Production of the chemokine RANTES by articular chondrocytes and role in cartilage degradation.

OBJECTIVE: To examine the expression of the chemokine RANTES and its receptors in normal and osteoarthritic (OA) human cartilage and to analyze its effects on chondrocyte function. METHODS: The expression of RANTES and its receptors were examined by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. The effect of RANTES on gene expression of other cytokines and on the release of mediators of cartilage degradation was also examined by PCR and enzyme-linked immunosorbent assay. RESULTS: The expression of RANTES was undetectable in normal chondrocytes until after stimulation with interleukin-1beta (IL-1beta) or IL-18. Cultures of normal cartilage also produced RANTES in response to IL-1beta, as demonstrated by immunohistochemistry. All OA cartilage samples analyzed expressed RANTES messenger RNA (mRNA); RANTES protein was detected by immunohistochemistry in the superficial and mid zones of the tissue. OA chondrocytes produced elevated levels of RANTES constitutively and after IL-1beta stimulation. Normal cartilage expressed the RANTES receptors CCR3 and CCR5, but not CCR1. CCR1 was expressed in OA cartilage, and CCR3 and CCR5 were increased. In normal chondrocytes, RANTES induced the expression of inducible nitric oxide synthase and IL-6. RANTES stimulated the release of matrix metalloproteinase 1 in normal and OA chondrocytes as effectively as IL-1beta. Treatment of normal articular cartilage with RANTES increased the release of glycosaminoglycans and profoundly reduced the intensity of Safranin O staining. CONCLUSION: Chondrocytes produce RANTES and express RANTES receptors. RANTES and CCR5 were markedly increased in OA and after in vitro treatment of normal chondrocytes with IL-1. Chondrocyte activation and cartilage degradation were identified as novel biologic and pathogenetic activities of this chemokine.

Regulation of YKL-40 production by human articular chondrocytes.

OBJECTIVE: YKL-40 (human cartilage glycoprotein 39) is one of the most abundant proteins secreted by cultured chondrocytes. The objectives of the present study were to identify regulators of YKL-40 production in cartilage and chondrocytes and to map the localization of YKL-40 in chondrocytes. METHODS: Human articular chondrocytes and cartilage explants (obtained from subjects at autopsy, from a tissue bank, and from osteoarthritis [OA] patients undergoing total joint replacement surgery) were stimulated with cytokines, growth factors, and other agents. YKL-40 expression was analyzed by Northern blot and polymerase chain reaction. YKL-40 secretion into the media was determined by enzyme-linked immunosorbent assay. RESULTS: YKL-40 production increased to very high levels during the early phase of chondrocyte monolayer culture and in normal cartilage explant cultures as a response to tissue injury. Spontaneous YKL-40 release was higher in OA than in normal cartilage explant cultures. In chondrocyte monolayer cultures, interleukin-1beta (IL-1beta) and transforming growth factor beta (TGFbeta) decreased the levels of secreted YKL-40, and this was associated with a reduction in YKL-40 messenger RNA levels. IL-1beta, but not TGFbeta, reduced YKL-40 production in cartilage explant cultures. Media from explants treated with cycloheximide had no detectable YKL-40, suggesting that the released protein was newly synthesized. Immunofluorescence microscopy showed YKL-40 staining in the Golgi system of the chondrocytes, but YKL-40 could not be detected in the extracellular matrix. CONCLUSION: The spontaneous increase in the production of YKL-40 in the early phase of culture appears to represent a cellular response to changes in the extracellular matrix environment. This, coupled with the profound suppressive effects of IL-1beta and TGFbeta on YKL-40 production, identifies a novel regulatory pattern for this major chondrocyte-derived protein.

Anticardiolipin antibodies from patients with the antiphospholipid antibody syndrome recognize epitopes in both beta(2)-glycoprotein 1 and oxidized low-density lipoprotein.

BACKGROUND: We recently suggested that many anticardiolipin antibodies bind only to oxidized cardiolipin (OxCL) and/or to OxCL-beta(2)-glycoprotein 1 (beta(2)GP1) adducts but not to a "reduced" cardiolipin that is unable to undergo oxidation. To test this hypothesis, we investigated 24 sera, 4 protein A-purified IgG fractions, and 3 human monoclonal antibodies that were all isolated from patients with antiphospholipid antibody syndrome (APS); testing was also performed in 7 controls. Two monoclonal antibodies (IS3 and IS4) were selected for binding to CL and one was selected for binding to beta(2)GP1 (LJB8). METHODS AND RESULTS: By chemiluminescent immunoassay, all APS sera samples bound only to OxCL and not to reduced CL, and the binding was inhibited >95% by OxCL but not reduced CL. All purified IgG fractions bound to beta(2)GP1 but only when the beta(2)GP1 was plated on microtiter wells coated with OxCL. All 3 monoclonal antibodies bound only to OxCL. On Western blots, IS4 and LJB8 bound to beta(2)GP1 as well as to delipidated apoB of oxidized LDL but not to native apoB. IS3 also bound to oxidized apoB on Western blot. Covalent modification of beta(2)GP1 with oxidation products of CL made it more antigenic for APS serum samples, for purified IgG fractions, and for the monoclonal antibodies. CONCLUSIONS: These data support the hypothesis that oxidation of CL is needed to generate epitopes for many anticardiolipin antibodies and that some of these epitopes are covalent adducts of OxCL with beta(2)GP1 or apoB.

The osteoprotegerin/receptor activator of nuclear factor kappaB/receptor activator of nuclear factor kappaB ligand system in cartilage.

OBJECTIVE: The receptor activator of nuclear factor kappaB (RANK) is a member of the tumor necrosis factor receptor family. It is activated by the secreted or cell surface-bound RANK ligand (RANKL). Osteoprotegerin (OPG) is a soluble nonsignaling receptor for RANKL and interferes with RANK activation. This receptor-ligand system regulates the differentiation of osteoclasts and dendritic cells. The present study examined human articular cartilage for the expression of these molecules and the role of RANKL in the regulation of chondrocyte function. METHODS: Normal and osteoarthritic (OA) human articular cartilage was used for explant tissue culture or for isolation of chondrocytes and cell culture. Expression of RANK, RANKL, and OPG was analyzed by immunohistochemistry, Western blotting, or reverse transcription-polymerase chain reaction. Recombinant RANKL was added to cartilage or chondrocyte cultures, and gene expression, collagenase and nitric oxide production, and NF-kappaB activation were determined. RESULTS: RANK, RANKL, and OPG messenger RNA (mRNA) were expressed in normal cartilage. By immunohistochemistry, RANK, RANKL, and OPG were detected in the superficial zone of normal cartilage. OA cartilage contained increased levels of OPG mRNA, and expression of the 3 proteins extended into the midzone of OA cartilage. OPG was detected by Western blotting, and was increased in response to interleukin-1beta stimulation. OPG, RANK, and RANKL protein were also detected in cultured chondrocytes. Addition of exogenous RANKL did not activate NF-kappaB, induce expression of genes encoding proinflammatory mediators in chondrocytes, or stimulate the production of collagenase and nitric oxide. CONCLUSION: These results demonstrate the expression of OPG, RANK, and RANKL in cartilage. However, RANKL does not activate human articular chondrocytes.

Functional analyses of patient-derived IgG monoclonal anticardiolipin antibodies using in vivo thrombosis and in vivo microcirculation models.

Antiphospholipid antibodies (aPL) have been associated with thrombosis and pregnancy losses in patients diagnosed with antiphospholipid syndrome (APS) and enhance thrombus formation in vivo in mice, but the mechanism of thrombosis by aPL is not completely understood. It has been proposed that aPL may affect endothelial cell (EC) function and/or induce their activation, transforming their anticoagulant surface into procoagulant, thus predisposing to thrombosis. It has been proposed that aPL may affect EC cell function and/or induce their activation, transforming their anticoagulant surface into procoagulant, thus predisposing to thrombosis. This study proposes to test the hypotheses that some IgG anticardiolipins (IgG aCL) with thrombogenic properties in mice, exert their effects through activation of endothelium. We studied seven patient-derived monoclonal aCL for their thrombogenic properties in an in vivo pinch-induced thrombosis model, and their functional activities in activating EC by analyzing in vivo leukocyte adhesion to endothelium in microcirculation in venules in exposed murine cremaster muscle and in vitro adhesion molecule expression in cultured EC. The binding of the monoclonal aCL to EC was also tested. In addition to the previous identified thrombogenic IS2, four of the five new more IgG monoclonal aCL (from two patients) were found to be thrombogenic. Of these five thrombogenic aCL, three caused more in vivo leukocyte adhesion to EC in microcirculation, as compared to that induced by the H2 control human monoclonal IgG, and enhanced expression of adhesion molecules (particularly VCAM-1) on cultured EC. These data show that about 2/3 patient-derived IgG monoclonal aCL are thrombogenic and suggest that some thrombogenic IgG aCL exert their effects through activating EC.

Characterization of IgG monoclonal anti-cardiolipin/anti-beta2GP1 antibodies from two patients with antiphospholipid syndrome reveals three species of antibodies.

Antiphospholipid antibodies (aPL), including antibodies detected in anti-cardiolipin (aCL) enzyme-linked immunosorbent assays and in lupus anticoagulant (LA) tests, are strongly associated with recurrent thrombosis and recurrent fetal loss, i.e. the antiphospholipid syndrome (APS). Although recent studies suggest that most APS-associated aCL are directed against the phospholipid (PL)-binding plasma protein beta2-glycoprotein 1 (beta2GP1), the precise nature of aCL binding specificities remains controversial. To address the issue of aCL specificity we generated five new monoclonal IgG aCL from two patients with APS. Characterization of these five aCL, as well as two previously published IgG aCL, revealed three patterns of reactivity: (1) four antibodies reacted strongly with human beta2GP1-cardiolipin (CL) complexes and weakly with human beta2GP1 alone; (2) two antibodies recognized bovine beta2GP1, but not human beta2GP1; (3) one antibody reacted with complexes of human beta2GP1 and CL, but not with human beta2GP1 alone. Only one monoclonal displayed weak LA activity. These patient-derived IgG monoclonal antibodies, and additional ones to be generated, may help define varying species of antibodies detected in aCL assays and identify the specific antibodies that may be pathogenic.

IL-18 is produced by articular chondrocytes and induces proinflammatory and catabolic responses.

IL-18, a cytokine originally identified as IFN-gamma-inducing factor, is a member of the IL-1 family of proteins. Because IL-1alpha and IL-1beta are important mediators in the pathogenesis of arthritis, the present study addresses the expression of IL-18 and its role in regulating in articular chondrocytes. IL-18 mRNA was induced by IL-1beta in chondrocytes. Chondrocytes produced the IL-18 precursor and in response to IL-1 stimulation secreted the mature form of IL-18. Studies on IL-18 effects on chondrocytes showed that it inhibits TGF-beta-induced proliferation and enhances nitric oxide production. IL-18 stimulated the expression of several genes in normal human articular chondrocytes including inducible nitric oxide synthase, inducible cyclooxygenase, IL-6, and stromelysin. Gene expression was associated with the synthesis of the corresponding proteins. Treatment of normal human articular cartilage with IL-18 increased the release of glycosaminoglycans. These finding identify IL-18 as a cytokine that regulates chondrocyte responses and contributes to cartilage degradation.

Generation and analysis of an IgG anti-platelet autoantibody reveals unusual molecular features.

Although serum transfer studies implicate IgG anti-platelet autoantibodies in the premature platelet destruction of idiopathic thrombocytopenic purpura (ITP), many characteristics of these putative pathogenic autoantibodies remain unclear. The inability to obtain relevant monoclonal autoantibodies from patients has prevented their molecular, genetic and functional studies as a homogenous population. We have generated a monoclonal IgG anti-platelet alpha IIb beta 3 autoantibody (termed G1) from an ITP patient. G1 binds human platelets (both resting and activated) and purified alpha IIb beta 3 with a Kd of 1.57 x 10(-8) M. G1 utilizes VH4 and V lambda 2 genes. The G1 VH region apparently has a 30 nucleotide insertion in its second complementarity determining region (CDR). Notably, somatic CDR insertion in the VH region has been observed only in one IgG rheumatoid factor, and not in any characterized polyreactive human autoantibodies reported in the literature. Combined these data suggest G1 may be a disease-relevant autoantibody. Further generation and study of monoclonal IgG anti-platelet antibodies are warranted to determine the significance of such unusual autoantibodies in the immunopathogenesis of chronic ITP.

IgG rheumatoid factors isolated by the surface-displaying phage library technique.

Our analysis of IgG rheumatoid factors (RFs) from three patients with rheumatoid arthritis (RA) revealed that most contained significant numbers of skewed mutations per V region, suggesting that these RFs arose from antigen-driven responses. To further study IgG RFs in RA, we used pComb3 vector to construct an IgG1,lambda combinatorial antibody library from a synovial fluid sample. After panning against human IgG, Fab fragments from 71/96 phage clones bound to Fc-coated wells. Sequence analysis of 20 randomly chosen Fc-binders showed that 17 (85%) clones had identical heavy (H) and light (L) chain V regions, represented by Humha311 and Humla211, respectively. Of the remaining three clones, two had the same Humla211 L chain, but each with a different H chain V region. All the putative germline V genes for these RFs also encode RF in RA patients. However, none of these RF V regions are similar to those of the two IgG RFs derived by the hybridoma technique from the same synovial fluid. The Humha311 H chain has two frameshifts: a one-base insertion upstream of the JH region and a four-base deletion near the end of the CH1 region, resulting in a mainly unconventional amino acid sequence in the CH1 region. In the future, it will be important to study the presence of IgG molecules with such unconventional CH1 amino acid sequences, and the effects of these amino acid sequences on the structures and immunological properties of the IgG molecules.

A monoclonal IgG anticardiolipin antibody from a patient with the antiphospholipid syndrome is thrombogenic in mice.

Antiphospholipid antibodies, including anticardiolipin antibodies (ACA), are strongly associated with recurrent thrombosis in patients with the antiphospholipid syndrome (APS). To date, reports about the binding specificities of ACA and their role(s) in causing and/or sustaining thrombosis in APS are conflicting and controversial. The plasmas of patients with APS, usually containing a mixture of autoantibodies, vary in binding specificity for different phospholipids/cofactors and vary in in vitro lupus anticoagulant activity. Although in vivo assays that allow assessment of the pathogenic procoagulant activity of patient autoantibodies have recently been developed, the complex nature of the mixed species prevented determination of the particular species responsible for in vivo thrombosis. We have generated two human IgG monoclonal ACA from an APS patient with recurrent thrombosis. Both bound to cardiolipin in the presence of 10% bovine serum, but not in its absence, and both were reactive against phosphatidic acid, but were nonreactive against purified human beta-2 glycoprotein 1, DNA, heparan sulfate, or four other test antigens. Both monoclonal autoantibodies lacked lupus anticoagulant activity and did not inhibit prothrombinase activity. Remarkably, one of the monoclonal antibodies has thrombogenic properties when tested in an in vivo mouse model. This finding provides the first direct evidence that a particular antiphospholipid antibody specificity may contribute to in vivo thrombosis.

The frequency of homozygous deletion of a developmentally regulated Vh gene (Humhv3005) is increased in patients with chronic idiopathic thrombocytopenic purpura.

Little is known of the genetic factors that may contribute to the development of chronic idiopathic thrombocytopenic purpura (cITP). We have previously shown that a developmentally regulated Vh gene (Humhv3005) is absent in 10/41 (24%) of patients with systemic lupus erythematosus while it is absent in only 7/88 (8%) of normal controls. This finding suggests that a homozygous deletion of an Ig variable (V) gene may alter the immune system and thus predispose the host to an autoimmune disorder. We have analyzed the same gene in 44 patients with cITP and found that Humhv3005 and like genes were absent in a higher percentage of patients (14 of 44, 31.8%) than they were absent in either normals (7/88, 8%, p = 0.002) or thrombocytopenic patients without cITP (6/53, 11.3%, p = 0.042); the hv3005 deletion frequency in the latter group did not differ from that in normals (P = 0.74). These data suggest that deletions of Humhv3005 and/or highly homologous Vh genes may predispose individuals to the development of cITP, and may contribute toward production of pathogenic antiplatelet antibodies.

Population and family studies of three disease-related polymorphic genes in systemic lupus erythematosus.

The contribution to systemic lupus erythematosus (SLE) of three lupus-associated polymorphisms (involving the C4A2 complement component, Humhv3005 and the T cell antigen receptor alpha chain gene) are investigated in 81 individuals from 14 multiplex SLE families, 41 unrelated lupus patients, and 88 unrelated healthy controls. The results show a strong association between C4A deletion and SLE in these families. While the current study confirms the previously reported association between hv3005 deletion and sporadic SLE, the study fails to support this association in familial SLE patients. Moreover, no correlation is detected between the occurrence of hv3005 deletion and C4A null alleles in lupus patients, suggesting that the effects of these genetic polymorphisms on predisposition to lupus are independent. The previously reported lupus-associated T cell receptor (TCR) alpha chain polymorphism is not detected in any of the individuals studied here. The combined data suggest that C4A null alleles predispose strongly to development of lupus, whereas the influence of hv3005 deletion is relatively weak. The results also suggest that contributions of weak susceptibility genes such as hv3005 to disease predisposition may be obscured by the effects of stronger genetic factors and thus need to be examined in patients lacking these factors.

Bacterial lipopolysaccharide copurifies with plasmid DNA: implications for animal models and human gene therapy.

During the course of gene therapy experiments in rodents, using intramuscular injections of plasmid DNA derived from Escherichia coli, we noted dose-related toxicity. This observation prompted a search for possible contaminants of DNA samples. We used the highly specific and sensitive limulus amoebocyte lysate assay (LAL), to monitor endotoxin bioactivity in DNA samples, and found plasmid DNA derived from standard E. coli bacterial strains, using traditional DNA isolation protocols, to be heavily contaminated with endotoxin, or lipopolysaccharide (LPA). Standard DNA isolation procedures resulted in the copurification of up to 500 micrograms/ml of LPS. LPS is a potent inducer of cytokines and other inflammatory mediators, and may complicate the use of naked DNA in gene therapy. The copurification of endotoxin with plasmid DNA also has important implications for in vitro transfection studies and microinjection of DNA into embryos. A simple and efficient protocol to reduce LPS contamination of plasmid DNA was developed. The conversion of intact bacteria to spheroplasts prior to the isolation of plasmid DNA, incubation with lysozyme, treatment with the detergent n-octyl-beta-D-thioglucopyranoside (OSPG) and polymyxin-B (PMB) chromatography, allowed the isolation of plasmid DNA containing less than 50 ng/ml LPS. This represents a 10,000-fold reduction in LPS contamination, compared to conventional methods of plasmid DNA purification, avoids potentially toxic reagents such as ethidium bromide, and produces a higher yield of plasmid DNA.

Defining the genetic origins of three rheumatoid synovium-derived IgG rheumatoid factors.

A major diagnostic marker in most rheumatoid arthritis (RA) patients is the rheumatoid factor (RF), an autoantibody that binds to the Fc region of IgG. To delineate the Ig genes and the underlying mechanism for RF production in RA patients, we applied a systematic approach to define the genetic origins of three IgG RFs derived from the synovial fluid of two RA patients. The results show that two of three IgG RF have substantial numbers of somatic mutations in their variable (V) regions, ranging from 13 to 23 mutations over a stretch of 291-313 nucleotides, resulting in a frequency of 4.4-7.8%. However, one IgG RF has only one mutation in each V region. This result indicates that an IgG RF may arise from a germline gene by very few mutations. The mutations occur mainly in the complementarity-determining regions (CDRs), and the mutations in the CDRs often lead to amino acid substitutions. Five of the six corresponding germline V genes have been found to encode either natural autoantibodies or autoantibodies in other autoimmune disorders; and three of the six V genes have been found in fetal liver. Taken together with other results, the data show that (a) several potentially pathogenic RFs in RA patients arise from natural autoantibodies, and (b) only a few mutations are required to convert the natural autoantibodies to IgG RFs.

Utilization of a potentially universal downstream primer in the rapid identification and characterization of V lambda genes from two new human V lambda gene families.

Polymerase chain reaction (PCR) has increased dramatically the speed of cloning and characterizing numerous genes. However, its application to identifying and analysing new germline Ig-variable (V) gene families has been hampered by the lack of sequence information in the downstream flanking regions of the concerned V genes, which are deleted during V(D)J rearrangements. To circumvent this problem, the possibility was explored that a degenerate downstream primer may be used in conjunction with a specific upstream primer, to clone members of new V lambda gene families, as much less is known about V lambda genes than Vh and Vk genes in humans. Firstly the feasibility and the specificity of a degenerate primer was examined by comparing it with an established downstream primer in amplifying known V lambda 1 genes. The results were positive. Thus, the degenerate primer was used to clone and characterize germline V lambda genes of the recently defined V lambda 8 and V lambda 9 gene families. This current strategy may help speed up the identification and characterization of all human V lambda genes. Moreover, a similar strategy can be applied to identify and characterize rapidly new V genes of either known or unknown Ig and T-cell receptor V gene families.

Genetic analysis of the variable region genes encoding a monospecific human natural anti-DNA antibody.

Recent evidence suggests that natural autoantibodies may play an integral role in the development of the normal immune repertoire. To explore the genetic origins of these antibodies, we have isolated and sequenced the variable (V) region genes encoding both the heavy (H) and light (L) chains of a natural anti-DNA antibody, Kim11.4. The genes appear to be derived from the VH4.18 (subgroup VHIV), JH5, Hum1L1 (subgroup V lambda I) and J lambda 3 germline genes. The origin of the H chain diversity gene is more obscure, being potentially derived from one or more of several germline genes, arranged in either the forward or reverse orientations. Both the Kim11.4 VH and VL genes share significant degrees of similarity with those utilized in other autoantibodies, indicating that at least some degree of V restriction may exist in human autoreactive B cells. The pattern of nucleotide differences between the Kim11.4 VH and VL genes and their putative germline counterparts suggests that the Kim11.4 genes may have undergone somatic mutation and arisen as a result of antigen selection.

Generation and molecular analyses of two rheumatoid synovial fluid-derived IgG rheumatoid factors.

OBJECTIVE: To study the Ig genes that encode IgG rheumatoid factor (IgG-RF) from rheumatoid synovial fluid. METHODS: We used rheumatoid synovial fluid B cells to generate IgG-RF-secreting hybridomas. We then characterized their binding properties and determined their nucleotide sequences. RESULTS: Two monospecific IgG-RFs were obtained. Sequence analysis of the RFs revealed a new V lambda gene family (designated V lambda 9) and extensive somatic diversification, including a duplication-insertion of 18 nucleotides (6 amino acid residues) into a hypervariable region. CONCLUSION: The data provide further support for an antigen-driven response in the sustained production of potentially pathogenic IgG-RFs in rheumatoid synovium.

Characterization of two highly homologous autoantibody-related VH1 genes in humans.

Previously, we showed that the Humha 1lr rearranged gene was almost identical to the consensus amino acid sequence of several G6 idiotype-positive rheumatoid factor (RF) heavy chains, and to the VH gene-encoded region of the fetally expressed 51P1 cDNA. The finding led us to suggest that the ha 1lr-corresponding germline gene encodes the heavy chains of many human IgM RFs. We now report the isolation of the proposed germline gene, designated Humhv1051; it is identical to the consensus sequence of the G6 heavy chain V regions. During this experiment, we also isolated unexpectedly an additional VH1 gene, termed Humhv1051K; it differs from hv1051 by one amino acid residue. Importantly, hv1051K is identical to Humha113, the rearranged VH1 gene of a natural anti-cardiolipin antibody.

Sequence analyses of three immunoglobulin G anti-virus antibodies reveal their utilization of autoantibody-related immunoglobulin Vh genes, but not V lambda genes.

Accumulated sequence analyses of the antibody repertoire have revealed that most autoantibodies and developmentally regulated antibodies share a small set of germline Ig-variable region (V) genes. The findings have prompted speculation that certain autoantibodies are of developmental importance and may be instrumental in maintaining homeostasis of the adult antibody repertoire. In order to evaluate this hypothesis critically, it is first necessary to determine the V gene usage in human antibodies against foreign substances. Unfortunately, only a few such antibodies have had their heavy and light chains characterized. To rectify the situation, we adapted the anchored polymerase chain reaction to clone and analyze rapidly the expressed V genes for three anti-virus IgG antibodies. The results show that all three heavy chain V (Vh) genes are highly homologous to the known autoantibody-related Vh genes. In contrast, two light chain V (VL) genes of the V lambda 1 subgroup are similar to a non-autoantibody-related germline V lambda 1 gene. Taken together with the reported Vh and VL sequences of several antibodies against viruses and bacteria, the data show that many antipathogen antibodies may use the same small set of Vh genes that encode autoantibodies, but diverse VL genes that are distinct from autoantibody-related VL genes. Thus, only a small portion of the potentially functional germline Vh genes are used recurrently to generate most antibodies in a normal antibody repertoire, regardless of their reactivities with either self or non-self.

Molecular characterization of the human immunoglobulin V lambda I germline gene repertoire.

To advance our understanding of the human immunoglobulin V lambda germline gene contribution to normal as well as autoimmune responses, we have isolated and sequenced six germline genes of the V lambda I subgroup. These genes can be divided into three sub-subgroups on the basis of greater than or equal to 93% nucleotide sequence homology and greater than or equal to 88% deduced amino acid sequence similarity. Examination of all cDNA and protein sequences available for expressed V lambda I genes supports the assignment of these three sub-subgroups. Sequence comparisons also suggest that germline gene members of two of these sub-subgroups, I-a and I-b, are preferentially utilized in the expressed V lambda I repertoire. This finding may be at least partially attributable to regulatory sequence abnormalities apparent in two of the other V lambda I germline genes (Humlv101 and Humlv104) which may interfere with their expression.