecific human monoclonal immunoglobulins and on 6 of 11 murine natural monoclonal auto-antibodies exhibiting a widespread anticytoskeletal protein-binding activity. Both the idiotopes were absent on two induced anti-tubulin antibodies exhibiting a monospecific anti-alpha- and anti-beta-tubulin specificity. Utilizing competitive and additivity immunoassays, we could show that the polyspecific human and mouse anticytoskeletal antibodies tested, whether bearing the TID 7.1 Id or not, appeared to compete in variable degrees for epitopes on the tubulin molecule recognized by the monoclonal IgA,K but distinct from the epitopes recognized by the induced monospecific anti-tubulin antibodies. The high incidence of the recurrent TID 7.1 idiotope in man and mouse suggests an important physiologic and perhaps regulatory function of this idiotope. Furthermore our data suggest that a restricted family of germ-line genes, highly conserved during phylogeny, may encode for these idiotope-bearing Ig molecules.

Characterization of Axolotl Heavy and Light Immunoglobulin Chains by Monoclonal Antibodies

Axolotl specific antibodies to 2,4-dinitrophenyl (DNP) were purified by affinity chromatography from the sera of animals immunized with 2,4,6-trinitrophenylated sheep red blood cells (TNP-SRBC). The purified anti-TNP/DNP antibodies, when analyzed by SDS-PAGE, were constituted of high molecular weight molecules, which in reducing conditions, were separated into heavy 72-88 kD and light 27-30 kD polypeptides. The axolotl heavy antibody chains strongly bound Concanavalin-A and migrate faster in SDS-PAGE after endoglycosidase-F (Endo-F) treatment. Using the same techniques, no carbohydrate components were detected onto light chains. Monoclonal antibodies (MAbs) were obtained against these purified axolotl immunoglobulins (Ig) and their specificities were studied by immunoblotting. MAbs 33.45.1 and 33.101.2 respectively recognized heavy and light chains determinants of the Ig molecule. These determinants were resistant to Endo-F digestion, suggesting that the two MAbs were not directed to polypeptide-associated N-linked high mannose or complex oligosaccharides. MAbs 33.45.1 and 33.101.2 were compared to 11.5.2, an anti-axolotl thymocytes MAb which was reactive for both axolotl leucocytes and soluble Ig. MAb 11.5.2 reacted in immunoblotting against several high molecular weight axolotl serum proteins, including heavy Ig chains. Light chains were not recognized. However, 11.5.2 did not further recognize Endo-F treated Ig, suggesting its specificity for a carbohydrate determinant of the heavy chain, and link to a large diversity of soluble or membrane glycoproteins.

Two M315 idiotopes defined by isologous monoclonal antibodies: one depends on germline and the other on mutated murine lambda 2 light chain sequences.

We have previously reported that T helper cells of BALB/c mice recognize the unique mutated sequence Phe94, Arg95, Asn96 of the lambda 2 L chain of isologous (BALB/c) myeloma protein 315. Here we study two Id (Id-315.1.4 and Id-315.TH) of the DNP-Lys binding M315 defined by two monoclonal isologous anti-Id Ab (Ab 2-1.4 and Ab 2-TH). Both Id were (1) totally expressed by Fv-315, but not by free unpaired V domains, (2) specifically dependent on VH-315, since lambda 2-315 recombined with four other H chains did not express the Id, (3) related to the hapten-binding site because their expression was blocked by the haptens DNP-Lys and DNP-Gly, and (4) topographically related because Ab 2-1.4 and Ab 2-TH competed with each other for binding to M315. The contribution of lambda chain V regions was studied with the aid of reconstituted Ig molecules of H-315 paired with lambda 1, lambda 2, and lambda 3 L chains. Id-315.TH was expressed equally well by reconstituted Ig containing three different lambda 2 chains (lambda 2-5-7, lambda 2-T952, and lambda 2-315), but its expression was profoundly reduced when H-315 was associated with lambda 3-SAPC15 or lambda 1-J558 L chains; it therefore depended upon amino acids encoded by germline lambda 2 genes. By contrast, Id-315.1.4 was only restored by the lambda 2-315 chain paired with H-315. Since lambda 2-5-7 and lambda 2-T952 differ from lambda 2-315 at positions 38, 94, 95, 96, and 98 or 99, respectively, Id-315.1.4 probably requires the unique mutated amino acids Phe94, Arg95, Asn96 of lambda 2-315. This resembles the effects on Id expression of previously reported unique amino acids of the D region. We failed to confirm that hyperimmunization of BALB/c mice with Ab 2-1.4 cross-linked to KLH induced M315-like Ab. The results are discussed in terms of the contribution of the third hypervariable loop of lambda chains to Id and the immunogenicity of isologous Ig.

Establishment and phenotypic characterization of three new human myeloma cell lines (U-1957, U-1958, and U-1996)

Three new human myeloma cell lines (U-1957, U-1958, and U-1996) have been established in vitro. The cell lines are Epstein-Barr virus (EBV) negative, monoclonal, and aneuploid and should thus represent malignant cell populations and not EBV-carrying non-neoplastic B lymphoblastoid cell lines. The myeloma origin of the cell lines is also suggested by their capacity for production of monoclonal complete immunoglobulin (Ig) molecules (U-1957 and U-1958) or IgG light chains (U-1996) of the same type as the myeloma protein in vivo. All the cell lines have morphological features of plasmablasts-plasma cells but appear to represent slightly different stages of B cell differentiation. Thus, the U-1958 has plasma cell morphology, expresses only PCA-1 and OKT-10 but no other B cell antigens, and secretes 1.5 micrograms/mL of IgG/10(6) cells/24 hours. The U-1957 has plasma cell morphology and expresses Fc receptors and the LB-1 antigen in addition to the PCA-1 and OKT-10 antigens. This line produces only minimal amounts of IgG, which appears not to be secreted. The U-1996, finally, is a kappa light chain producer, has a plasmablast morphology, and expresses LB-1 in addition to the PCA-1 and OKT-10 antigens. All three cell lines are chromosomally heterogeneous and contain several markers with a 14q+ abnormality as a common characteristic abnormality. These new myeloma lines have been in continuous culture for approximately 3 years and are instrumental in studies of various aspects of the biology of human myeloma.

Establishment and Phenotypic Characterization of Three New Human Myeloma Cell Lines (U-1957, U-1958, and U-1996)

AbstractThree new human myeloma cell lines (U-1957, U-1958, and U-1996) have been established in vitro. The cell lines are Epstein-Barr virus (EBV) negative, monoclonal, and aneuploid and should thus represent malignant cell populations and not EBV-carrying non-neoplastic B lymphoblastoid cell lines. The myeloma origin of the cell lines is also suggested by their capacity for production of monoclonal complete immunoglobulin (Ig) molecules (U-1957 and U-1958) or IgG light chains (U-1996) of the same type as the myeloma protein in vivo. All the cell lines have morphological features of plasmablasts-plasma cells but appear to represent slightly different stages of B cell differentiation. Thus, the U-1958 has plasma cell morphology, expresses only PCA-1 and OKT-10 but no other B cell antigens, and secretes 1.5 μg/mL of lgG/106 cells/24 hours. The U-1957 has plasma cell morphology and expresses Fc receptors and the LB-1 antigen in addition to the PCA-1 and OKT-10 antigens. This line produces only minimal amounts of IgG, which appears not to be secreted. The U-1996, finally, is a κ light chain producer, has a plasmablast morphology, and expresses LB-1 in addition to the PCA-1 and OKT-10 antigens. All three cell lines are chromosomally heterogeneous and contain several markers with a 14q + abnormality as a common characteristic abnormality. These new myeloma lines have been in continuous culture for ~3 years and are instrumental in studies of various aspects of the bioloay of human myeloma. © 1987 by Grune & Stratton, Inc. 0006-4971/87/6906-0009$3.00/0

Assembly and secretion of heavy chains that do not associate posttranslationally with immunoglobulin heavy chain-binding protein.

Heavy chain-binding protein (BiP) associates posttranslationally with nascent Ig heavy chains in the endoplasmic reticulum (ER) and remains associated with these heavy chains until they assemble with light chains. The heavy chain-BiP complex can be precipitated by antibody reagents against either component. To identify sites on heavy chain molecules that are important for association with BiP, we have examined 30 mouse myelomas and hybridomas that synthesize Ig heavy chains with well characterized deletions. Mutant Ig heavy chains that lack the CH1 domain could not be demonstrated to associate with BiP, whereas mutant Ig heavy chains with deletions of the CH2 or CH3 domain were still able to associate with BiP. In two light chain negative cell lines that produced heavy chains with deletions of the CH1 domain, free heavy chains were secreted. When Ig assembly and secretion were examined in mutants that did not associate with BiP, and were compared with normal parental lines, it was found that the rate of Ig secretion was increased in the mutant lines and that the Ig molecules were secreted in various stages of assembly. In one mutant line (CH1-) approximately one-third of the secreted Ig molecules were incompletely assembled, whereas the Ig molecules secreted by the parental line were completely assembled. Our data show the CH1 domain to be important for association with BiP and that when this association does not occur, incompletely assembled heavy chains can be secreted. This implies a role for BiP in preventing the transport of unassembled Ig molecules from the ER.

Analysis of the interaction of antibodies with immunoglobulin idiotypes on neoplastic B lymphocytes: implications for immunotherapy.

We have examined the reactions of a panel of nine monoclonal anti-idiotype antibodies with the surface immunoglobulin in situ on guinea pig L2C leukemic lymphocytes. Equilibrium binding constants were shown to range between 10(7) and 10(8) M-1 for univalent Fab' gamma fragments and between 10(8) and 10(9) M-1 for intact IgG. Saturation of the cell surface binding sites was achieved with 2.9 X 10(5) Fab' gamma molecules/cell and 1.2 X 10(5) IgG molecules/cell for each antibody, a result that is consistent with a bivalent mode of interaction for the IgG. Despite these overall similarities in binding characteristics antibodies showed striking differences in their ability to clear Ig from the cell surface by antigenic modulation in vitro. This suggested differences in the readiness with which the antibodies cross-linked neighboring surface Ig molecules. Such an interpretation was supported by differences in the times required to achieve bivalent binding at 0 degree C, and in the rates at which labeled antibody dissociated from the cell surface in the presence or absence of an excess of unlabeled antibody. The data are consistent with there being two functionally distinct types of anti-idiotype antibody: those that form predominantly intra-Ig bridges, with each antibody Fab being linked to an Fab on one target molecule ("monogamous" binding) and not favoring modulation; and those that form predominantly inter-Ig bridges ("bigamous" binding) and favor modulation. The nature of interaction is presumably dictated by the orientation of the particular idiotope concerned. This distinction could be of great importance in the therapeutic use of anti-idiotype to ablate B cell neoplasms.

The role of immunoglobulin heavy chain binding protein: in immunoglobulin transport

More than ten years ago a heavy chain binding protein (BiP) was described which is associated with immunoglobulin heavy chains (HC) within the endoplasmic reticulum (ER) [which is the site of Immunoglobulin (Ig) assembly]. Recently, Linda Hendershot and her collegues suggested that BiP might combine with nascent HC as they enter the ER and hold them there until assembly with light chain (LC) occurs. In the absence of LC synthesis or assembly, the HC would remain associated with BiP and would eventually be degraded internally. They now propose a means for BiP to block the transport of unassembled Ig molecules. Transport of protein from ER to the Golgi apparatus seems to be mediated by transport signals inherent to the protein molecule itself. Ig transport signals have been thought to be on the LC because LC can be secreted alone while HC cannot under normal circumstances. When BiP is displaced by LC, completed Ig molecules are transported. They use this model to explain regulated transport of Ig molecules during B-cell development, and suggest that BiP may be post-translationally associated with the nascent chains of other membrane and secretory proteins before folding or subunit assembly.

Differentiation of murine B-cell progenitors in agar culture: determination of the developmental potential of clonable pre-B cells.

The differentiation of stem cells into mature B lymphocytes comprises a sequence of developmental steps which leads to cells expressing unique immunoglobulin (Ig) molecules. Much has been learned about intermediate stages in this pathway through the application of a variety of experimental approaches to both transformed cell lines and populations of normal B-cell precursors.

Involvement of the D segment (DQ52) nearest to the JH region in immunoglobulin gene rearrangements of lymphoid-cell precursors.

The specific antigen recognition molecules expressed by B and T lymphocytes, the immunoglobulins (Ig) and the T-cell receptor for antigen (TCR) are coded for by genes which are assembled in an ordered series of somatic DNA recombination events during lymphocyte differentiation. In B lymphocytes the heavy chain gene of the Ig molecule (IgH) is the first to be assembled and this occurs by two successive DNA rearrangements in which first a diversity segment (D) and then a variable gene segment (V) are joined, usually by a process of intrachromosomal deletion, to a joining segment (J) to form a complete variable region sequence (for a review, see Alt et al. 1986). A similar sequence of DNA recombinations leads to the assembly of variable region sequences from V, D and J gene segments in the TCR genes (for a recent review, see Kronenberg et al. 1986).

Biological Activities Residing in the Fc Region of Immunoglobulin

This chapter discusses studies involving lymphocyte activation and immune regulation by peptides derived from IC and Fc fragments of immunoglobulin (Ig). The cellular and subcellular events involved in lymphocyte activation by these biologically active peptides are discussed. There are five major classes of mammalian Ig: IgG, IgM, IgA, IgD, and IgE. The fundamental structure of the Ig molecule consists of two identical light (L) and two identical heavy (H) chains. IgG can be cleaved into smaller fragments by a variety of enzymes other than papain. Pepsin cleaves Ig on the carboxy terminal side of the inter-H chain disulfide bonds, resulting in the formation of a large F (ab')2 fragment. The Fc fragment is degraded by pepsin into heterogeneous peptides. The Fc region of the Ig molecule is involved in a wide variety of biological effector functions that include control of catabolic rate, C fixation, binding to FcR, anaphylaxis, opsonization, placental and gut transfer, and immune regulation. Because the cellular events in lymphocyte activation by Fc fragments and IC appear to be identical, perhaps immunoregulation by these agents occurs via similar pathways. The release of biologically active peptides derived from the Fc fragment of antibody may be part of a potent nonspecific in vivo immunoregulatory network. In addition to the release of Fc region peptides, IC formation also results in C activation with the subsequent release of C fragments (C3a and C5a) that possess immunoregulatory properties.