Cellular Defense Response Research Articles

IL-1β INDUCES SERINE PROTEASE INHIBITOR 3 (SPI-3) GENE EXPRESSION IN RAT PANCREATIC β-CELLS. DETECTION BY DIFFERENTIAL DISPLAY OF MESSENGER RNA

Immune-mediated β-cell damage induces diverse intracellular signals, leading to transcription of different genes which may either contribute to β-cell repair and/or defence or lead to cell death. The cytokine interleukin-1β (IL-1) is a potential mediator of β-cell dysfunction and damage in type 1 diabetes mellitus. To understand the molecular actions of this cytokine upon β-cells, this study aimed at the cloning of genes induced in FACS-purified rat pancreatic β-cells by a 6- or 24-h exposure to IL-1 by using differential display of mRNA with reverse transcription-polymerase chain reaction (DDRT-PCR). Among these cytokine-induced genes, a gene encoding for rat serine protease inhibitor (SPI-3) was isolated. SPI-3 may be involved in cellular defence responses against inflammatory stress. RT-PCR analysis confirmed that SPI-3 mRNA expression in rat β-cells is increased by IL-1 at an early stage (2h), with maximal accumulation during 6–12h and decline after 24h. Similar observations were made in mouse pancreatic islets and in the rat insulinoma cell line RINm5F. IFN-γ neither increased SPI-3 gene expression nor potentiated its induction by IL-1 in rat β-cells. The stimulatory effects of IL-1 on SPI-3 mRNA expression were decreased by co-incubation with an inhibitor of gene transcription (actinomycin D), an inhibitor of protein synthesis (cycloheximide) or an inhibitor of NF-κB activation (PDTC). On the other hand, a blocker of inducible nitric oxide synthase (iNOS) activity (NG-methyl-L-arginine) did not prevent IL-1-induced SPI-3 expression. Thus, SPI-3 mRNA expression following IL-1 exposure depends on gene transcription, protein synthesis and activation of the nuclear transcription factor NF-κB, but it is independent of NO formation.

Phagocytosis inhibition of clam and mussel haemocytes by Perkinsus atlanticus secretion products.

The in vitro effects of the secretion products (SP) released by cultured Perkinsus atlanticus on the phagocytic activity of two shellfish species, Ruditapes decussatus and Mytilus galloprovincialis were determined. Haemocytes from clam and mussel were incubated with Perkinsus SP, and the number of phagocytosed particles were counted. The particles assayed were zymosan, Escherichia coli and Vibrio tapetis. Phagocytosis of zymosan was measured at 15 and 21°C, and different incubation times in SP were tested. In all cases, phagocytosis was inhibited in SP-treated haemocytes. Phagocytosis of zymosan was inhibited to a greater extent in haemocytes from mussel than from clam. No differences were found between phagocytosis at 15 and 21°C, and the decrease in the phagocytic activity was evident within 30min of haemocyte incubation in SP. Clam haemocyte phagocytosis of V. tapetis was inhibited to a greater extent than for zymosan and E. coli, while phagocytosis by the mussel haemocytes was inhibited to the same extent for the two bacterial species tested. Otherwise, the phagocytic index was higher for clam haemocytes than for mussel haemocytes in all the experiments. SP contained high protein concentration and acid phosphatase and protease activity. These results suggest that the products secreted by Perkinsus atlanticus may have a negative effect on the cellular defence response of these bivalve molluscs.

Epigenetic silencing of plant transgenes as a consequence of diverse cellular defence responses.

Linked and unlinked copies of transgenes and related endogenous genes in plants can be epigenetically silenced by homology-based mechanisms that operate at either the transcriptional or post-transcriptional level. Transcriptional inactivation is associated with promoter homology and meiotically heritable methylation. Post-transcriptional silencing requires homology in protein-coding regions and is fully reversed during meiosis. Recently, the notion that both of these processes reflect the action of different host defence systems has been strengthened: (i) Obvious parallels have emerged between promoter homology-dependent silencing/methylation of transgenes and paramutation of endogenous genes that contain transposable elements in their promoters: (ii) remarkable similarities have been observed between post-transcriptional silencing involving transgenes and natural forms of virus resistance in nontransgenic plants. These results and others implicate two distinct cellular defence responses in transgene silencing. One is active in the nucleus and is manifested by transgene methylation, a reaction that might have originated as a means to oppose the spread of transposable elements. A second line of defence resides in the cytoplasm and operates through enhanced RNA turnover, a process that might help plants overcome viral infection.

A simple systems approach to neural-immune communication.

The marine mollusc Aplysia californica is emerging as a useful model system to study neuralimmune communication at the mechanistic level because it has a well characterized nervous system that is easily accessible and it shares with mammals similar basic cellular defensive responses to wounded or non-self, i.e. the accumulation of defense cells (haemocytes) at the target site. Loose ligation of peripheral nerves in Aplysia induces a cellular defense response as evidenced by the accumulation of numerous haemocytes around the ligature. The excitability of nociceptive sensory neurons having axons close to the responding haemocytes is significantly increased. Haemocytes also accumulate at regions of axonal injury. The finding that human recombinant IL-1 beta can enhance the expression of injury-induced sensory hyperexcitability coupled with the detection of (ir)IL-1 in Aplysia haemolymph raises the interesting possibility that cytokines released from activated haemocytes attracted to an injured nerve or to a foreign body close to peripheral nerves may modulate nociceptive sensory function. The feasibility of using results from simple system such as Aplysia to formulate testable hypotheses in more complex systems is also discussed.

Isolation of cDNAs representing dithiolethione-responsive genes.

Dithiolethiones inhibit tumorigenicity elicited by many structurally diverse carcinogens in numerous target tissues. These protective actions are associated with the induction of several carcinogen detoxification enzymes, some of which have only recently been discovered. In order to identify additional novel inducible detoxification response genes, a cDNA library was prepared from liver of rats treated with 1,2-dithiole-3-thione (D3T) and was screened by a differential hybridization method. Complementary DNA clones for several known D3T-inducible genes were isolated, such as epoxide hydrolase, aflatoxin B1-aldehyde reductase, quinone reductase and multiple subunits of glutathione S-transferase. Clones representing genes not previously associated with detoxification were isolated, including those for ferritin heavy and light subunits, ribosomal proteins L18a and S16 and two novel genes, termed dithiolethione-inducible genes (or DIG-1 and DIG-2). Levels of mRNA recognized by each clone were increased from 2- to 31-fold, with maximum induction between 6 and 30 h after treatment with D3T. Except for epoxide hydrolase, the kinetics of induction of each mRNA was coordinate with increased rates of gene transcription. However, based on the time of response to D3T, at least two sets of responsive genes were identified. One set of genes, including glutathione S-transferase Yp, aflatoxin B1-aldehyde reductase, quinone reductase and DIG-1, had low constitutive and highly inducible expression (approximately 20-fold) and the other, including glutathione S-transferase Ya and Yb, epoxide hydrolase, ferritin heavy and light subunits, ribosomal proteins L18a and S16 and DIG-2, had relatively high constitutive and modestly inducible expression (approximately 5-fold). The simplest explanation for this differential expression of D3T-inducible genes is that multiple regulatory mechanisms govern their response. The transcriptional activation of ferritin, ribosomal protein, DIG-1 and DIG-2 genes in conjunction with those of carcinogen detoxification enzymes suggests that they participate in the pleiotropic cellular defense response to dithiolethiones that inhibits chemically produced tumorigenesis.

Eicosanoids mediate nodulation responses to bacterial infections in larvae of the tenebrionid beetle, Zophobas atratus

Nodulation is the temporally and quantitatively predominant cellular defense response to bacterial infection in insects and other invertebrates. Inhibition of eicosanoid biosynthesis in larvae of the tenebrionid beetle, Zophobas atratus, immediately prior to intrahemocoelic injections with heat killed preparations of the bacterium, Serratia marcescens, strongly reduced the nodulation response. Separate treatments with specific inhibitors of phospholipase A 2, cyclooxygenase, and lipoxygenase reduced nodulation, supporting the view that nodule formation is a complex process involving both cyclooxygenase and lipoxygenase products. The inhibitory effects of the phospholipase A 2 inhibitor, dexamethasone, on nodulation were apparent 1 h after infection, and the effects increased, relative to controls, over 24 h. The dexamethasone effects were expressed in a dose-dependent manner, and they were reversed by treating bacteria injected insects with the eicosanoid-precursor polyunsaturated fatty acid, arachidonic acid (C20:4n-6). Treatments with the saturated fatty acid, 16:0, which is not an eicosanoid precursor, did not reverse the dexamethasone effects on nodulation. The insects contain low levels of three eicosanoid precursor polyunsaturated fatty acids in six different tissues, and fat body preparations are competent to produce both cyclooxygenase and lipoxygenase products. These findings strongly support the identification of nodulation as a specific insect cellular defense mechanism that is mediated by eicosanoids.

Phenotypic expression of Pseudomonas syringae avr genes in E. coli is linked to the activities of the hrp-encoded secretion system.

The specific recognition of elicitors produced by plant pathogenic bacteria carrying avirulence (avr) genes is postulated to initiate cellular defense responses in plants expressing corresponding resistance genes. The biochemical functions of most avr genes, however, are not known. A heterologous system was developed to phenotypically express Pseudomonas syringae avr genes in Escherichia coli cells that required the P. syringae hrp cluster. E. coli MC4100 transformants carrying the plasmic-borne P. syringae pv. syringae Pss61 hrp cluster and p. syringae pv. glycinea avrB expressed from a triple lacUV5 promoter gained the ability to elicit the hypersensitive response in soybean cultivars expressing Rpg1 and in an Arabidopsis thaliana accession expressing RPM1. Inactivation of energy transducing or outer membrane components of the hrp-encoded secretion system blocked phenotypic expression expression of avrB in E. coli, but deletions abolishing harpinPSS production had little effect on the production of the AvrB phenotype by the E. coli transformants. Phenotypic expression of avrA, AvrPto, avrRpm1, avrRpt2, and avrPph3 in E. coli was also shown to require the hrp cluster. The results indicate that generation of the Avr phenotype in P. syringae strains is specifically dependent on the secretion activities of the hrp cluster.

Antimicrobial factors in solitary ascidians

Solitary ascidians possess a wide range of antimicrobial agents in their bloodand other tissues. A number have been purified and characterised with a view to developing novel pharmacological or commercially useful products. The most well known are the didemnins isolated from whole body homogenates of Caribbean ascidians belonging to the genus Trididemnum. Others include the halocyamines from Halocynthia roretzi, piclavines from Clavelina picta and lissoclinotoxins from Lissoclinum perforatum. For most species, however, little is known about the spectrum of activity, tissue location or mode of action of these compounds, and no details have been forthcoming about their biosynthesis, release or internal regulation. Recent work on Ciona intestinalis has shown that potent antibacterial activity against a range of Gram positive and Gram negative bacteria resides within the circulating blood cells, principally the morula cells. Activity is constitutive, unrelated to lysozyme and does not involve agglutination or direct lysis of the bacterial cell wall. Instead, it appears to be clue to at least two proteins with molecular masses of ca 8–12 kDa and 60–70 kDa respectively. Since the morula cells are known to participate in a variety of cellular host defence responses, there can be little doubt that these proteins aid in the neutralisation of bacterial, and possibly other micro-organisms, which may gain access to the tissues. However, in some ascidian species, peptides with antibiotic properties in vitro have been shown to have other biological effects; for example protection against predation, digestion or prevention of surface epibiosis. There is great scope for finding further novel antimicrobial proteins in the ascidian group, and areas where further research is needed include an analysis of their biochemical and phylogenetic relationship to other biologically active peptides, an understanding of their induction and modulation in vivo and an assessment of the ways they exert their biological effects on susceptible micro-organisms in vitro.

Eicosanoids mediate insect nodulation responses to bacterial infections.

We propose that nodule formation is mediated by eicosanoids in insects. Nodulation is the temporally and quantitatively predominant cellular defense response to bacterial infection in insects and other invertebrates. Inhibition of eicosanoid biosynthesis in larvae of the tobacco hornworm Manduca sexta immediately prior to intrahemocoelic infections with the bacterium Serratia marcescens strongly reduced the nodulation response. Inhibition of eicosanoid biosynthesis also reduced formation of cellular aggregates at 1 hr postinfection, which indicates that eicosanoids mediate early stages of nodulation. Separate treatments with specific inhibitors of phospholipase A2, cyclooxygenase, and lipoxygenase reduced nodulation, which supports the view that nodule formation is a complex process involving prostaglandins and lipoxygenase products. The inhibitory effects of the phospholipase A2 inhibitor dexamethasone on nodulation were apparent by 1 hr after infection, and the effects increased, relative to controls, over 24 hr. The dexamethasone effects were expressed in a dose-dependent manner, and they were reversed by treating infected insects with eicosanoid-precursor polyunsaturated fatty acids. Treatments with the saturated fatty acid 16:0, which is not an eicosanoid precursor, did not reverse the dexamethasone effects on nodulation. These findings strongly support the identification of nodulation as a specific insect cellular defense mechanism that is mediated by eicosanoids.

Induction of a cellular defense reaction is accompanied by an increase in sensory neuron excitability in Aplysia

The complexity of vertebrate immune and nervous systems makes detailed cellular analysis of neuroimmune interactions a challenging prospect. The immune systems of invertebrates, although much less complex than their vertebrate counterparts, share basic cellular defense responses to wounded self or nonself. We have developed a simple model system to study neuroimmune interactions using an invertebrate preparation well suited to detailed cellular analysis. Loose ligation of peripheral nerves in Aplysia induced a cellular defense reaction evidenced by the accumulation of large numbers of amebocytes at the ligation site. From 5 to 30 d after ligation, the excitability of the soma of sensory neurons having axons in ligated nerves was increased compared to contralateral sensory neurons with axons in nonligated nerves. Spike threshold and afterhyperpolarization were reduced, and spike amplitude and duration were increased. Spike accommodation was also decreased such that sensory neurons on the ligated side fired more spikes to a 1 sec intracellular depolarizing pulse than control sensory neurons. These effects are unlikely to be accounted for by ligation-induced injury of sensory axons since both morphological and electrophysiological evidence indicated that the axons in ligated nerves were healthy and able to conduct action potentials. Amebocytes activated by the presence of nonself may release factors that lead to a central sensitization of sensory neurons with axons in close proximity to the amebocytes.

Induction of a cellular defense reaction is accompanied by an increase in sensory neuron excitability in Aplysia.

The complexity of vertebrate immune and nervous systems makes detailed cellular analysis of neuroimmune interactions a challenging prospect. The immune systems of invertebrates, although much less complex than their vertebrate counterparts, share basic cellular defense responses to wounded self or nonself. We have developed a simple model system to study neuroimmune interactions using an invertebrate preparation well suited to detailed cellular analysis. Loose ligation of peripheral nerves in Ap/ysia induced a cellular defense reaction evidenced by the accumulation of large numbers of amebocytes at the ligation site. From 5 to 30 d after ligation, the excitability of the soma of sensory neurons having axons in ligated nerves was increased compared to contralateral sensory neurons with axons in nonligated nerves. Spike threshold and afterhyperpolarization were reduced, and spike amplitude and duration were increased. Spike accommodation was also decreased such that sensory neurons on the ligated side fired more spikes to a 1 set intracellular depolarizing pulse than control sensory neurons. These effects are unlikely to be accounted for by ligation-induced injury of sensory axons since both morphological and electrophysiological evidence indicated that the axons in ligated nerves were healthy and able to conduct action potentials. Amebocytes activated by the presence of nonself may release factors that lead to

Water mold infections of freshwater fish: Recent advances

Various investigations have identified morphological and physiological differences among water mold isolates. There is some evidence that isolates taken from fish lesions are more infective for live fish and are more resistant to host defenses, compared to environmental isolates. Many other characteristics, including propagule retention on fish, propagule morphology, propagule response to nutrients, fungal isozyme patterns, and antigenic relatedness of fungal isolates, have been examined. However, the significance of these characteristics to pathogenicity is uncertain, either because the putative pathogenic marker does not consistently differentiate pathogenic from saprophytic isolates, or the adaptive significance of the marker is unclear. Water molds grow rapidly in dead tissue, yet do not easily cause disease in healthy, unstressed hosts. They are usually relegated to the skin and superficial muscle. The fact that they are common secondary invaders of wounds implies that in these instances there is a significant compromise of host defenses. Trauma, nutritional deficiencies, endocrine changes, and stressful water quality have been implicated. There may be major differences in how different fish species respond to such stresses. Both humoral and cellular defensive responses to invading water molds have been observed. The type of response may modulate the gross clinical signs of the resulting infection.

Influence of Beauveria bassiana on the cellular defense response of the beet armyworm, Spodoptera exigua

Fifth instar Spodoptera exigua larvae were found to be highly susceptible to hemocoelic challenge of low dosages (50–500 cells/larvae) of Beauveria bassiana blastospores. At higher dosages (5 × 10 3–5 × 10 4 cells/larvae), fungal challenge caused cessation in larval development and death within 2–3 days postinjection. A dosage of 5 × 10 2 blastospores/larvae, producing synchronous larval mortality within 72 hr, was selected for phagocytic studies. Total and differential hemocyte counts revealed that infection by B. bassiana caused a dramatic reduction in the major hemocyte class, the granulocyte, by 36 hr postchallenge. Fungal infection was also observed to inhibit filopodial formation and spreading of granulocytes by 24 hr postchallenge. At intervals during the injection cycle, the phagocytic competence of circulating hemocytes were evaluated with a second injection of fluorescent-labeled fungal cells. Results of these assays demonstrated that as the disease progressed, an increasing number of hemocytes were unable to phagocytose labeled fungal cells. Interestingly, at the 36 hr intervals, hemocytes were able to phagocytose a portion of fluorescent-labeled cells but did not recognize the in vivo circulating hyphal body cells. In summary, B. bassiana appears to possess a multifaceted capability for both suppressing and eluding the cellular defense response of S. exigua larvae.

Effect of alkylating agents on the clearance of Staphylococcus aureus from murine lungs.

Alkylating agents have several effects on cellular host defense responses which could increase either the frequency or the severity of pulmonary infections. In addition, some of these agents directly injure lung parenchyma and could have effects on intrapulmonary killing processes independent of any effect on phagocyte number and function. We have used a murine model for staphylococcal clearance to evaluate the effect of cyclophosphamide and mechlorethamine on intrinsic lung defenses. Single doses of mechlorethamine (40 micrograms IV) or of cyclophosphamide (150 mg/kg IP) reduce peripheral blood neutrophil counts and spleen weights on day 3 after injection; with the exception of neutrophil counts in mechlorethamine-treated mice, these parameters returned to normal by days 10-12. Both drugs reduced the number of alveolar macrophages recoverable by bronchoalveolar lavage on days 10-12 but not day 3. Mechlorethamine delayed the clearance of Staphylococcus aureus 502A from the lung on both days 3 and 12, but cyclophosphamide did not alter clearance on either day 3 or 10. The defect in clearance in mechlorenthamine-treated mice resolved by 3 wk after drug administration. These results demonstrate that alkylating agents do not have uniform effects on antibacterial processes in the murine lung. Since the mechlorethamine effect on staphylococcal elimination appears independent of its effect on macrophage numbers, these results suggest that staphylococcal clearance also depends on nonphagocytic host defense factors.

Biochemical Aspects of Insect Immunology

Insects are among the oldest and the most successful groups of animals occupy­ ing this planet. An essential feature of the success of insects has been their ability to invade and to exploit a diverse range of ecological niches. Insects have evolved and, in many situations, thrived in environments replete with potential­ ly parasitic and pathogenic competitors. Disease in insect populations may reduce vigor, productivity, and ultimately survival of individuals and thus may contribute to the regulation of population levels. The ubiquitous presence of both infectious organisms and metazoan parasites in ecosystems occupied by insects has exerted a strong selection pressure for insects resistant to infection. Thus modem species of insects may represent the most extensive summary of successful defensive strategies against infection in the animal kingdom. In­ deed, several early studies of antimicrobial defense mechanisms in animals were performed using insect models (52). The defensive arsenal of insects, like that of man, contains both passive structural barriers against infection and a cascade of active responses to organ­ isms that gain access to the hemocoel following injury to the integument. These responses include both rapid changes in the circulating hemocyte population and the induced synthesis of new hemolymph proteins. In addition, active hemostatic mechanisms repair wounds in the integument and limit the extent of further infection. Much is known of the cellular and humoral defensive responses of insects to infective agents. However, this knowledge is often fragmentary and descriptive and is derived from a spectrum of species. Biochemical information on more than one aspect of insect defensive response for a single species is sufficient in