Clinical studies of multiple endocrine neoplasia type 1 (MEN1) [published erratum appears in QJM 1996 Dec;89(12):957-8]

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by the combined occurrence of parathyroid, pancreatic islet and anterior pituitary tumours. To facilitate a screening programme for MEN1, we investigated 709 people (364 males and 345 females, age range 1-84 years) from 62 MEN1 families, and 36 non- familial MEN1 patients. Of those investigated, 220 (95 males and 125 females, age range 8-79 years) suffered from MEN1. Parathyroid, pancreatic and pituitary tumours occurred in 95%, 41% and 30% of the patients, respectively. Parathyroid tumours were the first manifestation of MEN1 in 87% of patients, and amongst the pituitary and pancreatic tumours, somatotrophinomas and gastrinomas were more common in patients above the age of 40 years, whilst insulinomas occurred more frequently in patients below the age of 40 years. Biochemical screening indicated that the penetrance of MEN1 by the ages of 20, 35 and 50 years was 43%, 85% and 94%, respectively, and that the development of MEN1 was confined to first-degree relatives in 91% of patients and to second-degree relatives in 9% of patients. These findings have helped to define a proposed screening programme for MEN1.      

CTLA-4 ligation blocks CD28-dependent T cell activation [published erratum appears in J Exp Med 1996 Jul 1;184(1):301]

CTLA-4 is a CD28 homologue believed to be a negative regulator of T cell function. However, the mechanism of this downregulatory activity is not well understood. The present study was designed to examine the effect of CTLA-4 ligation on cytokine production, cell survival, and cell cycle progression. The results demonstrate that the primary effect of CTLA-4 ligation is not the induction of apoptosis. Instead, CTLA-4 signaling blocks IL-2 production, IL-2 receptor expression, and cell cycle progression of activated T cells. Moreover, the effect of CTLA-4 signaling was manifested after initial T cell activation. Inhibition of IL-2 receptor expression and cell cycle progression was more pronounced at late (72 h) time points after initial activation. The effects of anti-CTLA-4 mAbs were most apparent in the presence of optimal CD28- mediated costimulation consistent with the finding that CTLA-4 upregulation was CD28-dependent. Finally, the addition of exogenous IL- 2 to the cultures restored IL-2 receptor expression and T cell proliferation. These results suggest that CTLA-4 signaling does not regulate cell survival or responsiveness to IL-2, but does inhibit CD28- dependent IL-2 production.     

Neisserial porins induce B lymphocytes to express costimulatory B7-2 molecules and to proliferate [published erratum appears in J Exp Med 1996 Sep 1;184(3):1197]

The neisserial porins are the major protein components of the outer membrane of the pathogenic Neisseria (N. meningitidis and N. gonorrhoeae). They have been shown to be able to enhance the immune response to poorly immunogenic substances (e.g., polysaccharides, peptides, glycolipids, etc.). To explore the basis of their potent adjuvant activity, the effect of the neisserial porins on T-B cell interactions and T cell costimulation was examined. Neisserial porins increased the surface expression of the costimulatory ligand B7-2 (CD86) but did not affect the expression of B7-1 (CD80). In addition, incubation with the neisserial porins increased the T lymphocyte costimulatory ability of B lymphocytes, which was inhibited by anti-B7- 2 but not anti-B7-1 monoclonal antibodies. Upregulation of B7-2 on the surface of B lymphocytes may be the mechanism behind the immunopotentiating activity of neisserial porins.     

Oligoclonal T lymphocytes in the cerebrospinal fluid of patients with multiple sclerosis [published erratum appears in J Exp Med 1988 Jul 1;168(1):459]

We have investigated the T cell populations in the cerebrospinal fluid (CSF) of chronic progressive multiple sclerosis (MS) patients. Individual T cells from the CSF and blood were cloned before expansion and their clonotypes were defined by analysis of rearranged T cell receptor beta chain and gamma chain genes. 87 T cell clones from blood and CSF of two patients with chronic progressive MS were examined for common TCR gene rearrangement patterns. In one patient, 18 of 28 CSF-derived T cell clones demonstrated common TCR gene rearrangements indicating oligoclonal T cell populations; in the blood, two patterns were found twice among 26 T cell clones. In another patient, 5 of 27 CSF-derived clones had common TCR gene rearrangement patterns. In contrast, no common beta chain rearrangement pattern was found among 67 T cell clones derived from the blood or CSF of a patient with subacute sclerosing panencephalitis, among 20 clones from the CSF of a patient with herpes zoster meningoencephalitis, or among 66 clones from a normal subject. A subject with atypical, fatal MS of 8-mo duration was also studied and did not have oligoclonal T cells in the CSF or blood. These results demonstrate that distinct oligoclonal T cell populations can be found in the CSF immune compartment of subjects with nonmalignant inflammatory disease and they can create a new avenue for the investigation of the specificity of the T cell response within the central nervous system.     

Organ-specific selection of viral variants during chronic infection [published erratum appears in J Exp Med 1988 Jul 1;168(1):457]

This study demonstrates organ specific selection of viral variants during chronic lymphocytic choriomeningitis virus (LCMV) infection in its natural host. Isolates with different biological properties were present in the central nervous system (CNS) and lymphoid tissues of carrier mice infected at birth with the wt Armstrong strain of LCMV. Viral isolates from the CNS were similar to the wt Armstrong strain and induced potent virus-specific cytotoxic T lymphocyte (CTL) responses in adult mice and the infection was cleared within 2 wk. In contrast, LCMV isolates derived from the lymphoid tissues caused a chronic infection in adult mice associated with suppressed CTL responses. Revertants with wt Armstrong phenotype were present in the CNS of mice infected with a spleen isolate showing unequivocally the importance of host tissues in the selection of viral variants. These results provide a possible mechanism by which viral variants emerge in nature and suggest that tissue- and cell-specific selection is an important aspect of virus evolution.     

The induction of experimental autoimmune myocarditis in mice lacking CD4 or CD8 molecules [corrected] [published erratum appears in J Exp Med 1994 Jan 1;179(1):371]

Experimental induction of most autoimmune diseases appears to depend on the activation of CD4+ T helper cells, while CD8+ lymphocytes may have a role in disease progression. To study the role of CD4+ and CD8+ T cell subsets in T cell-dependent autoimmunity, mice lacking CD4 or CD8 molecules after gene targeting were injected with cardiac myosin to induce organ specific autoimmune myocarditis. Mice homozygous for the CD8 mutation (CD8-/-) developed significantly more severe disease as compared to CD4+/-CD8+/- controls. Surprisingly, CD4-/- mice developed autoimmune myocarditis with infiltration of TCR alpha beta +CD4-CD8- T cells in the heart tissue and appearance of autoantibodies. These data demonstrate that the lack of CD4+ or CD8+ T cells has no significant influence on the initiation of autoimmune myocarditis. CD4+ and CD8+ cells regulate disease severity and these results may explain the occurrence of autoimmunity in CD4 immunodeficiencies.     

Soluble interleukin-2 receptor: elevated levels in serum and synovial fluid of patients with rheumatoid arthritis

Soluble interleukin-2 receptor (sIL-2R) levels were quantitated in the serum and synovial fluid (SF) of patients with rheumatoid arthritis (RA) and degenerative joint disease (DJD). A sandwich immunoassay, employing two monoclonal antibodies against distinct epitopes on the IL-2R, was utilized for measurement. We found a striking elevation of sIL-2R in RA SF as compared with DJD SF (RA, 1319 +/- 135; DJD, 416 +/- 59; p less than 0.001). RA serum sIL-2R levels were also significantly elevated over DJD levels. There was no interaction between rheumatoid factor (RF) and sIL-2R. RA patients with elevated sIL-2R levels had significantly longer disease duration, higher c-reactive protein (CRP) levels in serum and SF, and higher RF levels in serum and SF. The groups were similar in regard to other laboratory variables. The presence of elevated levels of sIL-2R in RA serum and SF confirms the presence of a heightened immune reactivity and in vivo activation of lymphocytes in RA.     

Inhibition of immunoglobulin gene rearrangement by the expression of a lambda 2 transgene [published erratum appears in J Exp Med 1989 Aug 1;170(2):619]

The rearrangement of Ig genes is known to be regulated by the production of H and kappa L chains. To determine whether lambda L chains have a similar effect, transgenic mice were produced with a lambda 2 gene. It was necessary to include the H chain enhancer, since a lambda gene without the added enhancer did not result in transgene expression. The lambda 2 transgene with the H enhancer was expressed in lymphoid cells only. The majority of the B cells of newborn transgenic mice produced lambda, whereas kappa + cells were reduced. Concomitantly, serum levels of kappa and kappa mRNA were diminished. By 2 wk after birth the proportion of kappa-expressing cells was dramatically increased. Adults had reduced proportions of B cells that produced lambda only, but the levels of lambda were still higher than in normal littermates. Also, kappa + cells were still lower than in normal mice. Analysis of hybridomas revealed that reduction of kappa gene rearrangement was the basis for the decreased frequency of kappa + cells. Furthermore, many cells also contained an unrearranged H chain allele. It was concluded that feedback inhibition by the lambda 2 together with endogenous H protein may have inhibited recombinase activity in early pre-B cells, leading to inhibition of both H chain and kappa gene rearrangement. Thus, lambda 2 can replace kappa in a feedback complex. The levels of serum lambda 1 and, to a lesser degree, of spleen lambda 1 mRNA were reduced in the lambda 2 transgenic mice. However, the proportion of hybridomas with endogenous lambda gene rearrangement was at least as high as in normal mice. It was therefore concluded that the suppression of functional lambda 1 may be a consequence of decreased selection of endogenous lambda-producing cells because of the excess of transgenic lambda. The escape of kappa-producing cells from feedback inhibition may be the result of several mechanisms that operate to varying degrees, among them: (a) kappa rearrangement during a period in which the recombinase is still active after appearance of a lambda 2/mu stop signal; (b) a B cell lineage that is not feedback inhibited at the pre-B cell stage; (c) subthreshold levels of transgenic lambda 2 in some pre-B cells; and (d) loss of the lambda 2 transgenes in rare pre-B cells.     

A shared idiotype among human anti-Ro/SSA autoantibodies [published erratum appears in J Exp Med 1990 Feb 1;171(2):591]

Idiotypes and antiidiotypes are thought to be important immune regulators and have provided clues for the origin and pathogenicity of autoantibodies. Many lupus and Sj?gren's syndrome patients, as well as most neonatal lupus infants with congenital heart block or dermatitis, have antibodies to the ribonucleoprotein Ro/SSA, which is one of a group of RNA-protein autoantigens commonly found in human lupus sera. To characterize the fine specificity of anti-Ro/SSA antibodies, a rabbit antidiotypic serum was prepared against polyclonal affinity purified anti-Ro/SSA F(ab')2. The resulting antiidiotype, anti-Id-Rol, is specific for the F(ab')2 fraction of the anti-Ro/SSA immunogen and its binding to anti-Ro/SSA is inhibited by purified Ro/SSA. These data indicate that the Id-Rol epitope on anti-Ro/SSA is associated with the antigen binding site of these same antibodies. The Id-Rol idiotype was present by ELISA in 3 of 12 additional anti-Ro/SSA preparations from precipitin-positive donor sera and in anti-Ro/SSA from one normal donor with low level antibody. This is the first shared idiotype to be found in the human autoantibodies binding to this RNA-protein antigen. Idiotypic differences between anti-Ro/SSA autoantibodies have the potential to explain the variation in pathologic associations found in individuals who develop this autoantibody specificity.     

Macrosialin, a macrophage-restricted membrane sialoprotein differentially glycosylated in response to inflammatory stimuli [published erratum appears in J Exp Med 1992 Jan 1;175(1):309]

Rat monoclonal antibody FA/11 has been used to identify macrosialin, a sialoglycoprotein confined to murine mononuclear phagocytes and related cells. Originally identified as a macrophage-associated glycoprotein predominantly localized in intracellular membranes (Smith, M.J., and G.L.E. Koch. 1987. J. Cell Sci. 87:113), the antigen is widely expressed on tissue macrophages, including those in lymphoid areas, and is expressed at low levels on isolated dendritic cells. Immuno-adsorption experiments reported here show that macrosialin is identical to the major 87-115-kD sialoglycoprotein previously identified by lectin blotting in exudate but not resident peritoneal macrophages (Rabinowitz, S., and S. Gordon. 1989. J. Cell Sci. 93:623). Resident peritoneal macrophages express low levels of macrosialin antigen in a glycoform that does not bind 125I wheat germ agglutinin or 125I peanut agglutinin; inflammatory stimuli upregulate expression of this antigen (up to 17-fold), in an alternative glycoform that is detected by these lectins. Pulse-chase experiments reveal a 44-kD core peptide that initially bears high-mannose chains (giving Mr 66 kD) and is subsequently processed to a mature protein of Mr 87-104 kD. Each glycoform contains N-linked glycan, as well as O-linked sugar structures that show alternative processing. Poly-N-acetyllactosamine structures are detected in the exudate cell glycoform only. This new marker for mononuclear phagocytes illustrates two strategies by which macrophages remodel their membranes in response to inflammatory stimuli. Its predominantly intracellular location and restricted cell distribution suggest a possible role in membrane fusion or antigen processing.     

The protein tyrosine kinase p56lck regulates thymocyte development independently of its interaction with CD4 and CD8 coreceptors [corrected] [published erratum appears in J Exp Med 1993 Sep 1;178(3):1135]

The lck gene encodes a lymphocyte-specific protein tyrosine kinase of the nonreceptor type that is implicated in signal transduction pathways emanating from the CD4 and CD8 coreceptors. Previous studies also support a role for p56lck in regulating T cell receptor beta gene rearrangements and, more generally, thymocyte development. Here we report that a mutant form of p56lck, which is incapable of interacting with CD4 or CD8, behaves indistinguishably from association-competent p56lck with respect to its ability to affect thymocyte maturation. The effects of p56lck remained specific in that the closely related src- family kinase p59hck was incapable of substituting for p56lck in arresting beta locus gene rearrangements. These data support the view that src-family kinases perform highly specialized and often nonoverlapping functions in hematopoietic cells, and that p56lck acts independently of its association with CD4 and CD8 to regulate thymocyte development.     

Cloning and characterization of a cDNA for murine macrophage inflammatory protein (MIP), a novel monokine with inflammatory and chemokinetic properties [published erratum appears in J Exp Med 1989 Dec 1;170(6):2189]

In the course of studies on cachectin/TNF being conducted in our laboratory, a novel macrophage product has been detected and characterized. Termed macrophage inflammatory protein or MIP, this protein appears to be an endogenous mediator of the inflammatory events induced by endotoxin. A cDNA cloned probe for this protein has been isolated from a lambda gt10 phage library prepared from poly(A)+ RNA obtained of endotoxin-induced RAW264.7 cells. The sequence codes for a 92 amino acid-long polypeptide, of which 69 amino acids correspond to the mature product. The sequence predicts a molecular weight of 7,889 and structural analysis of the protein indicates a characteristic signal sequence alpha-helix and a hydrophobic core. Sequence data also confirm no sequence similarity to any other protein listed in the Dayhoff data base.     

Dynamic changes in cytokine levels in serum and synovial fluid following filtration leukocytapheresis therapy in patients with rheumatoid arthritis

We attempted to determine whether various cytokine levels in the serum and synovial fluid (SF) of rheumatoid arthritis (RA) patients are influenced by the performance of filtration leukocytapheresis (LCP). The filtration LCP procedure that used a Cellsorba® column (LCP group: n=22; responder subgroup: n=17, non‐responder subgroup: n=5) or sham apheresis (control group; n=7) was repeated three times at 1‐week intervals. Serum (LCP group, n=22; control group, n=7) and SF (LCP group, n=6; control group, n=3) samples were collected before and after LCP. Levels of tumor necrosis factor α (TNFα), interleukins (IL‐1β, IL–2, IL‐6, IL‐8, IL‐10, and IL‐15), granulocyte–macrophage colony–stimulating factor (GM‐CSF), monocyte chemoattractant protein–1 (MCP‐1), RANTES were measured by an enzyme‐linked immunosorbent assay. Serum TNFα, IL‐15, and RANTES were significantly reduced only in the LCP group. Serum IL‐10 significantly increased only in the LCP group. In the LCP subgroup, serum IL‐15, GM‐CSF, and RANTES levels were reduced significantly, while serum IL‐10 levels increased significantly only in the responder group after treatment. Serum TNFα levels were reduced significantly in both subgroups. Changes in serum IL‐10 correlated positively with the improvement of patient's assessment of pain and global severity, and physician's assessment of global severity. These results indicate that the removal of leukocytes from the peripheral blood of RA patients provokes dynamic changes in some cytokine levels in the serum and/or synovial fluid. These changes may explain some of the mechanisms by which the articular symptoms are improved by filtration LCP. J. Clin. Apheresis. 16:74–81, 2001. © 2001 Wiley‐Liss, Inc.     

Involvement of p59fynT in interleukin-5 receptor signaling [published erratum appears in J Exp Med 1995 Oct 1;182(4):1179]

Previous studies implicate the nonreceptor protein tyrosine kinase (PTK) p59fyn in the propagation of signals from the B cell antigen receptor. To elucidate the functions of this kinase, we examined B cell responsiveness in mice engineered to lack the hematopoietic isoform of p59fyn. Remarkably, antigen receptor signaling was only modestly defective in fynTnull B cells. In contrast, signaling from the interleukin (IL)-5 receptor which ordinarily provides a comitogenic stimulus with antiimmunoglobulin, was completely blocked. Our results document the importance of p59fynT in IL-5 responses in B cells, and they support a general model for cytokine receptor signal transduction involving the simultaneous recruitment of at least three families of PTK.     

In vivo expression of perforin by CD8+ lymphocytes during an acute viral infection [published erratum appears in J Exp Med 1989 Dec 1;170(6):2191]

CTL and NK cells cultured in vitro have been shown to contain a cytolytic pore-forming protein (PFP/perforin/cytolysin). To date, it has not been determined whether perforin is expressed by CTL that have been primed in vivo. Here, we have infected mice with two strains of lymphocytic choriomeningitis virus (LCMV), one of which mainly produces choriomeningitis and, the other, hepatitis. Brain and liver cryostat sections obtained from LCMV-infected mice were stained for various lymphocyte markers, including perforin. We were able to detect a large accumulation of perforin antigen in CD8+/Thy-1+/asialo GM1+/CD4- lymphocytes, which in fact represent the main infiltrating cell type found in brain and liver sections obtained during the late acute stage of LCMV infection. Perforin was also detected in a smaller population of CD8-/asialo GM1+/NK 1.1+/F480- cells, presumably corresponding to NK cells. Perforin-positive cells were found to have the morphology of blasts or large granular lymphocytes (LGL). These observations, together with in vitro studies performed in the past, indicate that perforin may be associated exclusively with LGL-like CTL blasts and NK cells. Our results demonstrate for the first time the presence of perforin in CTL that have been primed in vivo and suggest that perforin-positive CTL may be directly involved in producing the immunopathology associated with the LCMV infection.     

Net inflammatory capacity of human septic shock plasma evaluated by a monocyte-based target cell assay: identification of interleukin-10 as a major functional deactivator of human monocytes [published erratum appears in J Exp Med 1996 Nov 1;184(5):2075]

We have developed a functional assay to study the inflammatory capacity of plasma collected from patients with severe gram-negative septic shock. In this assay, elutriation-purified, cryo-preserved human monocytes from one healthy donor are combined with plasma from patients with severe persistent septic shock for 5 h. Subsequently, the plasma is removed, medium added, and procoagulant activity (PCA) and secretion of tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) measured after 18-h incubation. Plasma from 10 patients (6 died) infected with Neisseria meningitidis previously shown to contain high levels of native lipopolysaccharide (LPS) (median 2,700 pg/ml), TNF- alpha, IL-6, IL-8, and complement activation products, had a low net spontaneous inflammatory capacity on the monocytes. The median levels of PCA, TNF-alpha, and IL-6 were 5, 0, and 4%, respectively, of the monocyte activities induced by normal plasma boosted with purified N. meningitidis (Nm)-LPS (2,500 pg/ml; net LPS-boosted capacity, 100%). The levels of PCA, TNF-alpha, and IL-6 obtained with plasma from shock patients were not different from those induced by plasma from 10 meningococcal patients without shock or with plasma from healthy persons. Boosting shock plasma with 2,500 pg/ml Nm-LPS had little effect on the monocyte activities since the median values of PCA, TNF- alpha, and IL-6 revealed a minimal increase from 5, 0, and 4% to 9, 2, and 6%, respectively. The shock plasmas revealed a strong LPS- inhibitory capacity that was largely absent in plasmas from 10 meningococcal patients without shock since the median levels of PCA, TNF-alpha, and IL-6 increased from 5, 0, and 0% to 135, 51, and 73%, respectively, after boosting with 2,500 pg/ml Nm-LPS. The LPS- inhibitory capacity was closely associated with the levels of IL-10. The median levels of IL-10 were 19,000 pg/ml in nine shock patients vs. 22 pg/ml in nine nonshock patients with systemic meningococcal disease. Removal of native IL-10 by immunoprecipitation restored the capacity of plasmas to induce monocyte activation either by native LPS or by boosting with Nm-LPS. IL-4 and TGF-beta were not detected in shock plasmas. In 24 patients with detectable meningococcal LPS ( > 10 pg/ml, 0.1 endotoxin units/ml), the levels of IL-10 were correlated to the levels of LPS (r = 0.79, P < 0.001). IL-10 declined from initiation of antibiotic therapy and paralleled the levels of native LPS. Decreasing levels of IL-10 in serially collected shock plasmas were directly related to increasing monocyte responsiveness after Nm-LPS boosting. These results suggest that IL-10 plays a major role in containing activation of monocytes and possibly other LPS-responsive cells during overwhelming meningococcemia.