Tumor necrosis factor alpha-induced eosinophil accumulation in rat skin is dependent on alpha4 integrin/vascular cell adhesion molecule-1 adhesion pathways.

Tumor necrosis factor alpha (TNFalpha) is a cytokine implicated in the pathogenesis of numerous chronic and acute inflammatory conditions. In the present study, we have characterized the ability of TNFalpha in inducing eosinophil accumulation in rat skin and have shown the inhibitory effects of anti-alpha4 integrin and anti-vascular cell adhesion molecule-1 (VCAM-1) antibodies on this response. The intradermal injection of recombinant human TNFalpha induced a slowly developing, dose-dependent accumulation of 111In-eosinophils in rat skin that was maximal at the dose of 10(-11) mol/site. Coadministration of TNFalpha with the soluble TNFalpha receptor (p55)-IgG fusion protein (TNFR-IgG) totally inhibited the 111In-eosinophil accumulation induced by the cytokine. The TNFalpha-induced 111In-eosinophil accumulation was not affected after pretreatment of rats with the platelet-activating factor (PAF) receptor antagonist UK-74,505 or the antihuman interleukin-8 monoclonal antibody (MoAb) DM/C7. In contrast, the intravenous administration of an anti-alpha4 integrin MoAb, HP2/1 (3.5 mg/kg), or an anti-VCAM-1 MoAb, 5F10 (2 mg/kg), greatly inhibited the 111In-eosinophil accumulation induced by TNFalpha (the responses detected at 10(-11) mol/site were inhibited by 78% and 50%, respectively). These results show that TNFalpha is an effective inducer of eosinophil accumulation in vivo, with this response being dependent on an interaction between alpha4 integrins and VCAM-1.

Role of interleukin 8 on leucocyte-endothelial cell adhesion in intestinal inflammation.

BACKGROUND: An important action of interleukin 8 (IL8) is stimulation of granulocytes. The object of this study was to assess the contribution of IL8 to the leucocyte-endothelial cell interactions associated with intestinal inflammation in the rat. METHODS: Two indomethacin injections (48 and 24 hours prior to the experiments) induced a longlasting ileitis in rats. The number of adherent and emigrated leucocytes, leucocyte rolling velocity, and shear rate were monitored in normal and inflamed mesenteric postcapillary venules. Some animals received a monoclonal antibody (MAb) against IL8 or CD11b/CD18 at 24 and 12 hours prior to the experiment. RESULTS: Indomethacin elicited a seven-fold increase in leucocyte adherence and a 5.4-fold increase in leucocyte emigration, while leucocyte rolling velocity was reduced by nearly 80%. The indomethacin induced increases in leucocyte adherence and emigration were significantly reduced (by 57% and 67%, respectively) while leucocyte rolling velocity was increased (to 63% of control) by the IL8-specific MAb. The level of inhibition seen with the IL8 MAb was similar to that associated with administration of a MAb directed against the leucocyte adhesion molecule CD11b/CD18. CONCLUSIONS: IL8 contributes to the leucocyte-endothelial cell interactions elicited in mesenteric venules by indomethacin.

Identification and characterization of rhesus macaque interleukin-8.

To establish a direct link between IL-8 and inflammation in vivo, we first isolated the gene encoding rhesus macaque IL-8. The open reading frame directs the translation of a 101 amino acid (aa) precursor, which is 94% identical to human IL-8. Rhesus IL-8 was expressed in bacteria and purified to homogeneity with ion-exchange chromatography. Pure rhesus IL-8 was biologically active as measured by its ability to bind specifically to either rhesus (Kd = 0.5 nM) or human (Kd = 2 nM) IL-8 receptors and to promote in vitro chemotaxis of rhesus (EC50 = 2 nM) or human neutrophils (EC50 = 4 nM). Moreover, a mouse monoclonal antibody, DM/C7, which neutralizes human IL-8 activity, also recognized and neutralized (IC50 = 0.5-3.0 microgram/ml) rhesus IL-8 in vitro. Systemic administration of DM/C7 completely inhibited the dermal inflammation of rhesus ears induced by the external application of phorbol myristoyl acetate. These observations reveal that rhesus IL-8 is structurally and functionally similar to human IL-8 and suggests that IL-8 plays a prominent role in a primate model of inflammation.

IL-1 is a potent inducer of eosinophil accumulation in rat skin. Inhibition of response by a platelet-activating factor antagonist and an anti-human IL-8 antibody.

The aim of the present study was to investigate directly and characterize the ability of IL-1 beta in inducing eosinophil accumulation in vivo. For this purpose, we studied the recruitment of 111In-labeled eosinophils in rat skin in response to intradermally injected rat rIL-1 beta. Rat rIL-1 induced a dose-dependent accumulation of 111In-labeled eosinophils, with the maximal response being detected at 5 x 10(-13) mol/site. This response was slow in onset, progressively increasing over the 4-h period investigated. Rat rIL-1 also induced a small level of edema, as measured by the local accumulation of i.v. 125I-labeled albumin, which developed with a time course similar to that of 111In-labeled eosinophil accumulation. Co-administration of the cytokine with the IL-1R antagonist, IL-1ra, or actinomycin D, significantly inhibited the 111In-labeled eosinophil accumulation, and reduced the edema formation, induced by rat rIL-1. In addition, the 111In-labeled eosinophil accumulation was significantly suppressed in animals treated with the PAF antagonist UK-74,505 or an anti-human IL-8 mAb DM/C7. These observations demonstrate for the first time that IL-1 beta is a potent inducer of eosinophil accumulation in vivo. Moreover, the results reveal that this activity of IL-1 beta is receptor mediated and dependent on the induction of proteins that may be involved in the local generation of secondary inflammatory mediators including PAF and an IL-8-like molecule. These findings are consistent with the view that endogenously generated IL-1 may play an important role in the recruitment of eosinophils at sites of allergic inflammation.

Interleukin 8 and mast cell-generated tumor necrosis factor-alpha in neutrophil recruitment.

In the reverse passive Arthus reaction in mouse skin and immune injury of mouse dermal basement membrane, neutrophil (PMN) infiltration in mast cell deficient WBB6F1-W/Wv (W/Wv) mice was only 40% of that in WBB6F1-(+)/+ (+/+) mice that had a normal mast cell repertoire. An anti-tumor necrosis factor-alpha (TNF-alpha) monoclonal antibody (mAb) decreased PMN infiltration by 35-80% in +/+ but not W/Wv mice. In addition, an anti-human interleukin-8 (IL-8) MAb, DM/C7, inhibited PMN infiltration of the skin induced by either intradermal administration of recombinant human IL-1 beta or immune complex deposition. In both models of immune complex injury, DM/C7 reduced PMN infiltration by 40-60% in +/+ mice but not W/Wv mice. PMN infiltration and the sensitivity of this infiltration to anti-TNF-alpha or DM/C7 MAb in W/Wv mice whose mast cell population had been restored was indistinguishable from the influx observed in +/+ mice. These data suggest that TNF-alpha, IL-8, and mast cells play a fundamental role in PMN recruitment following immune complex injury.

Inhibition of lung inflammatory reactions in rats by an anti-human IL-8 antibody.

IL-8 belongs to the family of chemotactic cytokines and may play an important role in the inflammatory response. In the current studies, a murine mAb (DM/C7) to human rIL-8 was found to have protective effects in inflammatory lung injury in rats. DM/C7 was nonreactive with the rat cytokine-induced neutrophil chemoattractant peptide. In vivo, DM/C7 blocked the glycogen-induced accumulation of neutrophils in rats and was highly protective against lung and dermal vascular injury after deposition of IgG immune complexes. The latter model of injury has recently been shown to be E-selectin dependent. The protective effects of DM/C7 correlated with reduced tissue accumulation of neutrophils, as measured by myeloperoxidase content. DM/C7 reacted with an epitope expressed by TNF-alpha-stimulated rat pulmonary artery endothelial cells and with the pulmonary vascular endothelium after intrapulmonary deposition of IgG immune complexes. In the model of IgG immune complex-induced lung injury, the protective effects of DM/C7 were abolished by prior absorption of the antibody with human rIL-8. Polyclonal antibody to cytokine-induced neutrophil chemoattractant peptide failed to protect against IgG immune complex-induced vascular injury even though this antibody blocked the in vitro chemotactic activity of cytokine-induced neutrophil chemoattractant. In the model of rapidly developing lung injury due to systemic activation of C after infusion of cobra venom factor, DM/C7 was not protective. As well, in the neutrophil-independent model of IgA immune complex-induced lung injury, treatment with DM/C7 was not protective. These data indicate that in inflammatory lung injury that is linked to E-selectin-dependent recruitment of neutrophils in rats, antibody to human IL-8 also blocks recruitment of neutrophils and thereby affords protection against lung injury. The data suggest the presence of an IL-8-like product in this model of lung injury.

D-cycloserine acts as a partial agonist at the glycine modulatory site of the NMDA receptor expressed in Xenopus oocytes.

In the Xenopus oocyte preparation, D-cycloserine (DCS) had the profile of a partial agonist at the glycine modulatory site of the NMDA receptor. Maximal NMDA responses in the presence of DCS were only 40-50% of those in the presence of glycine. Glycine and D-serine were agonists at this site. The actions of DCS were competitively antagonized by HA-966, a known glycine antagonist.

Glycine antagonist action of 1-aminocyclobutane-1-carboxylate (ACBC) in Xenopus oocytes injected with rat brain mRNA.

ACBC has been reported to have the binding profile of an antagonist at the glycine site of the NMDA receptor. In Xenopus oocytes injected with rat brain mRNA, we have confirmed the antagonist action of ACBC on NMDA responses. ACBC and HA-966, a known glycine antagonist, blocked NMDA responses in a non-competitive manner and blocked the potentiation of NMDA responses by glycine in a competitive manner. We conclude that ACBC blocks NMDA responses via a competitive interaction at the glycine modulatory site.

cAMP stimulates transcription of the beta 2-adrenergic receptor gene in response to short-term agonist exposure.

In addition to conveying cellular responses to an effector molecule, receptors are often themselves regulated by their effectors. We have demonstrated that epinephrine modulates both the rate of transcription of the beta 2-adrenergic receptor (beta 2AR) gene and the steady-state level of beta 2AR mRNA in DDT1MF-2 cells. Short-term (30 min) exposure to epinephrine (100 nM) stimulates the rate of beta 2AR gene transcription, resulting in a 3- to 4-fold increase in steady-state beta 2AR mRNA levels. These effects are mimicked by 1 mM N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate (Bt2cAMP) or foskolin but not by phorbol esters. The half-life of the beta 2AR mRNA after addition of actinomycin D (46.7 +/- 10.2 min; mean +/- SEM; n = 5) remained unchanged after 30 min of epinephrine treatment (46.8 +/- 10.6 min; mean +/- SEM; n = 4), indicating that a change in transcription rate is the predominant factor responsible for the increase of beta 2AR mRNA. Whereas brief exposure to epinephrine or Bt2cAMP does not significantly affect the total number of cellular beta 2ARs (assessed by ligand binding), continued exposure results in a gradual decline in beta 2AR number to approximately 20% (epinephrine) or approximately 45% (Bt2cAMP) of the levels in control cells by 24 hr. Similar decreases in agonist-stimulated adenylyl cyclase activity are observed. This loss of receptors with prolonged agonist exposure is accompanied by a 50% reduction in beta 2AR mRNA. Transfection of the beta 2AR promoter region cloned onto a reporter gene (bacterial chloramphenicol acetyltransferase) allowed demonstration of a 2- to 4-fold induction of transcription by agents that elevate cAMP levels, such as forskolin or phosphodiesterase inhibitors. These results establish the presence of elements within the proximal promoter region of the beta 2AR gene responsible for the transcriptional enhancing activity of cAMP and demonstrate that beta 2AR gene expression is regulated by a type of feedback mechanism involving the second messenger cAMP.

Genetic regulation of beta-adrenergic receptors.

Several members of the family of receptors coupled to G proteins have been cloned in recent years. From the primary sequence information furnished by these clones, a characteristic seven-membrane spanning topography has emerged as the prototype for this class of receptors, in analogy with the opsin visual pigments. Cloned genes for the various receptors provide important tools for probing the regulation of their expression. For example, the underlying genetic basis for the expression of adrenergic receptor subtypes can now be explored. The physiological regulation of receptors by heterologous hormones, such as steroids and thyroid hormones, has long been suspected to involve changes in the expression of the relevant adrenergic receptor genes. At least for the glucocorticoids, beta 2AR expression is controlled at the level of transcription. The hormonal regulation of other adrenergic receptor subtypes is currently being explored.

Identification of a putative structural gene for cathepsin D in Caenorhabditis elegans.

Mutants of Caenorhabditis elegans having about 10% of wild-type activity of the aspartyl protease cathepsin D have been isolated by screening. Mutant homozygotes have normal growth rates and no obvious morphological or developmental abnormalities. The mutant gene (cad-1) has been mapped to the right extremity of linkage group II. Heterozygous animals (cad-1/+) show intermediate enzyme levels and animals heterozygous for chromosomal deficiencies of the right extremity of linkage group II have 50% of wild-type activity. Cathepsin D purified from a mutant strain has a lower activity per unit mass of pure enzyme. These data suggest that cad-1 is a structural gene for cathepsin D.

Expression of the differentiation-induced gene for fatty acid-binding protein is activated by glucocorticoid and cAMP.

We have isolated and characterized a fragment of the gene encoding adipose fatty acid-binding protein (gene 422) from a 3T3-L1 adipocyte genomic library. The 5'-flanking sequence of the 422 gene contains potential regulatory regions for adipose-specific expression. At position -120 there is a fat-specific element that occurs in several genes expressed as preadipocytes differentiate, and at position -393 there is a glucocorticoid regulatory element core sequence. Chimeric constructs were prepared by ligating 858 base pairs or 248 base pairs of 5'-flanking sequence and 22 nucleotides of 5'-untranslated sequence of the 422 gene to the bacterial gene encoding chloramphenicol acetyltransferase (CAT); these constructs (delta 858.CAT and delta 248.CAT) were transfected into 3T3-L1 preadipocytes. When differentiation was initiated by the adipogenic agents methylisobutylxanthine (a cAMP phosphodiesterase inhibitor), dexamethasone, and insulin, expression of both constructs increased, reaching maximal levels within 24 hr. Both constructs were maximally induced 48 hr before appreciable accumulation of the endogenous 422 mRNA. Expression of delta 858.CAT, but not of delta 248.CAT, was induced by dexamethasone, which correlates with deletion of the potential glucocorticoid regulatory element. Expression of both constructs was induced by 8-bromoadenosine 3',5'-cyclic monophosphate, thus implicating the first 248 base pairs of 5'-flanking sequence of the 422 gene in the response to cAMP. Indirect effects by the adipogenic factors on CAT protein or mRNA synthesis and turnover were ruled out, since replacing the 5'-flanking region of the 422 gene constructs with viral promoters abolished the effects of dexamethasone and 8-bromoadenosine 3',5'-cyclic monophosphate on CAT expression. We conclude that the first 858 base pairs of 5'-flanking sequence of the 422 gene contains elements that mediate activation by dexamethasone and cAMP.

Cloning of the cDNA and genes for the hamster and human beta 2-adrenergic receptors.

The adenylate cyclase-stimulatory beta 2-adrenergic receptor has been purified to apparent homogeneity from hamster lung. Partial amino acid sequence obtained from isolated CNBr peptides was used to clone the gene and cDNA for this receptor. The predicted amino acid sequence for the hamster beta 2-adrenergic receptor revealed that the protein consists of a single polypeptide chain of 418 aa with consensus N-glycosylation and phosphorylation sites predicted by previous in vitro data. The most striking feature of the receptor protein however, is that it contains seven stretches of hydrophobic residues similar to the proposed seven transmembrane segments of the light receptor rhodopsin. Significant amino acid homology (30-35%) can be found between the hamster beta 2-adrenergic receptor and rhodopsin within these putative membrane spanning regions. Using a hamster beta 2-adrenergic receptor probe, the gene and cDNA for the human beta 2-adrenergic receptor were isolated, revealing a high degree of homology (87%) between the two proteins from different species. Unlike the genes encoding the family of opsin pigments, of which rhodopsin is a member, the genes encoding both hamster and human beta 2-adrenergic receptors are devoid of introns in their coding as well as 5' and 3' untranslated nucleotide sequences. The cloning of the genes and the elucidation of the aa sequences for these G-protein coupled receptors should help to determine the structure-function as well as the evolutionary relationship of these proteins.

Delineation of the intronless nature of the genes for the human and hamster beta 2-adrenergic receptor and their putative promoter regions.

The beta 2-adrenergic receptor is the first adenylate cyclase-coupled receptor to be cloned. We provide here a detailed characterization of its complete gene in both the human and hamster which reveals several unusual and provocative features. The genes are present in a single copy, are intronless, and are bounded by homologous 18-bp (base pair) direct repeats. These findings suggest that the beta 2-adrenergic receptor may have arisen as a processed gene for another related gene. Genomic Southern blots done at reduced stringency in fact reveal additional weak signals. The human and hamster gene sequences 5' to the principal site of transcription initiation are highly homologous and share many characteristics of promoters for housekeeping genes. Moreover, there is present in the human genome a long (777 bp) open reading frame which is in frame with the beta-adrenergic receptor coding block and which ends only 234 bp 5' to the initiator methionine of the receptor. An unusual cDNA has been found, transcribed from a putative second more 5' promoter which contains the 5' half of the beta-adrenergic receptor as well as 1065-bp 5' to the receptor coding region, including the entire upstream long open reading frame (sufficient to encode a putative protein of Mr approximately 28,000).

cDNA for the human beta 2-adrenergic receptor: a protein with multiple membrane-spanning domains and encoded by a gene whose chromosomal location is shared with that of the receptor for platelet-derived growth factor.

We have isolated and sequenced a cDNA encoding the human beta 2-adrenergic receptor. The deduced amino acid sequence (413 residues) is that of a protein containing seven clusters of hydrophobic amino acids suggestive of membrane-spanning domains. While the protein is 87% identical overall with the previously cloned hamster beta 2-adrenergic receptor, the most highly conserved regions are the putative transmembrane helices (95% identical) and cytoplasmic loops (93% identical), suggesting that these regions of the molecule harbor important functional domains. Several of the transmembrane helices also share lesser degrees of identity with comparable regions of select members of the opsin family of visual pigments. We have localized the gene for the beta 2-adrenergic receptor to q31-q32 on chromosome 5. This is the same position recently determined for the gene encoding the receptor for platelet-derived growth factor and is adjacent to that for the FMS protooncogene, which encodes the receptor for the macrophage colony-stimulating factor.

Cloning of the gene and cDNA for mammalian beta-adrenergic receptor and homology with rhodopsin.

The adenylate cyclase system, which consists of a catalytic moiety and regulatory guanine nucleotide-binding proteins, provides the effector mechanism for the intracellular actions of many hormones and drugs. The tissue specificity of the system is determined by the particular receptors that a cell expresses. Of the many receptors known to modulate adenylate cyclase activity, the best characterized and one of the most pharmacologically important is the beta-adrenergic receptor (beta AR). The pharmacologically distinguishable subtypes of the beta-adrenergic receptor, beta 1 and beta 2 receptors, stimulate adenylate cyclase on binding specific catecholamines. Recently, the avian erythrocyte beta 1, the amphibian erythrocyte beta 2 and the mammalian lung beta 2 receptors have been purified to homogeneity and demonstrated to retain binding activity in detergent-solubilized form. Moreover, the beta-adrenergic receptor has been reconstituted with the other components of the adenylate cyclase system in vitro, thus making this hormone receptor particularly attractive for studies of the mechanism of receptor action. This situation is in contrast to that for the receptors for growth factors and insulin, where the primary biochemical effectors of receptor action are unknown. Here, we report the cloning of the gene and cDNA for the mammalian beta 2AR. Analysis of the amino-acid sequence predicted for the beta AR indicates significant amino-acid homology with bovine rhodopsin and suggests that, like rhodopsin, beta AR possesses multiple membrane-spanning regions.

Evidence for an increase in transcription of specific mRNAs during differentiation of 3T3-L1 preadipocytes.

Differentiation of 3T3-L1 preadipocytes in culture is accompanied by alterations in the abundance of several mRNAs and by the appearance of many new adipocyte-specific mRNAs. To investigate the processes responsible for these alterations, the kinetics of accumulation of several specific mRNAs were compared with their respective rates of nuclear runoff transcription. The mRNAs for fructose-1,6-bisphosphate aldolase and an unidentified 4800-base mRNA increase in abundance only moderately (2-4-fold) during differentiation. Runoff transcription by nuclei isolated from 3T3-L1 cells during the course of differentiation revealed very little or no change in the rates of transcription of these mRNAs. Similar results were obtained for the beta, alpha-actin and beta-tubulin mRNAs where no difference in nuclear runoff transcription rates were observed even though a 2-fold decrease in the steady-state levels of these mRNAs accompanies differentiation. In contrast, the steady-state levels of mRNAs for 3T3-L1 P2 protein, an adipocyte homologue of myelin P2 protein, and an unidentified 5000-base mRNA increased dramatically (greater than 20-fold) during adipose conversion. These large increases in abundance were correlated with marked rises (greater than 10-fold) in nuclear runoff transcription rates for these mRNAs during differentiation of 3T3-L1 preadipocytes. No change in runoff transcription activity for these mRNAs was detected by nuclei from control nondifferentiating 3T3-C2 cells. These results strongly suggest that an increased rate of specific transcription is primarily responsible for the accumulation of these mRNAs during preadipocyte differentiation.

Expression of specific mRNAs during adipose differentiation: identification of an mRNA encoding a homologue of myelin P2 protein.

To identify and characterize specific mRNAs that increase in abundance during differentiation of mouse 3T3-L1 preadipocytes, a cDNA library was constructed from poly(A)+RNA isolated from differentiated 3T3-L1 adipocytes. Mixed probe isotope ratio selection and RNA blot analyses have identified several unique cDNA clones that represent mRNA species expressed either exclusively or at dramatically increased levels in differentiated cells. Further characterization of one such clone (pAL422) revealed that the corresponding mRNA, detectable only after differentiation, is approximately the same length (600 +/- 150 bases) as the cDNA insert (672 bases). The complete nucleotide sequence of the cDNA insert in pAL422 revealed a single long open reading frame that encodes a 132 amino acid polypeptide (the 422 protein) of 14.6 kDa. These and other results suggest that this cDNA may represent a nearly full-length copy of the mRNA. Computer-assisted analyses showed that the 422 protein shares 69% and 64% homology with myelin P2 proteins from rabbit and bovine peripheral nerves, respectively, as well as 23% and 30% homology with fatty-acid binding proteins from rat liver and intestine, respectively. Moreover, the mRNA hybrid selected by pAL422 DNA directs the in vitro translation of an approximately equal to 13 kDa polypeptide, and this protein is specifically immunoprecipitated by antiserum against bovine myelin P2. These observations strongly suggest that the 422 protein is a structural, and possibly functional, analog of myelin P2.

Fatty acylation of proteins during development of sea urchin embryos.

Developing embryos of the sea urchin, Strongylocentrotus purpuratus, incorporate [3H]palmitic acid into at least 20 proteins. The [3H]palmitic acid associated with these proteins is released by alkaline hydrolysis or by treatment with hydroxylamine but not by extensive extraction with chloroform:methanol, indicating that the fatty acids are covalently attached to protein. The finding that the fatty acid is released by hydroxylamine or beta-mercaptoethanol at neutral or even slightly acidic pH suggests that this moiety may be attached to the polypeptide via a thiol ester bond. Concanavalin A-agarose chromatography and endo-beta-N-acetylglucosaminidase H digestion revealed that 14 of the proteins containing covalently linked fatty acid also contain at least one asparagine-linked oligosaccharide chain. With one exception, all of the fatty acylated proteins are tightly associated with membranes. The rate of incorporation of [3H]palmitic acid into the proteins is developmentally regulated. Between fertilization and the onset of gastrulation (approximately 30 h), embryos exhibit a linear, 5.5-fold increase in the rate of incorporation of fatty acid into polypeptide. Incorporation increases an additional 25% during gastrulation, and then remains constant throughout subsequent development to the pluteus stage (approximately 90 h). These findings demonstrate that the fatty acylation of proteins and glycoproteins is not limited to higher organisms, since it occurs during differentiation and embryonic development of a relatively simple invertebrate.

Quantitative measures of aging in the nematode Caenorhabditis elegans: II. Lysosomal hydrolases as markers of senescence.

In an attempt to provide additional quantitative markers of senescence in the nematode Caenorhabditis elegans, we have identified age-dependent increases in four lysosomal enzymes: acid phosphatase, beta-N-acetyl-D-glucosaminidase, beta-D-glucosidase, and alpha-D-mannosidase. These enzymes were judged to be lysosomal on the basis of their resemblance to analogous mammalian lysosomal enzymes with regard to subcellular fractionation, lectin binding, Km, molecular weights, inhibitor sensitivities, and pH optima. In nematode populations which had a median lifespan of 8.9 +/- 0.7 days and a maximum lifespan of 14-16 days, we observed the following increases in acid hydrolase activities per animal from day 3 (early adulthood) to day 10: (1) up to 2.5-fold for acid phosphatase; (2) 8-fold for beta-N-acetyl-D-glucosaminidase; (3) 9-fold for beta-D-glucosidase; and (4) 4-fold for alpha-D-mannosidase. Three forms of acid phosphatase and two forms of beta-D-glucosidase were separated by ion-exchange chromatography, but in each case only one form of the enzyme was primarily responsible for the age-dependent increase in total activity: acid phosphatase I increased 18-fold, while beta-D-glucosidase I increased 100-fold. By contrast, there were only slight age-dependent changes in choline acetyltransferase, acetylcholinesterase, or alpha-D-glucosidase activities after early adulthood. The age-dependent increases in acid phosphatase, beta-N-acetyl-D-glucosaminidase, beta-D-glucosidase, and alpha-D-mannosidase activities are sufficiently large and reproducible to be useful quantitative markers of senescence in C. elegans.