A quarter century of granzymes.

Granzymes (Grs) were discovered just over a quarter century ago. They are produced by cytotoxic T cells and natural killer cells and are released upon interaction with target cells. Intensive biochemical, genetic, and biological studies have been performed in order to study their roles in immunity and inflammation. This review summarizes research on the family of Grs.

Specificity and function of activating Ly-49 receptors.

Inhibitory Ly-49 receptors allow murine natural killer (NK) cells to kill cells with aberrant class I MHC expression while sparing normal cells. This is accomplished by their recognition of specific class I MHC products and prevention of NK-cell lysis of cells that present a normal repertoire of class I MHC ligands--"the missing self hypothesis". However, Ly-49 receptors that lack the cytoplasmic immunoreceptor tyrosine-based inhibitory motif, which is required for inhibition of killing, have also been described. These receptors were found to stimulate NK killing and are therefore referred to as activating Ly-49 receptors. Interestingly, the activating receptors have class I MHC-binding domains that are nearly indistinguishable from those of the inhibiting receptors, and binding to class I MHC has now been demonstrated for three activating receptors. Presently, there is no defined physiological role for activating Ly-49 receptors. Here we present an overview of current knowledge regarding the diversity, structure and function of activating Ly-49 receptors with a focus on class I MHC specificity, and we discuss their potential role(s) in natural resistance.

Attenuation of endothelin-1 induced vasoconstriction by 17beta estradiol is not sustained during long-term therapy in postmenopausal women with coronary heart disease.

OBJECTIVES: The goal of this study was to determine the long-term effects of estrogen replacement therapy on the response to endothelin-1 (ET-1) in postmenopausal women with coronary heart disease. BACKGROUND: It is thought that the vasoconstrictor ET-1 is involved in the development and progression of atherosclerosis. Estrogen replacement may slow the development of atherosclerosis in postmenopausal women. METHODS: Nineteen of 20 postmenopausal women randomized to either three months of 2 mg oral estradiol or placebo completed the double-blind placebo-controlled protocol. Change in forearm blood flow (FBF) in response to a 60 min brachial arterial infusion of ET-1 (5 pmol/min) was measured before randomization, after one month of randomized therapy and after three months of therapy using venous occlusion plethysmography. RESULTS: Estrogen treatment had no effect on baseline FBF. Systolic and diastolic blood pressure and heart rate did not change in response to estrogen therapy or ET-1. Before randomization, in response to ET-1, FBF was reduced by -21.9% (mean response over 60 min) in the placebo group and -19.0% in the estradiol group (p = 0.67). After one month of therapy, the response was attenuated in the estrogen group, -10.0%, compared with the placebo group, -23.6 (difference in means 13.6%, 95% confidence interval [0.7%, 26.6%], p = 0.041). After three months of therapy, there was no difference in response between the placebo group, -27.0%, and estrogen group, -30.2% (p = 0.65). CONCLUSIONS: In postmenopausal women with coronary heart disease, estrogen therapy inhibits the vasoconstrictor response to ET-1 after one month of therapy. This effect is lost after three months of therapy, suggesting that tachyphylaxis to one potentially beneficial action of estradiol develops during chronic treatment.

Ly-49W, an activating receptor of nonobese diabetic mice with close homology to the inhibitory receptor Ly-49G, recognizes H-2D(k) and H-2D(d).

The diversity and ligand specificity of activating Ly-49 receptors expressed by murine NK cells are largely unknown. We cloned a new Ly-49-activating receptor, expressed by NK cells of the nonobese diabetic mouse strain, which we have designated Ly-49W. Ly-49W is highly related to the known inhibitory receptor Ly-49G in its carbohydrate recognition domain, exhibiting 97.6% amino acid identity in this region. We demonstrate that the 4D11 and Cwy-3 Abs, thought to be Ly-49G specific, also recognize Ly-49W. Rat RNK-16 cells transfected with Ly-49W mediated reverse Ab-dependent cellular cytotoxicity of FcR-positive target cells, indicating that Ly-49W can activate NK-mediated lysis. We further show that Ly-49W is allo-MHC specific: Ly-49W transfectants of RNK-16 only lysed Con A blasts expressing H-2(k) or H-2(d) haplotypes, and Ab-blocking experiments indicated that H-2D(k) and D(d) are ligands for Ly-49W. Ly-49W is the first activating Ly-49 receptor demonstrated to recognize an H-2(k) class I product. Ly-49G and Ly-49W represent a new pair of NK receptors with very similar ligand-binding domains, but opposite signaling functions.

A low-level expression of human MUC1 mucin enhances lethality of murine tumor cells.

We report here the development of a mouse mammary adenocarcinoma cell line containing full-length human MUC1 cDNA that can be more lethal than the parental cell line. The metastatic murine mammary adenocarcinoma cell line 410.4 was transfected with cDNA coding for a 42-tandem-repeat version of human MUC1. Two cell lines were selected, one for stable, high expression in vitro of cell-surface MUC1 (GZHi) and one for stable, low expression in vitro of cell-surface MUC1 (GZLo). Following subcutaneous challenge of CB6F1 mice with various doses of tumor cells, GZHi tumors showed loss of MUC1 expression; negligible amounts of serum MUC1 mucin were detected and the mice survived longer than mice challenged with GZLo or wild-type (410.4) tumor cells. Mice challenged with GZLo tumor cells had shorter survival times than mice challenged with either GZHi or 410.4 tumor cells. GZLo-challenged mice that showed rapidly increasing serum MUC1 mucin levels several weeks prior to death had a shorter survival than mice without detectable rising MUC1 serum levels. Surprisingly, SCID-BEIGE mice challenged with GZLo cells also survived for a shorter time than those challenged with either GZHi or 410.4 cells. This suggests that MUC1 mucin may also enhance the aggressiveness of GZLo tumors by non-immune mechanisms.

Ly-49P activates NK-mediated lysis by recognizing H-2Dd.

Little is known regarding the ligand specificity of Ly-49 activating receptor subfamily members expressed by NK cells. A new Ly-49 activating receptor related to Ly-49A in its extracellular domain, designated Ly-49P, was recently cloned from 129 strain mice. We independently cloned an apparent allele of Ly-49P expressed by nonobese diabetic and nonobese diabetes-resistant mouse strain NK cells. We found it to be reactive with the A1 Ab thought to recognize a polymorphic epitope expressed only by the Ly-49A inhibitory receptor of the C57BL/6 strain. Rat RNK-16 cells transfected with Ly-49P mediated reverse Ab-dependent cellular cytotoxicity of FcR-positive target cells, indicating that Ly-49P can activate NK-mediated lysis. We determined that RNK-16 lysis of Con A blasts induced by Ly-49P was MHC dependent, resulting in efficient lysis of H-2Dd-bearing targets. We found that the Dd alpha1/alpha2 domain is required for Ly-49P-mediated RNK-16 activation, as determined by exon shuffling and transfection. Thus, Ly-49P is the second activating Ly-49 receptor demonstrated to induce NK cytotoxicity by recognizing a class I MHC molecule.

Bradykinin B(2) receptor antagonism attenuates blood pressure response to acute angiotensin-converting enzyme inhibition in normal men.

The physiological effects of angiotensin-converting enzyme (ACE) inhibition may be in part mediated by bradykinin. We investigated the effect of coadministration of the specific bradykinin B(2) receptor antagonist icatibant on hemodynamic and neurohormonal responses to acute intravenous ACE inhibition in normal men on a normal sodium diet. We performed a 4-phase, double-blind, double-dummy, placebo-controlled study in 12 male volunteers. The bradykinin antagonist icatibant (10 mg IV) was coadministered over the first 15 minutes of a 2-hour infusion of the ACE inhibitor perindoprilat (1.5 mg IV). Perindoprilat inhibited ACE activity and elicited the expected changes in active renin concentration and angiotensin peptides. Over the 3 hours after the start of drug infusion, perindoprilat lowered and icatibant increased mean arterial blood pressure (each P<0.0005 versus placebo). Coadministration of icatibant attenuated the mean arterial blood pressure response to perindoprilat (P<0.0005) but had no effect on neurohormonal responses to perindoprilat. Our study indicates that the bradykinin B(2) receptor antagonist icatibant attenuates the short-term blood pressure-lowering effect of acute ACE inhibition in normal men on a normal sodium diet. Bradykinin B(2) receptor antagonism alone increases resting blood pressure. Bradykinin may be involved in the control of blood pressure in the resting state in humans.

Comparison of the AT1-receptor blocker, candesartan cilexetil, and the ACE inhibitor, lisinopril, in fixed combination with low dose hydrochlorothiazide in hypertensive patients.

AIM: To compare candesartan cilexetil and lisinopril in fixed combination with hydrochlorothiazide with respect to antihypertensive efficacy and tolerability. METHODS: This was a double-blind (double-dummy), randomised, parallel group comparison in patients with a mean sitting diastolic blood pressure 95-115 mm Hg on prior antihypertensive monotherapy. Treatments were candesartan cilexetil/hydrochlorothiazide 8/12.5 mg once daily (n = 237) and lisinopril/hydrochlorothiazide 10/12.5 mg once daily (n = 116) for 26 weeks. The primary efficacy variable was change in trough sitting diastolic blood pressure. RESULTS: Changes in mean sitting diastolic blood pressure did not differ significantly between the groups (mean difference 0.5 mm Hg; 95% confidence interval -1.6, 2.7, P = 0.20). No significant differences between the groups was found for other haemodynamic variables (sitting systolic blood pressure, standing blood pressure, sitting/erect heart rate, and proportion of responders and controlled patients). Both drugs were well tolerated but the proportion of patients with at least one adverse event was significantly greater in the lisinopril group (80% vs 69%, P = 0.020). The proportion of patients spontaneously reporting cough (23.1% vs 4.6%) and discontinuing therapy due to adverse events (12.0% vs 5.9%) was also higher in the lisinopril group compared with the candesartan cilexetil group. CONCLUSIONS: The fixed combinations of candesartan cilexetil and hydrochlorothiazide 8/12.5 mg and lisinopril and hydrochlorothiazide 10/12.5 mg once daily are equally effective as antihypertensive agents. The fixed combination containing candesartan cilexetil is better tolerated than that containing lisinopril.

Bacterial modulation of antigen processing and presentation.

This review examines the mechanisms by which bacteria influence the antigenic processing of endogenous and exogenous antigens presented by class I, class II, and nonclassical MHC molecules. Consequent effects on presentation of bacterial antigens, the ability of bacteria to evade host defences, and the potential induction of autoimmunity are discussed.

Generation and characterization of a monoclonal antibody that recognizes an activation antigen expressed by a majority of adherent lymphokine-activated killer cells.

In an attempt to generate murine natural killer (NK) cell-specific monoclonal antibodies (MAbs) by immunizing Balb/c mice with C57BL/6 (B6) A-LAKs, we have isolated a hybridoma, CS/NicT.2, which secretes an IgM that recognizes a majority of B6 and B6 Rag-1-/- A-LAKs. The CS/NicT.2 antigen is highly expressed by A-LAKs, but only at extremely low levels on resting splenocytes, suggesting that its expression is tightly associated with IL-2 activation. Among the cell lines examined, only CTLL-2 expresses the CS/NicT.2 antigen at relatively high levels. A low level of CS/NicT.2 staining is also detected on resting allo-specific cytotoxic T lymphocytes (CTL) clones, AB.1 and C11. In addition, a similar low level of CS/NicT.2 staining is detected on the T-helper cell line HT-2. The CS/NicT.2 antigen is upregulated by ionomycin but not by phorbol 12-myristate 13-acetate (PMA). For the CTL clones examined, CS/NicT.2 staining is also dramatically increased by anti-TCRbeta or anti-CD3epsilon stimulation. Protease treatments of CTLL-2 show that this antigen is proteinase K sensitive, but relatively resistant to trypsin digestion. Furthermore, the CS/NicT.2 antigen exhibits a relatively fast turnover rate as assessed by proteinase K and cycloheximide treatments of CTLL-2, suggesting that the CS/NicT.2 antigen may have a short half-life on the cell surface.

Generation of a monoclonal antibody that recognizes a polymorphic epitope of C57BL/6 Ly-49G2.

To facilitate the study of natural killer (NK) cell functions, we have generated a panel of hybridomas that secrete antibodies recognizing B6 NK cell surface markers. One of these hybridomas, Cwy-3, secretes a mouse IgG1 specific for the B6 allele of Ly-49G2 (Ly-49G2B6). In addition, this monoclonal antibody (MAb) fails to stain A-LAKs derived from all other mouse strains tested. This suggests that Cwy-3 recognizes a polymorphic epitope of Ly-49G2B6. More importantly, this MAb exhibits no cross-reactivity to other known B6 Ly-49 family members. Therefore, Cwy-3 is in sharp contrast to the two known anti-Ly-49G2 MAbs, which are reported to recognize a nonpolymorphic epitope of Ly-49G2, and react with other Ly-49 members. In this regard, Cwy-3 will offer significant advantages over the cross-reactive anti-Ly-49G2 MAbs in fully defining the specificity and function of the Ly-49G2B6 NK cell receptor. With this novel Ly-49G2-specific MAb, we have isolated an EL-4 subline expressing a significant density of Ly-49G2 receptors.

Ly-49CB6 NK inhibitory receptor recognizes peptide-receptive H-2Kb.

NK-mediated cytotoxicity involves two families of receptors: activating receptors that trigger lysis of the target cells being recognized and inhibitory receptors specific primarily for MHC I on the target cell surface that can override the activating signal. MHC I molecules on the cell surface can be classified into molecules made stable by the binding of peptide with high affinity or unstable molecules potentially capable of binding high affinity peptide (hence, peptide receptive) and being converted into stable molecules. It has been previously shown that the Ly-49A inhibitory receptor recognizes stable Dd molecules. We show in this study that the inhibitory receptor Ly-49CB6 recognizes peptide-receptive Kb molecules, but does not recognize Kb molecules once they have bound high affinity peptide.

The murine nonclassical class I major histocompatibility complex-like CD1.1 molecule protects target cells from lymphokine-activated killer cell cytolysis.

Classical class I major histocompatibility complex (MHC) molecules, as well as the nonclassical class I histocompatibility leukocyte antigen (HLA)-E molecule, can negatively regulate natural killer (NK) cell cytotoxicity through engagement of NK inhibitory receptors. We show that expression of murine (m)CD1.1, a nonpolymorphic nonclassical MHC class I-like molecule encoded outside the MHC, protects NK-sensitive RMA/S target cells from adherent lymphokine-activated killer cell (A-LAK) cytotoxicity. Passage of effector cells in recombinant interleukin (rIL)-2 enhanced protection by mCD1.1, suggesting an expansion of relevant A-LAK population(s) or modulation of A-LAK receptor expression. Murine CD1. 1 conferred protection from lysis by rIL-2-activated spleen cells of recombination activating gene (Rag)-1(-/-) mice, which lack B and T cells, demonstrating that mCD1.1 can protect RMA/S cells from lysis by NK cells. An antibody specific for mCD1.1 partially restored A-LAK lysis of RMA/S.CD1.1 transfectants, indicating that cell surface mCD1.1 can confer protection from lysis; therefore, mCD1.1 possibly acts through interaction with an NK inhibitory receptor. CD1.1 is by far the most divergent class I molecule capable of regulating NK cell activity. Finally, mCD1.1 expression rendered RMA/S cells resistant to lysis by A-LAK of multiple mouse strains. The conserved structure of mCD1.1 and pattern of mCD1.1 resistance from A-LAK lysis suggest that mCD1.1 may be a ligand for a conserved NK inhibitory receptor.

Invasion by Salmonella typhimurium induces increased expression of the LMP, MECL, and PA28 proteasome genes and changes in the peptide repertoire of HLA-B27.

We have analyzed proteasomal adaptation and associated changes in the B27-bound peptide repertoire in response to cellular invasion with Salmonella. The peptide repertoire of HLA-B27 complexes was analyzed by two different methods: (i) high-pressure liquid chromatography (HPLC) profiles of newly synthesized peptides eluted from B27 following metabolic labeling with arginine and (ii) reactivities with two B27 monoclonal antibodies, Ye-2 and B27.M2, sensitive to peptide-induced conformational changes. LMP, MECL, and PA28 expression was analyzed by reverse transcription-PCR (RT-PCR) of mRNA and by Western blot analysis for LMP2. Invasion of HLA-B27-transfected HeLa cells by Salmonella typhimurium induced significant changes in the reactivities of HLA-B27 with these two antibodies, which was accompanied by significant quantitative and qualitative changes in the HPLC profile of peptides eluted from HLA-B27. We also observed increases in the RT-PCR values for the LMP2, LMP7, and MECL proteasome subunit genes, as well as the proteasomal activator PA28alpha and -beta genes, and increased expression of the LMP2 protein by Western blotting. Upregulation of LMP2, but not LMP7, gene expression showed a close correlation with the changes in antibody reactivities observed upon bacterial invasion. We observed similar changes in reactivity with the Ye-2 or the B27.M2 antibody of lymphoblastoid cells upon gamma interferon treatment, which significantly correlated with the increased RT-PCR values for the LMP2 gene. This was accompanied by consistent HPLC profile changes for eluted peptides. Thus, Salmonella invasion leads to serologically recognizable changes in the B27-bound peptide repertoire, which may include peptides of host origin potentially through modulation of proteasome LMP2 subunit expression and, as a consequence, proteasomal activities.

Evidence for sulfate modification of H-2Dd on N-linked carbohydrate(s): possible involvement in Ly-49A interaction.

Murine class I molecules are ligands for Ly-49 molecules, a family of regulatory receptors expressed on murine NK cells. Since soluble sulfated mono- and polysaccharides interfere with the interaction of Ly-49A, a C-type lectin, and its class I ligand, Dd, it is possible that the oligosaccharides on class I molecules are sulfated and participate in Ly-49A binding. In this report, we show that H-2Dd expressed by activated T cells and various tumor cell lines is sulfated, as demonstrated by immunoprecipitation of Dd following Na235SO4 labeling. The 35SO4(-2) label on Dd expressed by a representative tumor cell, NZB1.1, is removed by peptide N-glycosidase F, but is resistant to endoglycosidase H treatment, indicating that the sulfate group is located on mature N-linked oligosaccharides. Two-dimensional SDS-PAGE analysis revealed that all major mature glycosylation variants of the Dd expressed by NZB1.1 are sulfated. Sodium chlorate, a potent inhibitor of ATP-sulfurylase, which prevents the formation of the sulfate donor, 3'-phosphoadenosine 5'-phosphosulfate, inhibited metabolic sulfation of Dd. NZB1.1 binds isolated Ly-49A immobilized on solid phase through an interaction by cell surface Dd, since cell adhesion was blocked by Abs directed against Dd or Ly-49A. Treatment of the Dd-expressing NZB1.1 tumor cells with sodium chlorate reduced their ability to bind immobilized Ly-49A, particularly when Ly-49A density was limiting. These results provide evidence for sulfation of H-2Dd oligosaccharide moieties, and suggest a role for this posttranslational modification in the interaction of Dd with Ly-49A.

Left ventricular hypertrophy, blood pressure and ACE genotype in untreated hypertension.

It has been suggested that the deletion polymorphism of the angiotensin-converting enzyme (ACE) genotype may be important in the development of left ventricular hypertrophy (LVH). In order to test this hypothesis we investigated the interaction between blood pressure (BP), LVH and ACE genotype in 86 previously untreated hypertensive patients. Each underwent two-dimensional and Doppler echocardiography and ACE genotyping. There were no significant differences in BP, the parameters of left ventricular structure (including left ventricular mass index) or diastolic function between the three genotype groups. Additionally, there were no significant differences in the relationship between systolic BP and left ventricular mass index among the three genotype groups (II genotype, r = 0.46, P = 0.02; ID genotype, r = 0.42, P = 0.01; DD genotype, r = 0.34, P = 0.10; F = 0.38). In contrast to some previous studies, we have found in this group of previously untreated hypertensive subjects no evidence to suggest that the deletion polymorphism of the ACE genotype is important in the development of LVH.

The efficacy and tolerability of candesartan cilexetil in an elderly hypertensive population.

This study was performed to evaluate the antihypertensive efficacy and tolerability of candesartan cilexetil 8-16 mg once-daily in comparison with placebo in elderly hypertensive patients. Forty-one hospital and general practice centres in the Netherlands and in the United Kingdom enrolled 350 patients over 65 years of age with essential hypertension (WHO grades I or II). Patients with supine diastolic BP in the range 95-114 mm Hg after 4- to 8-week placebo run-in period (n = 193) were randomised to double-blind therapy with candesartan cilexetil or placebo. The initial dose of candesartan cilexetil 8 mg or placebo was doubled after 6 weeks if supine diastolic blood pressure (BP) exceeded 90 mm Hg. Mean (95% confidence interval) placebo-corrected reduction in supine diastolic BP after 12 weeks' treatment with candesartan cilexetil was 7.5 mm Hg (3.6-11.4; P < 0.001); the corresponding reduction in supine systolic BP was 13.6 mm Hg (6.9-20.2; P < 0.001). Placebo-corrected mean reduction in supine diastolic BP 2 and 4 h after the first dose of candesartan cilexetil were 2.2 mm Hg (-1.3 to +5.8; P = 0.219) and 4.0 mm Hg (-0.4 to +7.6; P = 0.027), respectively. Candesartan cilexetil had almost no influence on heart rate and did not affect the normal orthostatic changes in BP. Adverse events were equally common in the two treatment groups. Candesartan cilexetil 8-16 mg once-daily is an effective antihypertensive agent in elderly patients. The onset of action is smooth with no exaggerated response after the first dose and there is no postural hypotension. Candesartan cilexetil is very well tolerated in elderly hypertensives.

Major histocompatibility complex class I-dependent cell binding to isolated Ly-49A: evidence for high-avidity interaction.

Ly-49A molecules negatively regulate a subset of mouse natural killer (NK) cells, preventing lysis of H-2Dd-expressing target cells. In the present report, we immunoaffinity-purified Ly-49A from the EL4 lymphoma using the A1 monoclonal antibody (mAb) and examined cell adhesion to immobilized Ly-49A. Adhesion was observed by cells expressing relatively high levels of H-2Dd, but not cells expressing very low or no cell surface Dd, while antibodies specific for Dd or Ly-49A inhibited the cell binding, indicating that Dd and Ly-49A mediate the observed adhesion. The density of immobilized Ly-49A was varied and confirmed by ELISA. Cell binding exhibited a threshold Ly-49A density requirement, and above this threshold, increases in Ly-49A density resulted in substantial increases in cell adhesion to a high maximum cell binding. The density of Ly-49A homodimers required to mediate cell adhesion was found to be quite low: 140-250 molecules/microm2. These results suggest that the avidity of Ly-49A for Dd is relatively high and indicate that small changes in Ly-49A density near the threshold result in large changes in stable Ly-49A receptor engagement. The relatively sharp threshold and marked density dependence presented here for Ly-49A receptor engagement may explain the observation that relatively small differences in Ly-49A expression level on NK cells result in significant differences in functional outcome, i.e. whether a target cell expressing a low level of Dd is spared from lysis or not.