Cellular immunity to the G1 domain of cartilage proteoglycan aggrecan is enhanced in patients with rheumatoid arthritis but only after removal of keratan sulfate

OBJECTIVE: To determine whether patients with rheumatoid arthritis (RA) express cellular immunity to the purified G1 globular domain of cartilage proteoglycan (PG) aggrecan and whether it is influenced by the removal of keratan sulfate (KS) chains from the molecule. METHODS: The G1 globular domain of PG was purified from mature bovine articular cartilage, digested with keratanase, and used in proliferation assays with peripheral blood lymphocytes (PBL) isolated from 43 patients with RA, 11 patients with nonarticular rheumatism (NAR), including soft tissue rheumatism and mechanical back pain, and 13 healthy age- and sex-matched control subjects. RESULTS: Removal of KS chains from the G1 globular domain resulted in significantly increased prevalence and values of cellular immune responses to G1 in RA patients compared with the control and NAR groups. In the majority of RA patients, KS chains on G1 significantly inhibited its immune recognition by PBL. There was no significant effect of KS removal on the immunity to G1 in patients with NAR and in the healthy control group. CONCLUSION: These results reveal that immune reactivity to the G1 globular domain of the cartilage PG aggrecan is enhanced in patients with RA but only when KS chains are removed. Thus, KS chains inhibit immune responses to this domain of aggrecan. Since immunity to the G1 globular domain of aggrecan induces an erosive polyarthritis in BALB/c mice after removal of KS chains, immunity to the G1 globular domain, cleaved by proteases to remove KS chains, may play a role in the pathogenesis of RA.     

Characterization of aggrecan retained and lost from the extracellular matrix of articular cartilage. Involvement of carboxyl-terminal processing in the catabolism of aggrecan

The catabolism of aggrecan in bovine articular cartilage explants is characterized by the release into the culture medium of high molecular weight aggrecan fragments, generated by the proteolytic cleavage of the core protein between residues Glu373 and Ala374 within the interglobular domain. In this study, the position of the carboxyl-terminus of these aggrecan fragments, as well as a major proteolytically shortened aggrecan core protein present in cartilage matrix, have been deduced by characterizing the peptides generated by the reaction of aggrecan core protein peptides with cyanogen bromide. It was shown that two out of three such peptide fragments having an amino terminus starting at Ala374 have their carboxyl terminus located within the chondroitin sulfate 1 domain. The third and largest aggrecan core protein peptide, with an amino terminus starting at Ala374, has a carboxyl terminus in a region of core protein between the chondroitin sulfate 1 domain and the chondroitin sulfate 2 domain. The carboxyl terminus of this peptide appeared to be the same as that of the proteolytically degraded aggrecan core protein, which is retained within the extracellular matrix of the tissue. Another two aggrecan fragments recovered from the medium of explant cultures with amino-terminal sequences in the chondroitin sulfate 2 domain at Ala1772 and Leu1872 were shown to have their carboxyl termini within the G3 globular domain. These results suggest that the catabolism of aggrecan between residues Glu373 and Ala374 in the interglobular domain by the putative proteinase, aggrecanase, may be dependent on prior proteolytic processing within the carboxyl-terminal region of the core protein.     

Cell-mediated autoimmunity in patients with Wegener's granulomatosis (WG)

Despite the well described infiltration of cells of the cellular immune system in vasculitic lesions and the granuloma formation in patients with WG, the role of T cell-mediated autoimmunity in WG is not clear. Reports of T cell proliferation in response to neutrophil azurophilic granule proteins are contradictory. In this study we have assessed the proliferation of T cells of WG patients to purified proteinase 3 (PR3) and to total azurophilic granule proteins in two different assays. In addition to the classical proliferation assay with isolated peripheral blood mononuclear cells, we have used a whole blood proliferation assay. In both assays we found proliferative responses to PR3 in patients with WG. The number of patients reacting to the azurophilic granule extract was higher than the patients reacting to the purified PR3, suggesting that other autoantigens may also be involved. We have identified epitopes of PR3 that may be potential targets of class I-restricted T cell responses in the context of HLA-A*0201, the most common MHC class I molecule. These epitopes were determined by the binding of synthetic PR3 peptides to HLA-A*0201 on the antigen-processing defective cell line, T2. In addition, T cell lines were established from tissue biopsies, obtained from WG patients, and assessed for cytolytic reactivity against T2 cells, preloaded with synthetic PR3 peptides. We conclude that T lymphocytes of WG patients have increased proliferative responses to purified PR3 and to a larger extent to non-fractionated proteins of azurophilic granules of polymorphonuclear neutrophilic leucocytes (PMN).     

Progressive polyarthritis induced in BALB/c mice by aggrecan from normal and osteoarthritic human cartilage

OBJECTIVE: To find an "unlimited" source of antigenic material (aggrecan) for arthritis induction in BALB/c mice; to analyze the specificities of immune reactions to aggrecan and type II collagen in 2 arthritis-susceptible murine strains, BALB/c mice for proteoglycan (aggrecan)-induced arthritis and DBA/1j mice for collagen-induced arthritis; to compare the histopathologic features of arthritis induced by purified aggrecans or total extracts of osteoarthritic (OA) cartilage; and to determine arthritis susceptibility in various BALB/c colonies. METHODS: Aggrecans from total extracts of human fetal, normal adult, OA, and rheumatoid cartilage samples and from osteophytes were isolated, purified by gradient centrifugation, deglycosylated, characterized, and tested for arthritis induction. Purified type II collagen and salt-soluble collagens from OA cartilage were denatured, stromelysin treated, and used for immunization and arthritis induction in arthritis-susceptible (DBA/1j and BALB/c) murine strains. RESULTS: Chondrocytes from OA cartilage synthesize predominantly fetal-type aggrecan, which is the most efficient antigenic material for arthritis induction in BALB/c mice. The critical autoimmune/arthritogenic T cell epitopes of aggrecan are located in the G1 domain. Although most of the aggrecan molecules are heavily degraded and lost from OA cartilage, the G1 domain-containing fragments accumulate in OA cartilage. The amount of G1-containing fragments is approximately twice as much in OA than in normal adult articular cartilage, and the arthritogenic epitope(s) remains intact in G1-containing fragments retained in cartilage. Thus, total extracts of OA cartilage (without additional purification), if deglycosylated appropriately, can be used as arthritogenic material in BALB/c mice. CONCLUSION: Predominantly G1 domain-containing fragments of aggrecan accumulate in OA cartilage, and these are the fragments which induce arthritis in BALB/c mice. Arthritis induction is highly specific for aggrecan epitopes and dictated by the genetic background of the BALB/c strain.     

Delineation of PLA2 epitopes using short or long overlapping synthetic peptides: interest for specific immunotherapy

BACKGROUND: Venom immunotherapy is definitely indicated in severe systemic anaphylactic reactions to bee stings, but is not devoided of risks of anaphylaxis. Safer methods of immunotherapy need to be developed. OBJECTIVE: To delineate phospholipase A2 T-cell epitopes using short 15mer vs long 40-60mer overlapping peptides, and to approach the potential interest of a venom immunotherapy based on the use of long peptides (1-60, 51-99, 90-134) mapping the whole phospholipase A2 molecule vs a restricted number of immunodominant epitopes. METHODS: Proliferation of a CD8+ T cell depleted peripheral blood mononuclear cell fraction and short-term T-cell lines from unselected bee venom hypersensitive patients in response to phospholipase A2 synthetic peptides. RESULTS: Whereas T-cell proliferation to 15mer overlapping peptides was weak, T-cell response to long overlapping peptides was in contrast vigorous in all patients, mostly directed to C-terminal peptide 90-134. Our results did not support the concept of rare dominant T-cell epitopes, and disclosed T-cell responses to multiple epitopes in several patients. No significant IgE-binding to long overlapping peptides was detected except in one patient against peptide 90-134. CONCLUSION: 15mer peptides might not be sensitive enough to fully delineate all potential T-cell epitopes scattered along the allergen. Since they do not bind IgE in vitro or only weakly, and taking into account a T-cell response frequently directed to multiple epitopes, long overlapping peptides may represent ideal tools for immunotherapy.     

Comparative acute toxicity and primary irritancy of the ethylidene and vinyl isomers of norbornene

Perlecan and aggrecan are proteoglycans that receive primarily heparan sulfate and chondroitin sulfate side chains, respectively. Their large multidomained core proteins have little or no homology to each other and their glycosaminoglycan (GAG) attachment sites are restricted to certain domains only. We examined the involvement of the non-GAG bearing domains in designating the GAG type added to the GAG attachment domain by preparing cDNA constructs that expressed perlecan/aggrecan chimeras as recombinant products in COS-7 cells and then determining the size and GAG composition of the recombinant products. The results showed that domain I of perlecan receives primarily (73-81%) heparan sulfate when coupled with domain II and III of perlecan, but when coupled with the G3 domain of aggrecan, it receives primarily (59-63%) chondroitin sulfate. Furthermore, the chondroitin sulfate attachment region of aggrecan received GAG side chains more readily when coupled to the G3 domain of aggrecan than when coupled to domains II and III of perlecan. The GAG side chains on all these recombinant products were small and similar in size. These findings indicate that the utilization of attachment sites for heparan and chondroitin sulfate or the sulfation of these GAGs can be influenced, in part, by non-GAG bearing domains.     

Comparison of primary sensitization of naive human T cells to varicella-zoster virus peptides by dendritic cells in vitro with responses elicited in vivo by varicella vaccination

Dendritic cells (DC) are potent APC during primary and secondary immune responses. The first objective of this study was to determine whether human DC mediate in vitro sensitization of naive CD4+ T cells to epitopes of the immediate early 62 (IE62) protein of varicella zoster virus (VZV). The induction of CD4+ T cell proliferative responses to eight synthetic peptides representing amino acid sequences of the VZV IE62 protein was assessed using T cells and DC from VZV-susceptible donors. The second objective was to compare in vitro responses of naive T cells with responses to VZV peptides induced in vivo after immunization with varicella vaccine. T cell proliferation was induced by three peptides, P1, P4, and P7, in 71-100% of the donors tested before and after vaccination using DC as APC. Monocytes were effective APC for VZV peptides only after immunization. Two peptides, P2 and P8, induced naive T cell proliferation less effectively and were also less immunogenic for T cells from vaccinated or naturally immune donors. T cell recognition of specific peptides was concordant between naive, DC-mediated responses, and postvaccine responses using monocytes as APC in 69% of comparisons (p = 0.05; chi2); the predictive value of a positive response to an IE62 peptide before immunization for T cell sensitization in vivo was 82%. These observations indicate that primary T cell responses detected in vitro using DC as APC may be useful to characterize the potential immunogenicity of viral protein epitopes in vivo.     

T cell responses to synthetic peptides of herpes simplex virus type 1 glycoprotein D in naturally infected individuals

To locate T cell determinants of glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1), proliferation assays of lymphocytes obtained from 10 healthy HSV-seropositive individuals were performed using 34 overlapping gD peptides as antigens. Despite large differences between individual responses to the peptides both in number of stimulating peptides and gD regions, three regions (1-54, 110-214, and 290-314) induced a response in 50% or more of the HSV-seropositives. T cells were less frequently stimulated by peptides of region 210-294. No correlation was found between serological data and proliferative responses to the peptides. The diversity in T cell response to the peptides suggests a lack of immunodominance, implying that a single peptide/region of gD, or a combination of peptides, will not be sufficient to serve as a basis for a future HSV-1 vaccine.     

Identification of T cells that respond to serovar-specific regions of the Chlamydia trachomatis major outer membrane protein in persons with serovar E infection

Protection from Chlamydia trachomatis infection is serovar-specific and T cell-dependent; however, the T cell epitopes identified to date have not been serovar-specific. The chlamydial major outer membrane protein (MOMP) contains serovar-specific B cell epitopes in four regions of the molecule whose amino acid sequence varies among serovars, the variable segments (VS). Serovar-specific T cell epitopes were sought by examining proliferation of blood mononuclear cells (PBMC) from Chlamydia-infected patients in response to VS peptides of serovar E MOMP. Serovar E-specific peptides from VS1, VS2, and VS4 stimulated PBMC to a greater extent in serovar E-infected than in non-E-infected subjects. Peptides containing constant regions of MOMP were recognized equally by all infected persons. The observed responses were attributable to T cells. T cell recognition of serovar-specific regions of MOMP is common and may contribute to the serovar-specific protection previously observed.     

Immunity to T cell receptor peptides in multiple sclerosis. III. Preferential immunogenicity of complementarity-determining region 2 peptides from disease-associated T cell receptor BV genes

Vaccination with synthetic TCR peptides from the BV5S2 complementarity-determining region 2 (CDR2) can boost significantly the frequency of circulating CD4+ peptide-specific Th2 cells in multiple sclerosis (MS) patients, with an associated decrease in the frequency of myelin basic protein (MBP)-reactive Th1 cells and possible clinical benefit. To evaluate the immunogenicity of CDR2 vs other regions of the TCR, we vaccinated seven MS patients with overlapping BV5S2 peptides spanning amino acids 1-94. Six patients responded to at least one of three overlapping or substituted CDR2 peptides possessing a core epitope of residues 44-52, and one patient also responded to a CDR1 peptide. Of the CDR2 peptides, the substituted (Y49T)BV5S2-38-58 peptide was the most immunogenic but cross-reacted with the native sequence and had the strongest binding affinity for MS-associated HLA-DR2 alleles, suggesting that position 49 is an MHC rather than a TCR contact residue. Two MS patients who did not respond to BV5S2 peptides were immunized successfully with CDR2 peptides from different BV gene families overexpressed by their MBP-specific T cells. Taken together, these results suggest that a widely active vaccine for MS might well involve a limited set of slightly modified CDR2 peptides from BV genes involved in T cell recognition of MBP.     

Clinical applications of registration and fusion of multimodality brain images from PET, SPECT, CT, and MRI

The aim was to assess the specificity and functional significance of liver-infiltrating and peripheral blood T cells in chronic hepatitis C. Peripheral blood mononuclear cells hepatitis C virus from 50 of 58 (86.2%) patients with chronic hepatitis C virus infection and 6 of 28 (21.4%) controls showed a proliferative T cell response to at least one of 16 synthetic peptides covering highly conserved regions of the core, envelope (El) and non-structural regions (NS4) of hepatitis C virus. However, six immunodominant peptides were exclusively recognized by the proliferating blood mononuclear cells from 46 patients with chronic hepatitis C virus infection (79.3%). Fine specificity and HLA-restriction were studied with 15 peptide-specific CD4+ T cell lines and 23 T cell clones isolated from liver tissue and peripheral blood of 12 patients with chronic hepatitis C. It was demonstrated that the peptide-specific response of CD4+ T cells was restricted to the presence of autologous accessory cells and HLA-DR and -DP molecules. Eight peptide-specific T cell lines and five T cell clones derived from liver tissue and peripheral blood, released interferon-gamma (200-6600 pg/ml) and tumor necrosis factor-alpha (100-400 pg/ml) and no or little interleukin-4 (< 140 pg/ml) after peptide-specific or mitogeneic stimulation, thus resembling a Th1-like cytokine profile. Patients with active liver disease showed significantly higher proliferative responses to hepatitis C virus core peptides than asymptomatic hepatitis C virus carriers or complete responders to interferon therapy. In conclusion, class II-restricted CD4+ T cell responses to some immunodominant epitopes within the hepatitis core region correlated with disease activity in chronic hepatitis C virus infection. Functionally, liver-infiltrating and peripheral blood T cells released Th1-like cytokines in response to the specific stimulus. Thus, it can be suggested that CD4+ T cells can mediate the pathogenesis of chronic hepatitis C virus induced liver disease.     

Critical roles of glycosaminoglycan side chains of cartilage proteoglycan (aggrecan) in antigen recognition and presentation

Systemic immunization of BALB/c mice with proteoglycan (aggrecan) from fetal human cartilage induces progressive polyarthritis, an experimental disease similar to human rheumatoid arthritis. The development of the disease in this genetically susceptible murine strain is based on cross-reactive immune responses between the immunizing fetal human and mouse self-proteoglycans. One of the cross-reactive and arthritogenic T cell epitopes (92GR/QVRVNSA/IY) is localized in the G1 domain of human/murine proteoglycan. Susceptible BALB/c mice, however, develop arthritis only if both the chondroitin sulfate (CS) and keratan sulfate (KS) side chains of the arthritogenic human proteoglycans are removed. The function of these two glycosaminoglycan side chains is opposite. The presence of a KS side chain in adult proteoglycan inhibits the recognition of arthritogenic T cell epitopes, prevents the development of T cell response, and protects animals from autoimmune arthritis. In contrast, the depletion of the CS side chain generates clusters of CS stubs and provokes a strong B cell response. These carbohydrate-specific B cells are the most important proteoglycan APC. Taken together, proteoglycan-induced progressive polyarthritis is dictated by three major components: genetic background of the BALB/c strain, highly specific T cell response to epitope(s) masked by a KS chain in aging tissue, and the presence of proteoglycan (CS stub)-specific B cells required for sufficient Ag presentation.     

Characterization of the elastin binding domain in the cell-surface 25-kDa elastin-binding protein of staphylococcus aureus (EbpS)

Our previous studies have established that a cell-surface 25-kDa elastin-binding protein of Staphylococcus aureus (EbpS) mediates binding of this pathogen to the extracellular matrix protein elastin. Results from binding assays examining the activity of various EbpS fragments suggested that the elastin recognition domain is contained within the first 59 amino acids. In this report, we have used functional analyses with synthetic peptides and recombinant truncated forms of EbpS to localize the elastin binding domain to a 21-amino acid region contained within residues 14-34 of EbpS. Further evidence for the importance of this domain was obtained by demonstrating that the inhibitory activity of anti-EbpS antibodies on staphylococcal elastin binding was neutralized when these antibodies were pre-absorbed with a truncated recombinant EbpS construct containing residues 1-34. Overlapping synthetic peptides corresponding to EbpS residues 14-36 were then generated and tested for elastin binding activity to define further the elastin binding domain, and results from these studies showed that sequences spanning amino acids Gln14-Asp23, Asp17-Asp23, and Thr18-Glu34 inhibit binding of Staphylococcus aureus to elastin. Our analyses indicate that the hexameric sequence Thr18-Asn-Ser-His-Gln-Asp23 is the minimal sequence common to all active synthetic peptides, proteolytic fragments, and recombinant constructs of EbpS. Furthermore, substitution of Asp23 with Asn abrogated the blocking activity of the synthetic peptides, demonstrating the requirement for a charged amino acid at this location. The composite data indicate that staphylococcal elastin binding is mediated by a discrete domain defined by short peptide sequences in the amino-terminal extracellular region of EbpS.     

Identification of T-cell epitopes on the Rhesus polypeptides in autoimmune hemolytic anemia

We have shown previously that the Rhesus (Rh) polypeptides are the commonest targets for pathogenic anti-red blood cell (RBC) autoantibodies in patients with autoimmune hemolytic anemia (AIHA). The aim of the current work was to determine whether activated T cells from such patients also mount recall responses to epitopes on these proteins. Two panels of overlapping 15-mer peptides, corresponding to the sequences of the 30-kD Rh proteins associated with expression of the D and Cc/Ee blood group antigens, were synthesized and screened for the ability to stimulate the in vitro proliferation of mononuclear cells from the peripheral blood or spleen of nine AIHA cases. Culture conditions were chosen that favor recall proliferation by previously activated T cells, rather than primary responses. In seven of the patients, including all four cases with autoantibody to the Rh proteins, two or more peptides elicited proliferation, but cells from eight of nine patients with other anemias and seven of nine healthy donors failed to respond to the panels. Multiple peptides were also stimulatory in two positive control donors who had been alloimmunized with Rh D-positive RBCs. Six different profiles of peptides elicited responses in the AIHA patients, and this variation may reflect the different HLA types in the group. Stimulatory peptides were identified throughout domains shared between, or specific to, each of the related 30-kD Rh proteins, but T cells that responded to nonconserved regions did not cross-react with the alternative sequences. Anti-major histocompatibility complex class II antibodies blocked the responses and depletion experiments confirmed that the proliferating mononuclear cells were T cells. Notably, splenic T cells that proliferated against multiple Rh peptides also responded to intact RBCs. We propose that pathogenic autoantibody production in many cases of AIHA is driven by the activation of T-helper cells specific for previously cryptic epitopes on the Rh proteins.     

Autoreactive T cells to platelet GPIIb-IIIa in immune thrombocytopenic purpura. Role in production of anti-platelet autoantibody

T cell proliferative responses to platelet membrane GPIIb-IIIa were examined in 14 patients with chronic immune thrombocytopenic purpura (ITP), 7 systemic lupus erythematosus (SLE) patients with or without thrombocytopenia, and 10 healthy donors. Although peripheral blood T cells from all subjects failed to respond to the protein complex in its native state, reduced GPIIb-IIIa stimulated T cells from three ITP patients and one SLE patient with thrombocytopenia, and tryptic peptides of GPIIb-IIIa stimulated T cells from nearly all subjects. The specificity of the responses for GPIIb-IIIa was confirmed by activation of GPIIb-IIIa-primed T cells by a recombinant GPIIbalpha fragment in secondary cultures. Characterization of T cell response induced by modified GPIIb-IIIa showed that the response was restricted by HLA-DR, the responding T cells had a CD4(+) phenotype, and the proliferation was accelerated only in ITP patients, suggesting in vivo activation of these T cells. In vitro IgG anti-GPIIb-IIIa synthesis in PBMC cultures was induced by modified GPIIb-IIIa specifically in ITP patients with platelet-associated anti-GPIIb-IIIa antibody. Anti-GPIIb-IIIa antibody produced in supernatants was absorbed by incubation with normal platelets. In summary, CD4(+) and HLA-DR-restricted T cells to GPIIb-IIIa are involved in production of anti-platelet autoantibody in ITP patients and are related to the pathogenic process in chronic ITP.     

Identification of human T cell epitopes in the Mycobacterium leprae heat shock protein 70-kD antigen

In a number of pathogens, heat shock proteins (hsp) stimulate humoral and cellular immune responses despite significant sequence identity with host hsp. The 70-kD hsp of Mycobacterium leprae, which shares 47% identity with human hsp70 at the protein level, elicited a T cell response in most Myco. bovis (bacille Calmette-Guérin (BCG)) vaccinees as well as leprosy and tuberculosis patients and their contacts. In order to locate T cell epitopes, DNA fragments encoding portions of the 70-kD hsp were expressed in the vector pGEX-2T and tested for T cell reactivity in an in vitro proliferative assay. Cultures of peripheral blood mononuclear cells (PBMC) from BCG vaccinees indicated that the C-terminal half of the molecule contained multiple T cell epitopes, as the T cells from a majority of Myco. leprae hsp70-reactive individuals responded to C-344. Lower proportions of patients with paucibacillary leprosy (36%) and tuberculosis patients (16%) responded to C-344. The smaller C-142 fragment which includes the terminal 70 residues unique to Myco. leprae and is the target for the human antibody response elicited a cellular response in few patients and no vaccinees. In order to map T cell epitopes, two series of synthetic peptides encompassing the region 278-502 were prepared. Using overlapping 12mer and 20mer peptides, this region of the molecule was found to contain several potential T cell epitopes. The longer peptides gave a clearer indication of reactive sequences including regions of the molecule which were not identified with the 12mer peptides. Fine mapping of reactive peptide pools using the 12mer peptides identified two T cell epitopes. Although both were located in regions of the molecule shared with Myco. tuberculosis, one appeared to be cross-reactive with the equivalent human sequence, and thus has the potential to initiate autoimmune responses.     

Epitope repertoire of human CD4+ T cells on tetanus toxin: identification of immunodominant sequence segments

Sequence regions of tetanus toxin-forming CD4+ cell epitopes in 8 HLA-disparate subjects were identified. Overlapping synthetic peptides corresponding to the complete tetanus toxin sequence were used to test, in a proliferation assay, unselected blood CD4+ cells or CD4+ cell lines propagated by stimulation with tetanus toxoid. The CD4+ cell lines recognized most peptides recognized by the blood CD4+ cells and they recognized additional peptides. Their responses were stronger than those of unselected blood CD4+ cells. Two peptides were recognized by all subjects: one largely overlapped a tetanus toxin sequence region previously identified as a "universal" T cell epitope. Thirteen other peptides elicited a CD4+ cell response in 6 or 7 of the 8 subjects, and another 10 elicited responses in 5 subjects.     

Analysis of type 1 and type 2 T cells in synovial fluid and peripheral blood of patients with rheumatoid arthritis

OBJECTIVE: It has been reported that CD4+ helper T cells play an important role in the pathogenesis of rheumatoid arthritis (RA). We evaluated the presence of intracellular cytokines interleukin 4 (IL-4) and interferon-gamma (IFN-gamma) produced by CD4+ and CD8+ T cells in the synovial fluid and peripheral blood of patients with RA at the single cell level. METHODS: We used 3 color flow cytometric analysis. Synovial fluid mononuclear cells (SFMC) and peripheral blood mononuclear cells (PBMC) were stimulated with phorbol myristate acetate (PMA) and calcium ionophore. The stimulated SFMC and PBMC were triple stained with conjugated mononuclear antibodies (Mab) against cytokines and surface antigens after fixation and permeabilization with a saponine buffer solution. The cells were analyzed for intracellular cytokines (IFN-gamma, IL-4) and surface antigens (CD3, CD4, CD8) using a flow cytometer. RESULTS: The CD4/CD8 ratio was significantly lower in SFMC than in PBMC. The positive rates of IFN-gamma producing cells among CD4+ T cells were significantly higher than those of IL-4 producing cells in both the SFMC and the PBMC of patients with active RA. In the SF of these patients, we also found CD8+ T cells that produce IL-4 alone, or both IL-4 and IFN-gamma. CONCLUSION: In the SF of patients with RA, CD4+ type 1 T cells, which may infiltrate into the synovium and cause pathogenic immune responses in the tissue, are predominant. We believe this cell type also induces migration and activation of CD8+ type 2 T cells into the active site of inflammation, which appears to downregulate the activity of CD4+ type 1 T cells, modulating the excess immune response.     

Identification of functional domains in murine granulocyte-macrophage colony-stimulating factor using monoclonal antibodies to synthetic peptides

Granulocyte-macrophage (GM)-CSF is an important hematopoietic cytokine that regulates proliferation and differentiation of macrophages, neutrophils, and eosinophils. In this study, we generated mAb to five synthetic peptides that correspond to regions along the murine GM-CSF molecule. The ability of anti-peptide mAb to bind to and inhibit biologic activity of murine (m) GM-CSF was determined. mAb with the highest neutralization titers were derived from mice immunized with peptide II, which correspond to amino acids 27 to 38 of mGM-CSF. Immunochemical studies showed that peptide II specifically blocked binding of anti-peptide II mAb to GM-CSF. mAb to two other peptides in the N-terminal half corresponding to residues 7 to 17 and 47 to 58, respectively, of mGM-CSF also inhibited GM-CSF-dependent proliferation and differentiation of murine bone marrow precursors for macrophages and granulocytes. Anti-peptide mAb also inhibited growth of a murine hematopoietic cell line FDCP1 and a murine T cell line HT-2, which was shown to be dependent on GM-CSF for growth in vitro. Biologic activity of both natural and recombinant mGM-CSF was neutralized by anti-peptide mAb. These findings indicate that epitopes in the N-terminal region of mGM-CSF are important for biologic activity, and the epitope defined by peptide II (residues 27 to 38) lies within a particularly important functional domain of the mGM-CSF molecule.