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Rab proteins influence vesicle trafficking pathways through the assembly of regulatory protein complexes. Previous investigations have documented that Rab11a and Rab8a can interact with the tail region of myosin Vb and regulate distinct trafficking pathways. We have now determined that a related Rab protein, Rab10, can interact with myosin Va, myosin Vb, and myosin Vc. Rab10 localized to a system of tubules and vesicles that have partially overlapping localization with Rab8a. Both Rab8a and Rab10 were mislocalized by the expression of dominant-negative myosin V tails. Interaction with Rab10 was dependent on the presence of the alternatively spliced exon D in myosin Va and myosin Vb and the homologous region in myosin Vc. Yeast two-hybrid assays and fluorescence resonance energy transfer studies confirmed that Rab10 binding to myosin V tails in vivo required the alternatively spliced exon D. In contrast to our previous work, we found that Rab11a can interact with both myosin Va and myosin Vb tails independent of their splice isoform. These results indicate that Rab GTPases regulate diverse endocytic trafficking pathways through recruitment of multiple myosin V isoforms.

By interacting with the cytoplasmic tail of a Golgi-processed form of transforming growth factor-alpha (TGFalpha), Naked2 coats TGFalpha-containing exocytic vesicles and directs them to the basolateral corner of polarized epithelial cells where the vesicles dock and fuse in a Naked2 myristoylation-dependent manner. These TGFalpha-containing Naked2-associated vesicles are not directed to the subapical Sec6/8 exocyst complex as has been reported for other basolateral cargo, and thus they appear to represent a distinct set of basolaterally targeted vesicles. To identify constituents of these vesicles, we exploited our finding that myristoylation-deficient Naked2 G2A vesicles are unable to fuse at the plasma membrane. Isolation of a population of myristoylation-deficient, green fluorescent protein-tagged G2A Naked2-associated vesicles was achieved by biochemical enrichment followed by flow cytometric fluorescence-activated vesicle sorting. The protein content of these plasma membrane de-enriched, flow-sorted fluorescent G2A Naked2 vesicles was determined by LC/LC-MS/MS analysis. Three independent isolations were performed, and 389 proteins were found in all three sets of G2A Naked2 vesicles. Rab10 and myosin IIA were identified as core machinery, and Na(+)/K(+)-ATPase alpha1 was identified as an additional cargo within these vesicles. As an initial validation step, we confirmed their presence and that of three additional proteins tested (annexin A1, annexin A2, and IQGAP1) in wild-type Naked2 vesicles. To our knowledge, this is the first large scale protein characterization of a population of basolaterally targeted exocytic vesicles and supports the use of fluorescence-activated vesicle sorting as a useful tool for isolation of cellular organelles for comprehensive proteomics analysis.

The CXCR2 chemokine receptor is a G-protein-coupled receptor that undergoes clathrin-mediated endocytosis upon ligand binding. The trafficking of CXCR2 is crucial for cells to maintain a proper chemotactic response. The mechanisms that regulate the recycling/degradation sorting decision are unknown. In this study, we used dominant-negative (T19N) and GTPase-deficient activated (Q63L) RhoB mutants, as well as RhoB small interfering RNA (siRNA) to investigate the role of RhoB in CXCR2 trafficking. Expression of either of the RhoB mutants or transfection of RhoB siRNA impaired CXCR2-mediated chemotaxis. Expression of RhoB T19N and transfection of RhoB siRNA impaired sorting of CXCR2 to the lysosome after 3 hours of CXCL8 stimulation and impaired CXCL8-induced CXCR2 degradation. In cells expressing the RhoB Q63L mutant, CXCR2 recycling through the Rab11a recycling compartment was impaired after 30 minutes of CXCL8 stimulation as was CXCL8-induced CXCR2 degradation. For cells expressing activated RhoB, CXCR2 colocalized with Rab4, a marker for the rapid recycling pathway, and with the mannose-6-phosphate receptor, which traffics between the trans-Golgi network and endosomes. These data suggest that CXCR2 recycles through alternative pathways. We conclude that oscillation of RhoB GTPase activity is essential for appropriate sorting decisions, and for directing CXCR2 degradation and recycling – events that are required for optimal chemotaxis.

Gastric parietal cells possess an amplified apical membrane recycling system dedicated to regulated apical recycling of H-K-ATPase. While amplified in parietal cells, apical recycling is critical to polarized secretory processes in most epithelial cells. To clarify putative regulators of apical recycling, we prepared immunoisolated parietal cell H-K-ATPase-containing recycling membranes from human stomachs and analyzed protein contents by tryptic digestion and mass spectrometry. We identified and validated by Western blots many of the proteins previously identified on immunoisolated rabbit tubulovesicles, including Rab11, Rab25, syntaxin 3, secretory carrier membrane proteins (SCAMPs), and vesicle-associated membrane protein (VAMP)2. In addition, we detected several previously unrecognized proteins, including Rab10, VAMP8, syntaxin 7, and syntaxin 12/13. We also identified the K+channel component KCNQ1. Immunostaining of human gastric mucosal sections confirmed the presence of each of these proteins in parietal cells and their colocalization with H-K-ATPase on tubulovesicles. To investigate the role of the identified soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins in apical recycling, we transfected them as DsRed2 fusions into an enhanced green fluorescent protein (EGFP)-Rab11a-expressing Madin-Darby canine kidney (MDCK) cell line. Syntaxin 12/13 and VAMP8 caused a collapse of the EGFP-Rab11a compartment, whereas a less dramatic effect was observed in cells transfected with syntaxin 3, syntaxin 7, or VAMP2. The five DsRed2-SNARE chimeras were also transfected into a MDCK cell line overexpressing Rab11-FIP2(129-512). All five of the chimeras were drawn into the collapsed apical recycling system. This study, which represents the first proteomic analysis of an immunoisolated vesicle population from native human tissue, demonstrates the diversity of putative regulators of the apical recycling system.

Rab11a, myosin Vb, and the Rab11-family interacting protein 2 (FIP2) regulate plasma membrane recycling in epithelial cells. This study sought to characterize more fully Rab11-FIP2 function by identifying kinase activities modifying Rab11-FIP2. We have found that gastric microsomal membrane extracts phosphorylate Rab11-FIP2 on serine 227. We identified the kinase that phosphorylated Rab11-FIP2 as MARK2/EMK1/Par-1Bα (MARK2), and recombinant MARK2 phosphorylated Rab11-FIP2 only on serine 227. We created stable Madin-Darby canine kidney (MDCK) cell lines expressing enhanced green fluorescent protein-Rab11-FIP2 wild type or a nonphosphorylatable mutant [Rab11-FIP2(S227A)]. Analysis of these cell lines demonstrates a new role for Rab11-FIP2 in addition to that in the plasma membrane recycling system. In calcium switch assays, cells expressing Rab11-FIP2(S227A) showed a defect in the timely reestablishment of p120-containing junctional complexes. However, Rab11-FIP2(S227A) did not affect localization with recycling system components or the normal function of apical recycling and transcytosis pathways. These results indicate that phosphorylation of Rab11-FIP2 on serine 227 by MARK2 regulates an alternative pathway modulating the establishment of epithelial polarity.

An increasing number of human disorders have been linked to mutations in genes of the secretory pathway. The chemically induced zebrafish crusher variant results in malformed craniofacial skeleton, kinked pectoral fins and a short body length. By positional cloning, we identified a nonsense mutation converting leucine to a stop codon (L402X) in the sec23a gene, an integral component of the COPII complex, which is critical for anterograde protein trafficking between endoplasmic reticulum and Golgi apparatus. Zebrafish crusher mutants develop normally until the onset of craniofacial chondrogenesis. crusher chondrocytes accumulate proteins in a distended endoplasmic reticulum, resulting in severe reduction of cartilage extracellular matrix (ECM) deposits, including type II collagen. We demonstrate that the paralogous gene sec23b is also an essential component of the ECM secretory pathway in chondrocytes. In contrast, knockdown of the COPI complex does not hinder craniofacial morphogenesis. As SEC23A lesions cause the cranio-lenticulo-sutural dysplasia syndrome, crusher provides the first vertebrate model system that links the biology of endoplasmic reticulum to Golgi trafficking with a clinically relevant dysmorphology.

The HIV-1 accessory gene product Vpu is required for efficient viral particle release from infected human cells. The mechanism by which Vpu enhances particle assembly or release is not yet defined. Here, we identify an intracellular site that is critical for Vpu-mediated enhancement of particle release. Vpu was found to co-localize with markers for the pericentriolar recycling endosome. Expression of dominant negative mutants of Rab11a and myosin Vb that disrupt protein sorting through the recycling endosome abrogated the ability of Vpu to augment particle release. Remarkably, the effects of blocking recycling endosome function on HIV particle release were demonstrable only in human cell lines known to be responsive to Vpu, while no effect on particle release was seen in African green monkey cells. Inhibition of recycling endosome function in human cells also blocked the ability of HIV-2 envelope to enhance particle release. These studies indicate that Vpu and HIV-2 envelope glycoprotein enhance particle release via a common mechanism that requires the activity of the pericentriolar recycling endosome.

Members of the Rab family of small GTPases are involved in multiple trafficking events in both endocytotic and biosynthetic pathways. To understand more fully the regulation of these events, a concerted effort is underway to ascertain the binding partners and regulators of Rabs. Here, we describe methods to assess binding of Rab11a with Rab11-FIP2 and other Rab11-FIPs utilizing a modified far-Western approach. We then broaden this application to assess binding of Rab11-FIP2 with myosin Vb and homodimerization of Rab11-FIP2.

Myosin Vb interacts with Rab11 family members and is the major motor protein identified in association with plasma membrane recycling systems in nonpolarized and polarized cells. Using yeast two-hybrid binary screens, we demonstrated that dominant active Rab25S21V fails to interact with myosin Vb, but does interact with Rab11-FIP2. Transfection of DsRed2-myosin Vb tail in MDCK cell lines stably transfected with wild-type or dominant active forms of Rab11a or Rab25 demonstrated that the distribution of Rab25S21V is only partially altered by expression of the myosin Vb tail. Finally, we demonstrate EGF-dependent sequestration of internalized EGF receptor by EGFP-myosin Vb in A431 cells. Expression of myosin Vb tail represents a powerful provocative test for the trafficking of cargoes through Rab11-containing plasma membrane recycling systems.

Members of the Rab family of small GTPases are involved in multiple trafficking events in both endocytotic and biosynthetic pathways. To understand more fully the regulation of these events, a concerted effort is underway to ascertain the binding partners and regulators of Rabs. Here, we describe methods to assess binding of Rab11a with Rab11‐FIP2 and other Rab11‐FIPs utilizing a modified far‐Western approach. We then broaden this application to assess binding of Rab11‐FIP2 with myosin Vb and homodimerization of Rab11‐FIP2.

Myosin Vb interacts with Rab11 family members and is the major motor protein identified in association with plasma membrane recycling systems in nonpolarized and polarized cells. Using yeast two‐hybrid binary screens, we demonstrated that dominant active Rab25S21V fails to interact with myosin Vb, but does interact with Rab11‐FIP2. Transfection of DsRed2‐myosin Vb tail in MDCK cell lines stably transfected with wild‐type or dominant active forms of Rab11a or Rab25 demonstrated that the distribution of Rab25S21V is only partially altered by expression of the myosin Vb tail. Finally, we demonstrate EGF‐dependent sequestration of internalized EGF receptor by EGFP‐myosin Vb in A431 cells. Expression of myosin Vb tail represents a powerful provocative test for the trafficking of cargoes through Rab11‐containing plasma membrane recycling systems.

AKAP350 is a multiply spliced family of 350–450-kDa protein kinase A-anchoring proteins localized to the centrosomes and the Golgi apparatus. Using AKAP350A as bait in a yeast two-hybrid screen of a rabbit parietal cell library, we have identified a novel AKAP350-interacting protein, transforming acidic coiled-coil-containing protein 4 (TACC4). Two-hybrid binary assays demonstrate interaction of both TACC3 and TACC4 with AKAP350A and AKAP350B. Antibodies raised to a TACC4-specific peptide sequence colocalize TACC4 with AKAP350 at the centrosome in interphase Jurkat cells. Mitotic cell staining reveals translocation of TACC4 from the centrosome to the spindle apparatus with the majority of TACC4 at the spindle poles. Truncated TACC4 proteins lacking the AKAP350 minimal binding domain found in the carboxyl coiled-coil region of TACC4 could no longer target to the centrosome. Amino-truncated TACC4 proteins could no longer target to the spindle apparatus. Further, overexpression of TACC4 fusion proteins that retained spindle localization in mitotic cells resulted in an increased proportion of cells present in prometaphase. We propose that AKAP350 is responsible for sequestration of TACC4 to the centrosome in interphase, whereas a separate TACC4 domain results in functional localization of TACC4 to the spindle apparatus in mitotic cells.

The gastric parietal cell in mammals is a highly differentiated cell with a specialized function: the secretion of HCl into the gastric lumen. To accomplish this highly regulated function, the parietal cell sequesters the H/K-ATPase responsible for pumping protons within a intracellular tubulovesicular membrane network (5). The tubulovesicular network lies deep to an elaborate infolding of the apical plasma membrane designated as the intracellular canaliculus. The intracellular canaliculus represents a target membrane surface for regulated fusion with tubulo vesicles (38). In response to either cAMP-dependent (histamine) or calcium-dependent (muscarinic cholinergic) agonists, tubulovesicle membrane fuse with the apical canalicular target membranes to deliver the H/K-ATPase to the luminal surface of activated parietal cells. Following the cessation of agonist stimulation, the H/K-ATPase is internalized and reassembled into the tubulovesicular system (12).

Myosin Va is associated with discrete vesicle populations in a number of cell types, but little is known of the function of myosin Vb. Yeast two-hybrid screening of a rabbit parietal cell cDNA library with dominant active Rab11a (Rab11aS20V) identified myosin Vb as an interacting protein for Rab11a, a marker for plasma membrane recycling systems. The isolated clone, corresponding to the carboxyl terminal 60 kDa of the myosin Vb tail, interacted with all members of the Rab11 family (Rab11a, Rab11b, and Rab25). GFP-myosin Vb and endogenous myosin Vb immunoreactivity codistributed with Rab11a in HeLa and Madin-Darby canine kidney (MDCK) cells. As with Rab11a in MDCK cells, the myosin Vb immunoreactivity was dispersed with nocodazole treatment and relocated to the apical corners of cells with taxol treatment. A green fluorescent protein (GFP)-myosin Vb tail chimera overexpressed in HeLa cells retarded transferrin recycling and caused accumulation of transferrin and the transferrin receptor in pericentrosomal vesicles. Expression of the myosin Vb tail chimera in polarized MDCK cells stably expressing the polymeric IgA receptor caused accumulation of basolaterally endocytosed polymeric IgA and the polymeric IgA receptor in the pericentrosomal region. The myosin Vb tail had no effects on transferrin trafficking in polarized MDCK cells. The GFP-myosin Va tail did not colocalize with Rab11a and had no effects on recycling system vesicle distribution in either HeLa or MDCK cells. The results indicate myosin Vb is associated with the plasma membrane recycling system in nonpolarized cells and the apical recycling system in polarized cells. The dominant negative effects of the myosin Vb tail chimera indicate that this unconventional myosin is required for transit out of plasma membrane recycling systems.

This chapter discusses the expression and properties of Rab25 in polarized Madin-Darby canine kidney cells. In Madin-Darby canine kidney (MDCK) cells, Rab25 levels are very low, whereas endogenous Rablla levels are relatively high. Therefore, to study Rab25 in MDCK cells, cell lines are isolated stably transfected with the wild-type sequence of rabbit Rab25. To detect Rab25, monoclonal antibodies are developed specific for Rab25 and nonreactive against Rab11a. A double-screening enzyme-linked immunosorbent assay (ELISA) protocolis developed to isolate monoclonal antibodies against Rab25 that do not react with Rablla. To visualize the three-dimensional distribution of Rab proteins in polarized cells, the stably transfected Rab25- expressing 2A3 cell line is grown to confluence on Transwell clear filters and maintained for three days following confluence. Out of 20 lines screened, six lines were found to contain a transfected Rab25 message. The 2A3 line utilized for characterization of the association of Rablla and Rab259 was one of the two lowest expressing cell lines. To compare the effects of stable overexpression of Rab25 on endogenous message levels, Rab25 and Rablla mRNA levels were compared between mock pCB6-transfected cells and the Rab25-transfected 2A3 line.

The maintenance of a barrier with controlled permeability is an important characteristic for multi-cellular organisms. In mammalian cells, the tight junction functions in that role allowing compartments with different solute composition to be separate, but not absolutely unconnected. The permeability of this paracellular zone needs to be controlled by both internal and external factors allowing for modulation of the permeability under certain circumstances. The purpose of this chapter is to introduce the reader to the molecular components of the mammalian tight junction. Also provided, is a brief description of how these junctional components interact with other members of the tight junction plaque and components of both the cytoskelton and signaling cascade.

Myosin Vb (MYO5B) is an actin based motor that localizes specific Rab small GTPases (Rab8a, Rab10, Rab11, and Rab25) to sub-apical domains in polarized epithelial cells. Microvillus Inclusion Disease (MVID) represents a pathophysiologic window into the apical trafficking process, because it arises as a result of inactivating mutations in MYO5B that leads to the loss of microvilli in intestinal enterocytes and chronic unremitting diarrhea in the affected newborns. Understanding how mutations in MYO5B lead to aberrant enterocyte phenotype will provide novel insights into the fundamental mechanisms governing apical recycling system trafficking, microvilli formation, and MVID. We hypothesize that MYO5B plays a crucial role in apical trafficking and polarization in enterocytes, and that defective MYO5B in MVID patients mislocalizes Rab small GTPases leading to disruption in apical transport and loss of polarity in enterocytes. To test this hypothesis we have established cellular models of aberrant enterocyte apical trafficking by stably knocking down MYO5B in CaCo2-BBE cells. The cells grown on permeable filters for 15 days were analyzed using immunostaining, Scanning EM, and Transmission EM to examine markers of apical and basolateral polarity, intracellular trafficking, and the establishment of apical microvilli. MYO5B KD knockdown (KD) caused a loss of microvilli and dispersal of Rab8a and Rab11acontaining vesicles from their usual subapical distribution to diffusely cytoplasmic in the cells. MYO5B KD elicited an increase in claudin-2 and a decrease in claudin-1 expression as well as a decrease in lateral membrane staining for p120-catenin (p120). MYO5B KD also caused a loss of apical active GTP-bound cdc42 in the cells. Rescue of theMYO5B KDwith synthetic MYO5B wild type elicited recovery of normal microvilli. However, re-expression of MYO5B(P660L), the Navajo MVID mutation, failed to rescue and in addition caused the formation of microvillus inclusions. Navajo MVID MYO5B(P660L) patient samples demonstrated a loss of p120 and a pseudostratified columnar epithelium at the villus tips where ezrin-staining microvillus inclusions were most predominant. Patient biopsies also showed aberrant localization of MYO5B, Rab8a, Rab11a, Rab11-FIP2, DPPIV, and Ezrin. Rab11a and MYO5B localized around microvillus inclusions, while Rab8a staining was diffuse. Importantly, while a severe loss of brush border was observed in cells at the tips of microvilli, the cells in the mid-villus appeared to have a preserved normal brush border. Our results suggest that MYO5B regulates the polarity of intestinal epithelial cells, thereby maintaining the functional brush border. The normal brush border seen in the proximal villi in the Navajo MVID patients suggests that the pathogenesis of MVID lies in a loss of polarity in enterocytes at the tips of intestinal villi.

Publisher Summary This chapter examines protein interactions in the tight junction and the role of membrane-associated guanylate kinase (MAGUK) proteins in regulating tight-junction organization and function. This chapter presents some of what is known about the proteins of the tight junction and their interactions. A very important insight is offered by recognition that both ZO-1 and ZO-2, cytoplasmic plaque proteins of the tight junction, are members of the membrane-associated guanylate kinase (MAGUK) homolog protein family. Other members of this family are located on the cytoplasmic surface of a variety of cell–cell contacts ranging from the septate junctions in Drosophila to vertebrate synapses and the lateral surface of epithelial cells. Growing evidence suggests MAGUK proteins are involved in organizing the structural and functional links between transmembrane proteins, signaling pathways, and the cortical cytoskeleton. Review of the experimental evidence addressing the functional role of MAGUK proteins leads us to speculate that the cytoplasmic surface of the tight junction can be considered a specialized case of protein interactions that are used to connect membrane events at many different regions of the cell cortex.

Cell walls from M+ and M- protein variants of group A streptococci were examined for their arthritogenicity in female Lewis rats. Intraperitoneal administration of both of these sonicated cell wall preparations caused a severe acute and chronic arthritis in recipient rats. Histological evaluation of the hind paw of these rats indicated synovial lining hyperplasia, cell infiltration in the subsynovial space, pannus formation, and erosions of bone and cartilage. Joint pathology was similar in the hind paws of rats immunized with cell walls prepared from either the M+ or the M- protein variants. Cell-mediated immunity was also similar when lymph nodes were exposed to cell walls derived from these two preparations. A recombinant M6 protein from streptococci did not elicit a proliferative response from lymph nodes prepared from arthritic rats. These observations indicate that the M protein that has previously been implicated in auto-immunity does not have a critical role in the pathogenesis of streptococcal cell wall arthritis in rats.

Recombinant preparations of TNF and lymphotoxin (LT) increase the expression of class I MHC antigens on cultured human endothelial cells (EC) without inducing expression of class II antigens. These actions are similar to those of rIFN-alpha or rIFN-beta. However, TNF and LT differ from IFN-alpha/beta in that the former synergize with IFN-gamma for class I regulation whereas the latter do not. Furthermore, LT or TNF do not affect IFN-gamma-mediated class II induction at optimal class I inducing concentrations (100 U/ml), whereas IFN-alpha and IFN-beta (at their optimal concentrations of 1,000 U/ml) are strikingly inhibitory. LT and TNF also can further increase expression of class I antigens on cells already maximally stimulated by IFN-alpha or IFN-beta. A recombinant preparation of IL-6 (formerly called 26-kD protein, IFN-beta 2, or B cell stimulating factor 2) was without effect on class I expression in EC. These data make it seem unlikely that the actions of LT or TNF on EC expression of MHC antigens are mediated through autocrine or paracrine production of IFN-alpha, IFN-beta or IL-6. More importantly, they suggest that LT or TNF are more likely to be immunostimulatory, whereas IFN-alpha or IFN-beta are more likely to be immunoinhibitory in vivo, a consideration of potential relevance for cytokine administration to various patient populations.

Kawasaki syndrome (KS) is characterized by diffuse vasculitis and marked T cell and B cell activation. In this study, sera from 16 patients with acute KS, 15 patients in the convalescent phase of KS, and 19 age-matched controls were assessed for complement dependent cytotoxic activity against 111In-labeled human umbilical vein endothelial (HUVE) cells, Neither sera from patients with KS nor sera from controls had cytotoxic effects on HUVE cells cultivated under standard conditions. Since activated T cells such as those present in acute KS secrete gamma interferon (gamma-IFN), we also examined the effects of sera from acute KS on HUVE cells preincubated with gamma-IFN. We report here that immunoglobulin M (IgM) antibodies in sera from patients with acute KS cause significant (P less than 0.01) killing of gamma-IFN-treated HUVE cells. Pretreatment with interleukin 2, gamma-IFN, or beta-IFN failed to render HUVE susceptible to lysis with acute KS sera. The observed effects were not mediated via immune complexes. The cytotoxic antibodies in acute KS seem to be directed against inducible monomorphic antigenic determinants present on gamma-IFN-treated HUVE cells but not on control or gamma-IFN treated autologous human dermal fibroblasts (HDF). Similarly, acute KS sera also induced lysis of gamma-IFN-treated human saphenous vein endothelial (HSVE) cells but not gamma-IFN treated human saphenous vein smooth muscle (HSVSM) cells. Since gamma-IFN induces the same level of class I and class II major histocompatibility complex (MHC) antigen expression on HDF, HUVE, HSVE, and HSVSM cells, our results suggest that the anti-endothelial cell antibodies in acute KS are directed to gamma-IFN-inducible molecules other than MHC determinants. These observations are further substantiated by the failure of human B cells or monocytes to absorb the anti-endothelial cell activity. Since most vasculitides, including acute KS, are characterized both by marked immune activation and the secretion of lymphokines, antibodies directed to gamma-IFN-inducible endothelial cell antigens may represent a general mechanism for vascular injury.

Recombinant human tumor necrosis factor (TNF), purified to greater than 99% homogeneity, increases surface expression of class I major histocompatibility complex (MHC) antigens to a maximum of 9-fold on cultured human endothelial cells (HEC) and human dermal fibroblasts (HDF). The increase is concentration dependent (peak 20-100 units/ml) and time dependent (nearly maximal by 4 days); expression remains elevated in the continued presence of TNF and requires greater than 7 days to return to basal levels upon TNF withdrawal. The increase in surface expression appears to result from increases in steady-state mRNA levels for the class I antigens, although the increase in mRNA is proportionately greater than for surface expression. No surface expression of or mRNA for class II MHC antigens is detectable in either control or TNF-treated HEC or HDF. These effects are similar to those produced by leukocyte or fibroblast (type I) interferons (IFNs). The protein synthesis inhibitor cycloheximide (CHX), when added coincidentally with type I IFNs, leads to superinduction of mRNA for class I MHC antigens and, unexpectedly, leads to the appearance of mRNA for class II MHC antigens. CHX has no effect by itself upon mRNA levels for class I or class II MHC antigens, nor does it modulate the increases in mRNA produced by immune (type II) IFN. Most interesting, CHX blocks the increase in mRNA for class I MHC antigens induced by TNF. Thus TNF appears to act on MHC gene expression through a newly synthesized protein intermediate. Our results provide direct evidence that TNF can modulate gene expression in normal (untransformed) cell types and contribute to understanding the complex nature of MHC gene regulation. Finally, they suggest that TNF may act in vivo as an immunoregulatory molecule.

Kawasaki syndrome (KS) is an acute febrile illness of early childhood characterized by diffuse vasculitis and marked immune activation. The present study was undertaken to determine whether the acute phase of KS is associated with circulating cytotoxic antibodies directed to target antigens induced on vascular endothelium by the monokines, IL-1, or tumor necrosis factor (TNF). Sera from 20 patients with acute KS, 11 patients in the convalescent phase of KS, and 17 age-matched controls were assessed for complement-dependent cytotoxic activity against 111In-labeled human endothelial cells (HEC), dermal fibroblasts, and vascular smooth muscle cells. Sera from patients with acute KS but not the other subject groups caused significant (p less than 0.01) complement-mediated killing of IL-1- or TNF-stimulated HEC. None of the sera tested had cytotoxicity against control HEC cultures or the other target cell types, with or without IL-1 or TNF pretreatment. Expression of the IL-1- or TNF-inducible target antigens on endothelial cells was rapid and transient, peaking at 4 h and disappearing after 24 h despite continued incubation with monokine. In contrast, we have previously shown that IFN-gamma requires 72 h to render HEC susceptible to lysis with acute KS sera. Serum adsorption studies demonstrated that IL-1- and TNF-inducible endothelial target antigens are distinct from IFN-gamma-inducible antigens. These observations suggest that mediator secretion by activated monocyte/macrophages could be a predisposing factor to the development of vascular injury in acute KS. Although our present observations have been restricted to KS, the development of cytotoxic antibodies directed to monokine-inducible endothelial cell antigens may also be found in other vasculitides accompanied by immune activation.