Novel potential diagnostic test for Mycobacterium tuberculosis complex using combined immunomagnetic separation (IMS) and PCR-CTPP.

AIMS: To exploit immunomagnetic separation combined with PCR with confronting two-pair primers (IMS-PCR-CTPP) as a rapid method for detection of Mycobacterium tuberculosis complex (MTC) and identification of Mycobacterium bovis from sputum specimens. METHODS AND RESULTS: Monoclonal antibody (mAb) against the mycobacterial antigen, 85B (Ag85B), was coupled with magnetic particles for specific immunomagnetic separation (IMS) of Mycobacterium spp. Immunofluorescence assay indicated the capability of mAb to bind to Ag85B in both the recombinant and the native form. The IMS combined with PCR-CTPP targeting the mycobacterial lep B gene was further implemented using 133 sputum samples with acid-fast bacilli grading from negative to 3+. The results showed the sensitivity and specificity of IMS-PCR-CTPP vs gold standard culture method were 89·9 and 88·6% respectively. CONCLUSIONS: The IMS-PCR-CTPP method shortens the time for tuberculosis (TB) diagnosis from months to a day. This method is also suitable for investigation of MTC and epidemiological study of Myco. bovis in sputum specimens. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first report emphasizing the combination of IMS and PCR-CTPP for the detection of MTC and simultaneous identification of Myco. bovis from sputum. It could be used for TB diagnosis in resource-limited countries with high TB burden.

EMMPRIN reduction via scFv-M6-1B9 intrabody affects α3ß1-integrin and MCT1 functions and results in suppression of progressive phenotype in the colorectal cancer cell line Caco-2.

Extracellular matrix metalloproteinase inducer (EMMPRIN) exhibits overexpression in various cancers and promotes cancer progression and metastasis via the interaction with its associated molecules. The scFv-M6-1B9 intrabody has a potential ability to reduce EMMPRIN cell surface expression. However, the subsequent effect of scFv-M6-1B9 intrabody-mediated EMMPRIN abatement on its related molecules, α3ß1-integrin, MCT1, MMP-2 and MMP-9, is undefined. Our results demonstrated that the scFv-M6-1B9 intrabody efficiently decreased α3ß1-integrin cell surface expression levels. In addition, intracellular accumulation of MCT1 and lactate were increased. These results lead to suppression of features characteristic for tumor progression, including cell migration, proliferation and invasion, in a colorectal cancer cell line (Caco-2) although there was no difference in MMP expression. Thus, EMMPRIN represents an attractive target molecule for the disruption of cancer proliferation and metastasis. An scFv-M6-1B9 intrabody-based approach could be relevant for cancer gene therapy.

A novel test tube method of screening for hemoglobin E.

INTRODUCTION: Hemoglobin (Hb) E is a ß-structural variant common worldwide. This Hb disorder can form a compound heterozygous state with the ß-thalassemia gene, leading to life-threatening hereditary hemolytic anemia, HbE/ß-thalassemia. Screening of HbE has proven to be a challenging practice in prevention and control of the HbE/ß-thalassemia. METHODS: A novel test tube method for HbE screening using diethyl aminoethyl (DEAE)-cellulose resin was described. With the developed system, HbE/A(2) did not bind to the resin and remained dissolved in the supernatant, whereas other Hbs completely bound to the resin. The red color of the supernatant observed in the test tube indicated the presence of HbE. Colorless or markedly pale color of the supernatant indicates the absence of HbE. RESULTS: Accuracy and efficiency of the established method in detecting HbE was comparable with the standard cellulose acetate electrophoresis method. The developed method is cheap and simple with no requirement of sophisticated equipment. The reagent could be stored at 4 °C for up to 5 months. Hemolysate samples aged up to 5 months were still suitable for this test. CONCLUSION: The described novel test tube method could be an alternative method of mass population screening for HbE, particularly in small health care facilities.

Na, K ATPase beta3 subunit (CD298): association with alpha subunit and expression on peripheral blood cells.

Beta3 subunit is described as one of the Na, K ATPase subunits. Recently, we generated a monoclonal antibody (mAb), termed P-3E10. This mAb was shown to react with the Na, K ATPase beta3 subunit or CD298. By immunofluorescence analysis using mAb P-3E10, it was found that all peripheral blood leukocytes express Na, K ATPase beta3. The presence of beta3 subunit on leukocytes is not in a quantitative polymorphic manner. Upon phytohemagglutinin or phorbol myristate acetate activation, the expression level of the Na, K ATPase beta3 subunit on activated peripheral blood mononuclear cells was not altered in comparison with those of unstimulated cells. Red blood cells (RBCs) of healthy donors showed negative reactivity with mAb P-3E10. However, more than 80% of thalassemic RBCs showed positive reactivity. By immunoprecipitation, moreover, a protein band of 55-65 kDa was precipitated from normal RBC membrane using mAb P-3E10. These results evidenced that the beta3 subunit of Na, K ATPase is expressed on RBC membrane but the epitope recognized by mAb P-3E10 is hidden in normal RBCs. Furthermore, we showed the association of beta3 subunit and alpha subunit of Na, K ATPase. This information is important for further understanding of the functional roles of this molecule.

Construction and characterization of phage-displayed leukocyte surface molecule CD99.

The phage display technique has been described for the production of various recombinant molecules. In the present report, we used this technique to display a leukocyte surface molecule, CD99. PCR subcloning of CD99 cDNA from the mammalian expression vector pCDM8 to the phagemid expression vector pComb3HSS was performed. The resulting phagemid, pComb3H-CD99, was transformed into Escherichia coli XL-1 Blue. CD99 was displayed on the phage particles following infection of the transformed E. coli with the filamentous phage VCSM13. Using sandwich ELISA, the filamentous phage-displayed CD99 was captured by a CD99 monoclonal antibody (mAb) then detected with anti-M13 conjugated to horseradish peroxidase, confirming that the CD99 molecule was displayed on the phage particles. The CD99-phages inhibited induction of Jurkat cell aggregation by CD99 mAb MT99/1. Proper folding of the displayed CD99 bioactive domain was inferred from this finding. Our results demonstrate that the phage display technique can be applied to the generation of full-length CD99 molecules. The phage carrying this cell surface protein will be useful for identification of its counter receptor or ligand.

Engagement of CD147 molecule-induced cell aggregation through the activation of protein kinases and reorganization of the cytoskeleton.

CD147 is a broadly expressed cell surface molecule of the immunoglobulin superfamily whose expression is up-regulated upon T cell activation. Engagement of CD147 by CD147 monoclonal antibodies (mAbs) has been shown to induce homotypic aggregation of U937 cells. To study intracellular signal transduction induced by the engagement of CD147 molecules, protein kinase C (PKC) and protein tyrosine kinase (PTK) inhibitors were used to inhibit cell aggregation. The results indicated that a PKC inhibitor, sphingosine, and a PTK inhibitor, herbimycin A, inhibited CD147 mAb-induced cell aggregation in a dose-dependent manner. In contrast to herbimycin A, a PTK inhibitor, genistein, enhanced cell aggregation. This discrepancy may be due to the differential effect of herbimycin A and genistein on the target cells. Effect of actin filament polymerization blocking agent, cytochalasin B, was also studied and it was found that cytochalasin B completely inhibited CD147 mAb-induced cell aggregation. This result implied that U937 cell aggregation induced by CD147 mAbs is associated with cytoskeleton reorganization. Thus, our observations suggest that cell aggregation induced by the engagement of CD147 with specific mAbs depend upon the activation of protein kinases and a functional cytoskeleton.

Production of polyclonal and monoclonal antibodies against CD54 molecules by intrasplenic immunization of plasmid DNA encoding CD54 protein.

DNA immunization, in theory, is of great interest as a source of specific antibodies against different antigens. In an attempt to produce polyclonal and monoclonal antibodies against cell surface molecules by using the DNA immunization strategy, intramuscular and intrasplenic routes of DNA injection were compared. Two to five, but not a single, intramuscular DNA immunizations induced anti-CD54 and anti-CD147 antibody production. In contrast, a single intrasplenic immunization of CD54-encoding DNA could induce anti-CD54 antibody production. To produce monoclonal antibody (mAb), spleen cells obtained from an intrasplenic CD54-encoding DNA immunized mouse were fused with myeloma cells using the standard hybridoma technique. A hybridoma secreting specific mAb to CD54 was established. The generated mAb reacted to CD54 protein expressed on transfected COS cells and various cell types, the same as using standard CD54 mAb MEM-111. Our results demonstrated that direct immunization of antigen-encoding DNA into spleen is an effective route for production of both polyclonal and monoclonal antibodies to cell surface molecules. This finding is very useful for the production of antibodies to cell surface molecules where the protein antigen is not available or difficult to prepare, but cDNA encoding the corresponding protein is available.

CD99 monoclonal antibody induce homotypic adhesion of Jurkat cells through protein tyrosine kinase and protein kinase C-dependent pathway.

CD99 is a 32 kDa cell surface glycoprotein which is involved in cell adhesion. Engagement of the CD99 molecule by CD99 monoclonal antibodies has been shown to induce homotypic aggregation of various cell types. By using a newly established CD99 monoclonal antibody, MT99/3, we show here that LFA-1/ICAM-1 independent cell adhesion pathways are activated via CD99. Engagement of the CD99 molecule by MT99/3 induced homotypic cell aggregation of Jurkat T-cells within 30 min reaching its maximal level within 4 h. The Jurkat cell aggregation was not blocked by addition of CD11a (LFA-1) and CD54 (ICAM-1) mAbs. Furthermore, MT99/3 treatment did not alter the expression of LFA-1 and ICAM-1 molecules. Induction of Jurkat homotypic aggregation by MT99/3 was, however blocked by the protein kinase C inhibitor, sphingosine, the protein tyrosine kinase inhibitor, genistein, and by actin filament polymerization blocking agent, cytochalasin B. Thus, these observations suggest that CD99 can mediate beta2-integrin independent cell adhesion that depends on activation of protein kinases and reorganization of the cytoskeleton.

Production of anti-CD14 monoclonal antibodies using CD14 expressing COS cells as immunizing antigen.

CD14 is a leukocyte surface molecule expressed on monocytes but not on lymphocytes. Recently, CD14 molecule was demonstrated to function as a receptor for endotoxin. CD14 specific monoclonal antibody (MAb), therefore, can be used to identify monocytes and study the host defense mechanism to bacterial endotoxin. To produce MAb against CD14 protein, in this study cDNA encoding CD14 protein and COS cell expression systems were used to prepare CD14 expressing COS cells. The CD14 transfectants were then used as antigen for mouse immunization. The spleen cells of the immunized mouse were then fused with myeloma cells by conventional hybridoma technique. By using this strategy, 5 hybridroma clones secreting antibody specific for CD14 molecule were generated within one fusion. The generated CD14 MAbs were strongly positive with monocytes, weakly positive with neutrophils but negative with lymphocytes. In addition, the generated CD14 MAb blocked the binding of lipopolysaccharide (LPS) to the CD14 molecules. These CD14 MAbs could be used to enumerate peripheral blood monocytes as well as using referent CD14 MAb. We, therefore, introduce an alternative method for preparation of antigen for production of monoclonal antibody. This type of antigen is a very effective antigen for the production of monoclonal antibodies against cell surface molecules.

Inhibition of PHA-induced cell proliferation by polyclonal CD4 antibodies generated by DNA immunization.

Although the role of CD4 molecule as associative binding element to MHC class II is well documented, their role in T cell activation is unclear. In the present report we used DNA immunization, which is currently shown to induce potent immune responses, to produce the polyclonal antibodies specific for the CD4 molecule and used the generated antibodies to characterize the CD4 function. A rabbit was pre-treated with bupivacaine hydrochloride for 24 h which was followed by intramuscular injection of DNA encoding CD4 protein (CD4-DNA) at weekly interval. By this procedure, CD4 antibodies were detected in the immunized serum after two DNA inoculations. The CD4 antibodies titer was up to 1:800 after five DNA inoculations. The rabbit polyclonal CD4 antibodies recognized both recombinant CD4 protein expressed on CD4-DNA transfected COS cells and native CD4 protein presented on peripheral lymphocytes and CD4+ cell lines. These generated CD4 antibodies could block the binding of standard CD4 mAb, Leu3a and 13B8.2, to the CD4 molecule. To characterize the function of CD4 molecule, PBMC were cultured in the presence of sub-optimal dose of PHA and the produced polyclonal CD4 antibodies. We found that the polyclonal CD4 antibodies strongly suppressed PHA induced cell proliferation. The inhibitory effect of CD4 antibodies may be due to their steric inhibition of the CD4-TCR/CD3 association or may interfere with the binding of CD4 to its ligand IL-16, resulting in the reduction of signal transduction and subsequent cellular responses. Our results indicate the possibility of utilizing DNA immunization to produce polyclonal antibodies against cell surface molecule.

CD147 monoclonal antibodies induce homotypic cell aggregation of monocytic cell line U937 via LFA-1/ICAM-1 pathway.

CD147 is a 50 000-60 000 MW glycoprotein of the immunoglobulin superfamily broadly expressed on haemopoietic cell lines and peripheral blood cells. In the present study, six monoclonal antibodies (mAbs) directed against the CD147 protein were generated. The antigen defined by the generated CD147 mAbs is widely expressed on haemopoietic cell lines, peripheral blood cells and is a lymphocyte activation-associated cell surface molecule. The generated CD147 mAbs precipitated a broad protein band from U937 cells of 45 000-65 000 MW under reducing conditions. Functional analysis indicated that the CD147 mAbs markedly induced homotypic cell aggregation of U937 cells, but not K562 cells. The CD147 mAb-induced cell aggregation was inhibited by leucocyte function-antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) mAbs. However, the expression of LFA-1 and ICAM-1 molecules on U937 was not altered by CD147 mAb treatment. The U937 cell aggregation induced by CD147 mAb was also inhibited by ethylenediamine tetra-acetic acid (EDTA), sodium azide and when incubated at 4 degrees. We therefore propose that the binding of CD147 mAb to CD147 molecule, which mimics the natural ligand binding, may generate intracellular signals that activate LFA-1/ICAM-1 intercellular adhesion pathway.

Production of monoclonal antibody to CD4 antigen and development of reagent for CD4+ lymphocyte enumeration.

A hybridoma secreting monoclonal antibody (mAb) specific to CD4 protein was generated. This monoclonal antibody, named MT4, was proved to be specific to CD4 protein as it reacted with CD4-DNA transfected COS cells, CD4+ cell lines and CD4+ lymphocytes. Furthermore, MT4 mAb inhibited the binding of standard CD4 monoclonal antibodies to CD4 proteins on CD4+ cells. To develop a home made reagent for CD4+ lymphocyte determination by flow cytometry, fluorescein isothiocyanate (FITC) was conjugated to MT4 mAb. To evaluate the developed reagent, 30 HIV infected and 30 healthy individuals were determined for CD4+ lymphocytes by using both a commercial Simultest reagent kit and home made FITC labeled MT4 mAb simultaneously. The study has shown that both percentages and absolute CD4+ lymphocyte counts obtained from both reagents were equivalent. The correlation coefficient for regression analysis was 0.995 and 0.996 for percentages and absolute CD4+ lymphocyte counts, respectively. The results suggest that home made FITC labeled MT4 reagent is an acceptable alternative reagent for monitoring CD4+ lymphocytes in blood samples by flow cytometry.

Production of mouse anti-CD4 antibodies by DNA-based immunization.

The intramuscular injection of plasmid DNA encoding an antigenic protein has been developed recently as a tool for immunization. DNA-based immunization was shown to generate immune responses against the encoded antigen in diverse animal species. In this report, we present the use of DNA-based immunization for the production of antibodies to CD4, a human leukocyte surface molecule. Mice were injected intramuscularly with eukaryotic expression vector containing cDNA encoding CD4 protein, termed CD4-DNA, and were subsequently assayed for anti-CD4 antibody production by indirect immunofluorescence. Sera collected from 2 of 3 inoculated mice reacted with CD4 expressing transfected COS cells and Sup-T1 cells. Anti-CD4 antibody activity was abolished by adsorption with CD4 molecule expressing cells. CD4+ cell depleted lymphocytes were also used to confirm the specificity of the anti CD4 antibodies present in immune serum. CD4-DNA immune serum reacted with approximately 1/3 of freshly isolated lymphocytes but to very few cells in the CD4+ cells-depleted preparation. CD4-DNA immunized sera was used to enumerate CD4+ cells in the peripheral blood of 6 healthy donors and 2 AIDS patients. The number of CD4+ cells estimated by DNA immunized sera was very similar to estimates using standard anti-CD4 monoclonal antibody Leu3a. DNA-based immunization is therefore capable of raising antibodies to human leukocyte surface antigens. This technology may be useful for producing antibodies to other cell surface antigens in mice or other animals.

The effect of thalidomide on the pathogenesis of human immunodeficiency virus type 1 and M. tuberculosis infection.

Tumor necrosis factor alpha (TNF-alpha), a cytokine produced during the host defense against infection, is associated with fevers, weakness, and progressive weight loss. Thalidomide inhibits the synthesis of TNF-alpha both in vitro and in vivo and may have clinical usefulness. We therefore initiated a pilot study of thalidomide treatment in patients with human immunodeficiency virus type 1 (HIV-1)-associated wasting with or without concomitant infection with tuberculosis. Thirty-nine patients were randomly allocated to treatment with either thalidomide or placebo in a double-blind manner for 21 days. Thirty-two patients completed the study. In patients with concomitant HIV-1 and tuberculosis infections, thalidomide therapy was associated with a reduction in both plasma TNF-alpha levels and HIV-1 levels. No significant reduction in either TNF-alpha or HIV- 1 levels was observed in patients with HIV-1 infection only. During the study period, patients receiving thalidomide treatment (n=16) showed a significant weight gain (mean +/- SEM: 6.5 +/- 1.2%; p<0.02) relative to placebo-treated patients (n=16). Patients with simultaneous HIV-1 and tuberculosis infections experienced a higher mean weight gain during thalidomide treatment than the group of patients with HIV-1 infection only. The results of this pilot study suggest that thalidomide may have a clinical role in enhancing weight gain and possibly reducing TNF-alpha and HIV-1 levels in patients with HIV-1 and concomitant mycobacterial infections.

Lack of augmenting effect of interferon-gamma on dengue virus multiplication in human peripheral blood monocytes.

The effect of interferon-gamma (IFN-gamma) on dengue virus multiplication in human peripheral blood monocytes was investigated. Enriched monocytes were treated with IFN-gamma and then infected with dengue virus type 2 either directly or in the presence of optimal infection-enhancing levels of antibodies. Pretreatment of monocytes from dengue-immune donors with 100 IU/ml of IFN-gamma caused 12- to 97-fold and 13- to 137-fold reduction of virus yields at 24 hr after infection in the absence and presence of an anti-flavivirus monoclonal antibody, respectively. IFN-gamma also diminished virus yields when infection of monocytes from a donor who lacked anti-dengue antibody was enhanced 40-fold. The percentage of infected monocytes in IFN-gamma-pretreated cultures was similarly reduced. Dominance of the antiviral effect of IFN-gamma in monocytes is in contrast to an augmenting effect previously observed in the promonocytic cell line U937.

Enhancement of human lymphocyte proliferative response to purified protein derivative by an anti-interleukin-2 receptor alpha chain antibody (CD25).

While it is clear that the beta subunit of interleukin-2 receptor (IL-2R) plays a pivotal role in IL-2-induced signal transduction, the function of the alpha subunit, other than modulating the association rate of IL-2, is still unknown. It has been reported that the interaction between IL-2 and the IL-2R alpha subunit of several IL-2-dependent murine T-cell lines may result in a negative regulatory signal. To confirm this finding, we investigated the effect of an anti-IL-2R alpha antibody, CD25-8D8, on the proliferative response of human peripheral blood lymphocytes. Lymphocytes from purified protein derivative (PPD)-positive donors were cultured with PPD and various concentrations of CD25-8D8 for up to 9 days, and [3H]thymidine uptake was measured. Whereas the proliferative response of human lymphocytes to PPD was suppressed by high concentrations of CD25-8D8, subinhibitory amounts of CD25-8D8 enhanced lymphocyte proliferation by 3.5-fold (range 2.2-6.2-fold) on the second day after maximal [3H]thymidine uptake had occurred. By itself, CD25-8D8 could not induce proliferation of washed 5-day PPD-activated lymphocytes during reculturing; instead, growth enhancement by CD25-8D8 was dependent on the presence of PPD-activated culture supernatant or moderate levels of exogenous IL-2. The enhancing effect of anti-IL-2R alpha antibody, observed in both murine and human systems, reinforces the possibility that binding of IL-2 to the IL-2R alpha chain plays a negative regulatory role in signal transduction.

CD1 molecule expression on human monocytes induced by granulocyte-macrophage colony-stimulating factor.

In this paper we demonstrate that granulocyte-macrophage CSF (GM-CSF) specifically induces the expression of CD1 molecules, CD1a, CD1b and CD1c, upon human monocytes. CD1 molecules appeared upon monocytes on day 1 of stimulation with rGM-CSF, and expression was up-regulated until day 3. Monocytes cultured in the presence of LPS, FMLP, PMA, recombinant granulocyte-CSF, rIFN-gamma, rTNF-alpha, rIL-1 alpha, rIL-1 beta, and rIL-6 remained negative. The induction of CD1 molecules by rGM-CSF was restricted to monocytes, since no such effect was observed upon peripheral blood granulocytes, PBL, and the myeloid cell lines Monomac1, Monomac6, MV4/11, HL60, U937, THP1, KG1, and KG1A. CD1a mRNA was detectable in rGM-CSF-induced monocytes but not in those freshly isolated. SDS-PAGE and immunoblotting analyses of CD1a mAb VIT6 immunoprecipitate from lysate of rGM-CSF-activated monocytes revealed an appropriate CD1a polypeptide band of 49 kDa associated with beta 2-microglobulin. Expression of CD1 molecules on monocytes complements the distribution of these structures on accessory cells, and their specific induction by GM-CSF strengthens the suggestion that CD1 is a family of crucial structures required for interaction between accessory cells and T cells.

Human leukocyte activation antigen M6, a member of the Ig superfamily, is the species homologue of rat OX-47, mouse basigin, and chicken HT7 molecule.

Peripheral granulocytes from rheumatoid arthritis and reactive arthritis patients were recently found to express higher levels of a newly defined Ag, termed M6, in comparison to granulocytes from healthy subjects. We present here the molecular characterization of M6 Ag and show that it is a novel human leukocyte activation-associated cell surface glycoprotein. Peripheral lymphocytes do not significantly express M6 Ag, however, it appears upon 3-day PHA-activated T blasts. On monocytes, which constitutively express M6 Ag, it is down-regulated on day 1 but re-induced on day 3 of granulocyte-macrophage CSF stimulation. SDS-PAGE analysis of M6 immunoprecipitates shows a single band of 54 kDa under nonreducing conditions that shifts to 65 kDa under reducing conditions. Endoglycosidase F treatment of M6 immunoprecipitate reveals that 50% of the M6 molecule is composed of N-linked carbohydrates. By modifying the COS cell cloning strategy, we have isolated cDNA clones encoding M6 Ag. M6 cDNA hybridizes with a single mRNA transcript of approximately 1.7 kb in Northern blotting. Comparison analysis of the M6 sequence indicates that M6 Ag is a member of the Ig superfamily and the species homologue of rat OX-47 Ag, mouse basigin (gp42), and chicken HT7 molecule. The highly conserved remarkable transmembrane domain suggests that the M6 Ag may be a component of a multichain complex in the plasma membrane.

Enumeration of interleukin-1 producing monocytes from human peripheral blood mononuclear leukocytes by agar plating technique.

An agar plating technique was developed for enumeration of IL-1-producing monocytes based on the principle that when IL-1-producing monocytes were cocultured with mouse thymocytes and PHA in semisolid agar medium in a plate, mouse thymocytes proliferated around IL-1-producing monocytes resulting in the clusters or colonies of cells. The IL-1-produced clusters or colonies of cells can be counted under a dissecting microscope. Optimal conditions were established for induction and development of IL-1-producing monocytes. The numbers of IL-1-producing monocytes ranged from 819 to 1930 cells/10(5) monocytes, with mean +/- SEM = 1344 +/- 182 cells/10(5) monocytes; the IL-1 activity ranged from 11.7 to 85.9 U/10(5) monocytes/ml, with mean +/- SEM = 42.8 +/- 11.2 U/10(5) monocytes/ml in seven normal subjects. The IL-1 activity per one monocyte ranged from 12.7 to 86.5 mU, with mean +/- SEM = 33.5 +/- 9.8 mU. The mean numbers of IL-1-producing monocytes and the mean IL-1 levels produced by monocytes from the same normal subjects were highly correlated (r = 0.981). The numbers of IL-1-produced colonies resulting from IL-1-producing monocytes could be completely abolished by incorporation of rabbit anti-human IL-1 in the semisolid agarose but not by rabbit anti-human IL-6 or anti-human TNF-alpha.

Molecular characterization and functional analysis of the leukocyte surface protein CD31.

The CD31 Ag is a surface glycoprotein of 130 kDa with a broad cellular distribution. We show that among peripheral human blood cells, it is expressed on monocytes, granulocytes, platelets, and a subpopulation of lymphocytes. Activation of granulocytes leads to down-regulation of CD31 molecule expression. Sequence analysis and quantitative measurements of the relatedness of the CD31 molecule to other known proteins demonstrate that it consists of six Ig constant domains and that each domain bears substantial similarity to Fc gamma R domains. We find, however, that the CD31 molecule does not bind Ig Fc domains. On human monocytes we demonstrate that CD31 mAb recognizing certain epitopes of the CD31 molecule induce the generation of reactive oxygen metabolites. No such effect was seen with human granulocytes. By using two CD31 mAb, termed 1B5 and 7E4, we analyzed the requirements for activation of the monocyte respiratory burst via CD31 Ag in more detail. We show that signal transduction occurs via formation of a CD31 Ag-mAb-Fc gamma R complex involving either Fc gamma RI (CD64) or Fc gamma RII (CDw32) molecules.