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Familial Mediterranean fever (FMF) is a recessive disorder characterized by episodes of fever and intense inflammation. FMF attacks are unique in their sensitivity to the microtubule inhibitor colchicine, contrasted with their refractoriness to the anti-inflammatory effects of glucocorticoids. The FMF gene, MEFV, was recently identified by positional cloning; it is expressed at high levels in granulocytes and monocytes. The present study investigated the subcellular localization of the normal gene product, pyrin. These experiments did not support previously proposed nuclear or Golgi localizations. Instead fluorescence microscopy demonstrated colocalization of full-length GFP- and epitope-tagged pyrin with microtubules; this was markedly accentuated in paclitaxel-treated cells. Moreover, immunoblot analysis of precipitates of stabilized microtubules with recombinant pyrin demonstrated a direct interaction in vitro. Pyrin expression did not affect the stability of microtubules. Deletion constructs showed that the unique N-terminal domain of pyrin is necessary and sufficient for colocalization, whereas disease-associated mutations in the C-terminal B30.2 (rfp) domain did not disrupt this interaction. By phalloidin staining, a colocalization of pyrin with actin was also observed in perinuclear filaments and in peripheral lamellar ruffles. The proposal is made that pyrin regulates inflammatory responses at the level of leukocyte cytoskeletal organization and that the unique therapeutic effect of colchicine in FMF may be dependent on this interaction. (Blood. 2001;98:851-859)

Familial Mediterranean fever (FMF) is a recessive disorder characterized by episodes of fever and neutrophil-mediated serosal inflammation. We recently identified the gene causing FMF, designated MEFV, and found it to be expressed in mature neutrophils, suggesting that it functions as an inflammatory regulator. To facilitate our understanding of the normal function of MEFV, we extended our previous studies. MEFV messenger RNA was detected by reverse transcriptase-polymerase chain reaction in bone marrow leukocytes, with differential expression observed among cells by in situ hybridization. CD34 hematopoietic stem-cell cultures induced toward the granulocytic lineage expressed MEFV at the myelocyte stage, concurrently with lineage commitment. The prepromyelocytic cell line HL60 expressed MEFV only at granulocytic and monocytic differentiation. MEFV was also expressed in the monocytic cell lines U937 and THP-1. Among peripheral blood leukocytes, MEFV expression was detected in neutrophils, eosinophils, and to varying degrees, monocytes. Consistent with the tissue specificity of expression, complete sequencing and analysis of upstream regulatory regions of MEFV revealed homology to myeloid-specific promoters and to more broadly expressed inflammatory promoter elements. In vitro stimulation of monocytes with the proinflammatory agents interferon (IFN) gamma, tumor necrosis factor, and lipopolysaccharide induced MEFV expression, whereas the antiinflammatory cytokines interleukin (IL) 4, IL-10, and transforming growth factor beta inhibited such expression. Induction by IFN-gamma occurred rapidly and was resistant to cycloheximide. IFN-alpha also induced MEFV expression. In granulocytes, MEFV was up-regulated by IFN-gamma and the combination of IFN-alpha and colchicine. These results refine understanding of MEFV by placing the gene in the myelomonocytic-specific proinflammatory pathway and identifying it as an IFN-gamma immediate early gene.

Many B-cell malignancies express the CD22 antigen on their cell surface. To kill cells expressing this antigen, the RFB4 monoclonal antibody (MoAb) has been linked chemically with either deglycosylated ricin A chain or truncated versions of Pseudomonas exotoxin. These immunotoxins exhibited selective cytotoxic activity for CD22+ cells and antitumor activity in nude mouse models bearing human B-cell lymphomas. To construct a recombinant immunotoxin targeted to CD22, we first cloned the variable portions of the heavy and light chains of RFB4. The cloned Fv fragments were joined by a newly created disulfide bond to form a disulfide stabilized (ds) construct. The RFB4 construct was combined by gene fusion with PE38, a truncated version of PE. The recombinant immunotoxin was then expressed in Escherichia coli, purified by column chromatography and tested for cytotoxicity activity. RFB4(dsFv)PE38 retained its binding activity for CD22, was very stable at 37 degrees C and exhibited selective cytotoxic activity for CD22+-cultured cell lines. Because of its favorable binding characteristics and potency for CD22-positive cell lines, RFB4(dsFv)PE38 was tested for antitumor activity in a nude mouse model of human lymphoma. CA46 cells were injected subcutaneously and then treated with the RFB4(dsFv)PE38 immunotoxin. Antitumor activity was dose responsive and was not evident when an irrelevant immunotoxin was administered on the same schedule.