Rochelle Hirschhorn

Adenosine: an endogenous inhibitor of neutrophil-mediated injury to endothelial cells.

Since adenosine and its analogue 2-chloroadenosine prevent neutrophils from generating superoxide anion in response to chemoattractants, we sought to determine whether these agents could inhibit neutrophil-mediated injury of endothelial cells. The chemoattractant N-formyl-methionyl-leucyl-phenylalanine (FMLP, 0.1 microM) enhanced the adherence of neutrophils to endothelial cells twofold (18 +/- 2% vs. 39 +/- 3% adherence, P less than 0.001) and caused substantial neutrophil-mediated injury to endothelial cells (2 +/- 2% vs. 39 +/- 4% cytotoxicity, P less than 0.001). 2-Chloroadenosine (10 microM) not only inhibited the adherence of stimulated neutrophils by 60% (24 +/- 2% adherence, P less than 0.001) but also diminished the cytotoxicity by 51% (20 +/- 4% cytotoxicity, P less than 0.002). Furthermore, depletion of endogenously released adenosine from the medium by adenosine deaminase-enhanced injury to endothelial cells by stimulated neutrophils (from 39 +/- 4% to 69 +/- 3% cytotoxicity, P less than 0.001). Indeed, in the presence of adenosine deaminase, even unstimulated neutrophils injured endothelial cells (19 +/- 4% vs. 2 +/- 2% cytotoxicity, P less than 0.001). These data indicate that engagement of adenosine receptors prevents both the adhesion of neutrophils and the injury they cause to endothelial cells. Adenosine inhibits injury provoked not only by cells that have been stimulated by chemoattractants but also by unstimulated cells. Based on this model of acute vascular damage we suggest that adenosine is not only a potent vasodilator, but plays the additional role of protecting vascular endothelium from damage by neutrophils.

Severe combined immunodeficiency and adenosine deaminase deficiency.

Because others had described a lack of the enzyme adenosine deaminase as associated with severe combined immunodeficiency, we surveyed kindreds with infants affected with such an immunodeficiency. Three infants in two families with severe combined immunodeficiency were found to have no detectable erythrocyte adenosine deaminase. Eleven family members heterozygous for adenosine deaminase deficiency were encountered among the first-degree relatives; adenosine deaminase deficiency and severe combined immunodeficiency were associated and inherited as autosomal recessive traits in both kindreds. Successful bone-marrow transplantation was carried out in two of these infants. Normal immunologic function was established in both children, but the deficiency of adenosine deaminase persisted in their erythrocytes. The enzyme deficiency did not impair the successful establishment of normal humoral and cellular immunity by transplants of bone-marrow cells from siblings who were either normal or heterozygous for adenosine deaminase deficiency.

Effect of cyclic 3',5'-adenosine monophosphate and theophylline on lymphocyte transformation.

Shortly after polypeptide hormones act on target tissues, the tissues undergo a series of morphologic changes that can be related to the rearrangement of vacuolar systems. Thus thyroid-stimulating hormone induces endocytosis of thyroglobulin, parathyroid hormone induces the bulk exocytosis of lysosomal hydrolases and hydrogen ions from osteoclasts, glucagon stimulates the formation of autophagic vacuoles in liver, and melanophore-stimulating hormone induces the rearrangement of melanosomes in skin (1, 2). Since cyclic 3′, 5′-adenosine monophosphate (cA MP) has been implicated as the “second messenger” for these effects of hormones upon target tissues, it appeared possible that adenine nucleotides might regulate other functions of the vacuolar system in various cell types. Evidence suggesting that increments in the level of intracellular cAMP may inhibit granule flow and merger has been obtained by Lichtenstein and Margolis (3), who found that both theophylline and dibutyryl cyclic AMP (dcAMP) inhibited the release of histamine from leukocytes. Indeed, further experiments with peripheral blood leukocytes showed that these compounds inhibited both the antigenic release of histamine and the phagocytic release of β-glucuronidase (4). Since redistribution of acid hydrolases follows enhanced endocytosis in cultures of human lymphocytes exposed to phytohemagglutinin (PHA) (5, 6), these findings suggested that the effects of PHA on lymphocytes might also be mediated by cyclic AMP. We have therefore studied the effect of cyclic AMP, its dibutyryl derivative and theophylline (an inhibitor of cyclic AMP degradation) (1, 2) upon the stimulation of lymphocytes. Materials and Methods. Separation and culturing of cells. Human peripheral blood lymphocytes were obtained by methods previously described. Briefly, heparinized blood was allowed to sediment spontaneously at 37°, the supernatant plasma was removed, treated with adenosine diphosphate to aggregate platelets and the filtrate was passed through a prewarmed nylon fiber column at 37°.

Overview of Biochemical Abnormalities and Molecular Genetics of Adenosine Deaminase Deficiency

ABSTRACT.: Approximately 20 years ago, Giblett and coworkers serendipitously discovered that in some patients with the syndrome of severe combined immunodeficiency, the disease is due to an inherited deficiency of the enzyme adenosine deaminase (ADA). This then led to the discovery that inherited deficiency of the next enzyme in the same pathway for purine salvage, purine nucleoside phosphorylase, results in a profound defect in cell-mediated immunity. These two disorders, sometimes termed “purinergic immunodeficiency disorders,” were the first of the inherited immunodeficiency disorders in which the specific molecular basis was determined. Although both are rare diseases, they are of importance for several reasons. First, they are among the few inherited disorders of which some children can be cured by a single treatment; second, they are ideally suited for gene therapy; and third, the pathologic mechanisms can tell us more about the nature of the immune system and have already allowed development of chemotherapy for some malignancies of the immune system. Knowledge of the specific defects has also facilitated diagnosis, counseling of families, and development of new approaches to therapy. This article focuses on ADA deficiency, briefly reviews the clinical and biochemical findings to provide a background for the next two articles in this supplement issue, and details work in progress with respect to several aspects of the specific molecular defects in different patients. Some of the generalizations that are beginning to emerge from studies of other disease loci as to the nature, diversity, and distribution of mutations at different genetic loci and the emerging correlations of phenotype with genotype are reviewed to provide a framework for examining the molecular defects in ADA deficiency. The status of our knowledge of the ADA locus with respect to analysis of mutations and possible correlations with genotype is then considered. It is important to note that although the ADA gene has been isolated and the gene product is known there are still unanswered questions.

ACID PHOSPHATASE-RICH GRANULES IN HUMAN LYMPHOCYTES INDUCED BY PHYTOHEMAGGLUTININ.

Human lymphocytes, cultured in the presence of phytohemagglutinin, undergo morphologic transformation and subsequent mitosis. Before mitosis (48 to 72 hours), a sharp increase in acid phosphatase activity occurs in cells stimulated with phytohemagglutinin. Histochemical examination of these cells demonstrates that innumerable granules containing acid phosphatase develop in the cytoplasm before mitosis. It is possible that enzymes present in granules which stain for acid phosphatase activity (lysosome-like) may play a role in phytohemagglutinin-stimulated cell division.

RESTORATION OF IN-VITRO LYMPHOCYTE RESPONSES WITH EXOGENOUS ADENOSINE DEAMINASE IN A PATIENT WITH SEVERE COMBINED IMMUNODEFICIENCY

Deficiency of adenosine deaminase (A.D.A.) occurs in an autosomal recessive form of severe combined immunodeficiency (S.C.I.D.). The role of this enzyme deficiency in the pathogenesis of the immune defects is not clear. A patient with A.D.A. S.C.I.D., studied during the first six weeks of life, was found to have B and T lymphocytes as well as 25% of normal lymphocyte responses to mitogens. This patient subsequently became severely lymphopenic with loss of mitogen responsiveness. Addition of calf-intestinal A.D.A. or human-erythrocyte A.D.A. to cultures of this patients lymphocytes restored their ability to proliferate when stimulated with mitogens. These data indicate that A.D.A. deficiency is causally related to the cellular immune defects observed in A.D.A. S.C.I.D. and suggests a possible role for enzyme replacement in the therapy of this disorder.

Erythrocyte Adenosine Deaminase Deficiency without Immunodeficiency: EVIDENCE FOR AN UNSTABLE MUTANT ENZYME

Inherited deficiency of the purine salvage enzyme adenosine deaminase (ADA) gives rise to a syndrome of severe combined immunodeficiency (SCID). We have studied a 2.5-yr-old immunologically normal child who had been found to lack ADA in his erythrocytes during New York State screening of normal newborns. His erythrocytes were not detectably less deficient in ADA than erythrocytes of ADA(-)-SCID patients. In contrast, his lymphocytes and cultured long-term lymphoid cells contained appreciably greater ADA activity than those from patients with ADA(-)-SCID. This residual ADA activity had a normal molecular weight and K(m) but was markedly unstable at 56 degrees C. His residual erythrocytes-ADA activity also appeared to have diminished stability in vivo. ADA activity in lymphoid line cells of a previously reported erythrocyte-ADA-deficient!Kung tribesman was found to contain 50% of normal activity and to exhibit diminished stability at 56 degrees C. ATP content of erythrocytes from both partially ADA-deficient individuals was detectably greater than normal (12.3 and 6.1 vs. normal of 2.6 nmol/ml packed erythrocytes). However, the dATP content was insignificant compared to that found in erythrocytes of ADA(-)-SCID patients (400-1,000 nmol/ml packed erythrocytes). The New York patient, in contrast to normals, excreted detectable amounts of deoxyadenosine, but this was <2% of deoxyadenosine excreted by ADA(-)-SCID patients. Thus, the residual enzyme in cells other than erythrocytes appears to be sufficient to almost totally prevent accumulation of toxic metabolites.

Phytohemagglutinin: Inhibition of the Agglutinating Activity by N-Acetyl-d-Galactosamine

The effect of simple sugars on the agglutinating activity of phytohemagglutinin was studied. N-Acetyl-d-galactosamine selectively inhibits the agglutination of leukocytes and erythro- cytes by phytohemagglutinin.

Amelioration of Neurologic Abnormalities after Enzyme Replacement in Adenosine Deaminase Deficiency

INHERITED deficiency of the purine salvage enzyme adenosine deaminase (ADA) results in the fatal infantile syndrome of severe combined immunodeficiency.1 2 3 4 The accumulation of the purine substr...

An Asp8Asn substitution results in the adenosine deaminase (ADA) genetic polymorphism (ADA 2 allozyme): occurrence on different chromosomal backgrounds and apparent intragenic crossover

We have now determined the molecular genetic basis for the common biochemical polymorphism at the adenosine deaminase (ADA) locus. The ADA*2 allele contains a G to A transition at nt22 (relative to the ATG) that results in substitution of asparagine for aspartic acid at codon 8 (Asp8Asn). Introduction of the nucleotide substitution into an ADA 1 cDNA and transfection into monkey kidney (Cos) cells confirmed that the mutation resulted in expression of an enzyme that comigrated with the naturally occurring ADA 2 allozyme. The substitution of neutral asparagine for anionic aspartic acid is consistent with the more cathodal electrophoretic migration of ADA 2 as compared with ADA 1. The nucleotide substitution was found on at least two different genetic backgrounds, suggesting independent recurrence of the mutation. Consistent with independent recurrence, the G to A transition is at a CpG dinucleotide and represents a type of mutation that occurs with high frequency. We have also unexpectedly identified a probable intragenic crossover in the very large first intron that is rich in repetitive DNA sequences.