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Oral interferon alpha (IFNα): Modulation of respiratory syncytial virus (RSV) infection in cotton rats
Abstracts
P8 October 4: LymphokinedCytokines
I 569
in Disease
Al 80
Al83 Oral Interferon alpha (IFNo): Modulation of Respiratory Syncytial Virus (RSV) Infection in Cotton Rats. Steven Krakowka, J. Cununins, G. Prince, V. Hemming. The Ohio State University 43210, Amarillo Cell Cuiture 79101, Virion Systems 20850, Uniformed Services University of Health Sciences 20814. The dose dependent effects of oral IFNo upon recoverable virus and histologic lesions in cotton rats inoculated with the Long strain of RSV was determined. In a pilot study, Cantell preparation IFNLu, administered in drinking water from post infection day (PID) -2 through 2, reduced plaque forming units (pm) in the lung (P < 0.05) as well as the number (P <0.05) and severity (P< 0.02) of pulmonary lesions when compared to controls not given IFNa. Here, cotton rats were treated orally with Hayashibara Biochemical Laboratories (HBL) natural human (nHu) IFNol and were terminated on PIDs 4 and 6. Evaluations were performed without knowledge of treatment groups. Oral interferon did not reduce viral pfu in nasal turbinates. Similarly, significant differences were not observed between treated rats and controls in the amount of virus in lung on PIDs 4 and 6. Both the severity and number of RSV-induced pulmonary lesions were reduced (P < 0.16) on PID 4 in a dose (low to high) dependent fashion. By PID 6, this improved score benefit was lost in all groups except those rats given 0.2 U (P
VIRAL
AND HUMAN IL-10 ARE CRITICAL FOR B CELL GROWTH TRANSFORMATION WITH EBV Isao Miyazaki, Roy Cheung and Michael Dosch Hosp. for Sick Children, Research Institute, Toronto, Ont., M5G 1X8 Epstein-Barr virus (EBV) is a human herpes virus associated with lymphatic and epithelial malignancies. We have demonstrated that viral IL10 (vIL10) is a latency gene and is critical for the transformation of resting B cells. Viral and human (h) IL-10 mRNA were detected in EBV infected B cells 6 hr and 24h post-infection, respectively. Blocking studies with anti-ILlO, vILl0 antisense reagents and drugs which block cellular activation events triggered by EBV now indicate that v&hIL-10 expression is regulated independently and that both are required for transformation. This was confirmed through the use of vILlO-deficient EBV whose vILl0 gene was disrupted by gene targeting. This virus failed to transform purified B cells unless exogenous IL10 was added. The rescue of transforming function could as well be achieved by addition of normal (tonsillar) T cells and this effect was as well inhibited by anti ILlO. Extending our observation to established EBVf and EBV- B cell lines, we found that v&hILlO were expressed at high levels only in the EBV+ clones, cells carrying vILlO- virus expressed hILl0 only. IL10 is secreted and required for B cell growth since anti-IL10 suppressed cell growth. IL10 is a marker of EBV transformed cells in general, as we found v&hILlO expression in nasopharyngeal tumors. vILl0 requirement early in transformation may reflect time constraints of the transformation process.
INFLAMMATORY CYTOKINE mRNA IN PATIENTS WITH CHRONIC RENAL FAILURE. R. McKenna, R Allan, A Fine, D Coy K. Bernstein J G Dodd University of Manitoba, Winnipeg, Manitoba R3A lR9 We have previously reported that peripheral blood mononuclear cells (PBMC) from patients with renal failure on hemodialysis (HD) but not on peritoneal dialysis (PD) have high production of tumor necrosis factor (TNF) and interleukin-6 (IL-6) when cultured either spontaneously or in the presence of autologous sera. In the present study we examined mRNA by Northern analysis to elucidate the regulation of cytokine expression in these patients. In all the groups tested i.e controls, HD, PD and renal failure non-dialysis patients we were unable to detect cytokine mPNA in unstimulated cells (i.e spontaneous production). Maximum cytokine mRNA was detected when the PBMC were stimulated for 4 hours with lipopolysaccharide (IOU&~). mRNA for TNF but not IL-6 was also detected when the PBMC of controls and PD but not HD patients were cultured in autologous sera (% of maximum LPS response, controls,36-48%; PD, 14 tolS%). Thus we conclude (1) that in controls and PD patients the regulation of PBMC TNF and IL-6 mRNA is different and (2) that in the HD patients TNF regulation may occur at a post transcriptional level.
INVOLVEMENT OFCYTOKINES Rubhana Raqibt,3, Alf Lindber$
ALTERED IL- IO LEVELS IN TRAUMA PATIENTS’ M@ AND T LYMPHOCYTES. C.Miller-Graziano, A.K.De, and K. Kodys, UMass Medical Center, Worcester, MA 01655. Depressed mitogen and elevated monocyte (M0) inflammatory cytokine responses are immunoaberrant hallmarks in severe trauma patients. Conflicting reports have appeared regarding IL-lo’s role in these immune and cytokine alterations. Here we assessed the levels of IL-10 (ELISA) in PBMC, adherence purified M0, and SRBC-rosette-purified T cells from 31 normals and 31 trauma patients with injury severity score >30. IL- 10 levels of PBMC, MI? and T cells were correlated to patients’ PBMC PHA responses, their APC independent T cell proliferation in response to anti-CD3 plus anti-CD4 and their MO production of hyperelevated TNFc( levels (LM bioassay). Trauma patients with depressed PBMC responses to PHA also had significantly decreased IL-10 levels in their stimulated PBMC supernates (p=.OO22) and their MDP stimulated isolated M0 population (p=.OOO4). However, patients with depressed PHA responses could have either normal or depressed T cell proliferation in absence of APC. T cell R-10 levels were depressed (p=O.O07) when APC-independent T cell proliferation was suppressed but normal or elevated when the T cell responses were normal or elevated. Decreased IL-10 levels correlated with depressed mitogen responses and depressed T cell proliferation and, therefore, could not be inducing the patients’ immunosuppression. Patients having high MO TNFa levels also had depressed II-10 levels, suggesting that loss of IL-10 production may contribute to the hyperinduction of MO TNFu in these patients. Rsearch supported by NIH
MOLECULAR MECHANISMS OF CYTOKINE-INDUCED HIV EXPRESSION IN CHRONICALLY INFECTED Ul CELLS. B. M. Saget, E. V. Granowitz, J. B. Angel, C. A. Dinarello and P. R. Skolnik. New England Medical Center Hospitals and Tufts University School of Medicine, Boston, MA. 02111, We have shown by p24 antigen production that 10 rig/ml IL-lb maximally induces HIV expression approximately 2-5 fold in a chronically-infected monoblastoid cell line (Ul). IL-lp-induced HIV replication is completely blocked by IL-I receptor antagonist (II-1Ra) at 1 &ml. Using electrophoretic mobility shift assays (EMSA) to measure the level of the cellular transcription factor NFKB, we have shown that IL-lp induces NFKB levels approximately two-fold above the unstimulated control. IL-lp-induced NFKB activity is decreased to unstimulated levels in the prescence of IL-1Ra. Northern analyses using a chemiluminescent HIV RNA probe demonstrate that 10 rig/ml IL-IO induces HIV mRNA which is subsequently blocked by IL-1Ra. We have also shown that phorbol myristate acetate (PMA)-induced HIV replication is blocked approximately 86% by TBP-1 (the soluble TNF receptor ~55) and approximatley 89% by a soluble TNF ~75 receptor chimera which contains two monomeric soluble TNF ~75 receptors fused to the constant region of the Fc portion of IgG. Using EMSA for NFrB activity and Northern analysis for HIV message, we have demonstrated that both the ~55 and ~75 soluble receptors block PMA-induced NFKB activity and PMA-induced HIV message.
A181
Al84
Infection
IN THE PATHOGENESIS
OF SHICELLOSIS
and Jan Andersson2s4
is accompanied by an mtestinal
with
of T cells and
Shigella activation the inflamed COIOIUC mucosa. We westigated the relation of cytokines to Shigeila mfection durmg awte (2 to 5 days after the onset of diarrhoea) and convalescent stages (30 days after onset) and the correlatmn of cytokine profile with histo1ogw.l severity shigellosls induces in in
macraphageswithin
Acute avast number ofproinflammatory cytokines in thecnlonic mucosa, m 100 fold higher cOncentrafiom sto& camp&an to plasma levels stool cytokine
pattern was dominated by IL-lb, IL-lra, TNF-a, IL-6, IL-8, GM-CSF with alack ofsecreted IL-4, IL-IO and TGFfil.3 IF-N-yshowed different kinetics wth depressed levels at the onset of dlarrhoea with a gradual contmuous rise m levels till the end of the study period Endemic confrols had persistent elevated ievels of IF7-y and IL-In in stools Productton of cytokmes detected at the single cell level in crjopreserved rectal tissues by immunahistochemistry showed extensive production oflL-la, IL-ID. IL-lra, TNF-a, IL-6. IL-S, IL-4. IL-IO, IFN-‘I, TNF-/I and TGFpl.3 Healthy controls showed huge production of IL-8 in the crypts with little or no reactwey of other cytokines. Severe hlsrology was associated with mcreased productmn of IL-I, Patients, clinically
IL-6, healthy
IFN-y and TNF-a m the rectum as compared to mdd histology 30 days afler the onset of disease exhibited acute and or chrome
_ producing
cells
between
acute
and convalescent
stages.
The results
suggest
that
bacterial
antigens such as Shlga toxin and endotoxm together with the released pro-inflammatory cytokines indue a synergistic effect un the marphogenesls ofmflammatory lesmns in shigellasis with a probable mununomodulatory role of INF-y in the convaiesccnt stage.
Al82
Al85
P8 October 4: LymphokinedCytokines
I 569
in Disease
Al 80
Al83 Oral Interferon alpha (IFNo): Modulation of Respiratory Syncytial Virus (RSV) Infection in Cotton Rats. Steven Krakowka, J. Cununins, G. Prince, V. Hemming. The Ohio State University 43210, Amarillo Cell Cuiture 79101, Virion Systems 20850, Uniformed Services University of Health Sciences 20814. The dose dependent effects of oral IFNo upon recoverable virus and histologic lesions in cotton rats inoculated with the Long strain of RSV was determined. In a pilot study, Cantell preparation IFNLu, administered in drinking water from post infection day (PID) -2 through 2, reduced plaque forming units (pm) in the lung (P < 0.05) as well as the number (P <0.05) and severity (P< 0.02) of pulmonary lesions when compared to controls not given IFNa. Here, cotton rats were treated orally with Hayashibara Biochemical Laboratories (HBL) natural human (nHu) IFNol and were terminated on PIDs 4 and 6. Evaluations were performed without knowledge of treatment groups. Oral interferon did not reduce viral pfu in nasal turbinates. Similarly, significant differences were not observed between treated rats and controls in the amount of virus in lung on PIDs 4 and 6. Both the severity and number of RSV-induced pulmonary lesions were reduced (P < 0.16) on PID 4 in a dose (low to high) dependent fashion. By PID 6, this improved score benefit was lost in all groups except those rats given 0.2 U (P
VIRAL
AND HUMAN IL-10 ARE CRITICAL FOR B CELL GROWTH TRANSFORMATION WITH EBV Isao Miyazaki, Roy Cheung and Michael Dosch Hosp. for Sick Children, Research Institute, Toronto, Ont., M5G 1X8 Epstein-Barr virus (EBV) is a human herpes virus associated with lymphatic and epithelial malignancies. We have demonstrated that viral IL10 (vIL10) is a latency gene and is critical for the transformation of resting B cells. Viral and human (h) IL-10 mRNA were detected in EBV infected B cells 6 hr and 24h post-infection, respectively. Blocking studies with anti-ILlO, vILl0 antisense reagents and drugs which block cellular activation events triggered by EBV now indicate that v&hIL-10 expression is regulated independently and that both are required for transformation. This was confirmed through the use of vILlO-deficient EBV whose vILl0 gene was disrupted by gene targeting. This virus failed to transform purified B cells unless exogenous IL10 was added. The rescue of transforming function could as well be achieved by addition of normal (tonsillar) T cells and this effect was as well inhibited by anti ILlO. Extending our observation to established EBVf and EBV- B cell lines, we found that v&hILlO were expressed at high levels only in the EBV+ clones, cells carrying vILlO- virus expressed hILl0 only. IL10 is secreted and required for B cell growth since anti-IL10 suppressed cell growth. IL10 is a marker of EBV transformed cells in general, as we found v&hILlO expression in nasopharyngeal tumors. vILl0 requirement early in transformation may reflect time constraints of the transformation process.
INFLAMMATORY CYTOKINE mRNA IN PATIENTS WITH CHRONIC RENAL FAILURE. R. McKenna, R Allan, A Fine, D Coy K. Bernstein J G Dodd University of Manitoba, Winnipeg, Manitoba R3A lR9 We have previously reported that peripheral blood mononuclear cells (PBMC) from patients with renal failure on hemodialysis (HD) but not on peritoneal dialysis (PD) have high production of tumor necrosis factor (TNF) and interleukin-6 (IL-6) when cultured either spontaneously or in the presence of autologous sera. In the present study we examined mRNA by Northern analysis to elucidate the regulation of cytokine expression in these patients. In all the groups tested i.e controls, HD, PD and renal failure non-dialysis patients we were unable to detect cytokine mPNA in unstimulated cells (i.e spontaneous production). Maximum cytokine mRNA was detected when the PBMC were stimulated for 4 hours with lipopolysaccharide (IOU&~). mRNA for TNF but not IL-6 was also detected when the PBMC of controls and PD but not HD patients were cultured in autologous sera (% of maximum LPS response, controls,36-48%; PD, 14 tolS%). Thus we conclude (1) that in controls and PD patients the regulation of PBMC TNF and IL-6 mRNA is different and (2) that in the HD patients TNF regulation may occur at a post transcriptional level.
INVOLVEMENT OFCYTOKINES Rubhana Raqibt,3, Alf Lindber$
ALTERED IL- IO LEVELS IN TRAUMA PATIENTS’ M@ AND T LYMPHOCYTES. C.Miller-Graziano, A.K.De, and K. Kodys, UMass Medical Center, Worcester, MA 01655. Depressed mitogen and elevated monocyte (M0) inflammatory cytokine responses are immunoaberrant hallmarks in severe trauma patients. Conflicting reports have appeared regarding IL-lo’s role in these immune and cytokine alterations. Here we assessed the levels of IL-10 (ELISA) in PBMC, adherence purified M0, and SRBC-rosette-purified T cells from 31 normals and 31 trauma patients with injury severity score >30. IL- 10 levels of PBMC, MI? and T cells were correlated to patients’ PBMC PHA responses, their APC independent T cell proliferation in response to anti-CD3 plus anti-CD4 and their MO production of hyperelevated TNFc( levels (LM bioassay). Trauma patients with depressed PBMC responses to PHA also had significantly decreased IL-10 levels in their stimulated PBMC supernates (p=.OO22) and their MDP stimulated isolated M0 population (p=.OOO4). However, patients with depressed PHA responses could have either normal or depressed T cell proliferation in absence of APC. T cell R-10 levels were depressed (p=O.O07) when APC-independent T cell proliferation was suppressed but normal or elevated when the T cell responses were normal or elevated. Decreased IL-10 levels correlated with depressed mitogen responses and depressed T cell proliferation and, therefore, could not be inducing the patients’ immunosuppression. Patients having high MO TNFa levels also had depressed II-10 levels, suggesting that loss of IL-10 production may contribute to the hyperinduction of MO TNFu in these patients. Rsearch supported by NIH
MOLECULAR MECHANISMS OF CYTOKINE-INDUCED HIV EXPRESSION IN CHRONICALLY INFECTED Ul CELLS. B. M. Saget, E. V. Granowitz, J. B. Angel, C. A. Dinarello and P. R. Skolnik. New England Medical Center Hospitals and Tufts University School of Medicine, Boston, MA. 02111, We have shown by p24 antigen production that 10 rig/ml IL-lb maximally induces HIV expression approximately 2-5 fold in a chronically-infected monoblastoid cell line (Ul). IL-lp-induced HIV replication is completely blocked by IL-I receptor antagonist (II-1Ra) at 1 &ml. Using electrophoretic mobility shift assays (EMSA) to measure the level of the cellular transcription factor NFKB, we have shown that IL-lp induces NFKB levels approximately two-fold above the unstimulated control. IL-lp-induced NFKB activity is decreased to unstimulated levels in the prescence of IL-1Ra. Northern analyses using a chemiluminescent HIV RNA probe demonstrate that 10 rig/ml IL-IO induces HIV mRNA which is subsequently blocked by IL-1Ra. We have also shown that phorbol myristate acetate (PMA)-induced HIV replication is blocked approximately 86% by TBP-1 (the soluble TNF receptor ~55) and approximatley 89% by a soluble TNF ~75 receptor chimera which contains two monomeric soluble TNF ~75 receptors fused to the constant region of the Fc portion of IgG. Using EMSA for NFrB activity and Northern analysis for HIV message, we have demonstrated that both the ~55 and ~75 soluble receptors block PMA-induced NFKB activity and PMA-induced HIV message.
A181
Al84
Infection
IN THE PATHOGENESIS
OF SHICELLOSIS
and Jan Andersson2s4
is accompanied by an mtestinal
with
of T cells and
Shigella activation the inflamed COIOIUC mucosa. We westigated the relation of cytokines to Shigeila mfection durmg awte (2 to 5 days after the onset of diarrhoea) and convalescent stages (30 days after onset) and the correlatmn of cytokine profile with histo1ogw.l severity shigellosls induces in in
macraphageswithin
Acute avast number ofproinflammatory cytokines in thecnlonic mucosa, m 100 fold higher cOncentrafiom sto& camp&an to plasma levels stool cytokine
pattern was dominated by IL-lb, IL-lra, TNF-a, IL-6, IL-8, GM-CSF with alack ofsecreted IL-4, IL-IO and TGFfil.3 IF-N-yshowed different kinetics wth depressed levels at the onset of dlarrhoea with a gradual contmuous rise m levels till the end of the study period Endemic confrols had persistent elevated ievels of IF7-y and IL-In in stools Productton of cytokmes detected at the single cell level in crjopreserved rectal tissues by immunahistochemistry showed extensive production oflL-la, IL-ID. IL-lra, TNF-a, IL-6. IL-S, IL-4. IL-IO, IFN-‘I, TNF-/I and TGFpl.3 Healthy controls showed huge production of IL-8 in the crypts with little or no reactwey of other cytokines. Severe hlsrology was associated with mcreased productmn of IL-I, Patients, clinically
IL-6, healthy
IFN-y and TNF-a m the rectum as compared to mdd histology 30 days afler the onset of disease exhibited acute and or chrome
_ producing
cells
between
acute
and convalescent
stages.
The results
suggest
that
bacterial
antigens such as Shlga toxin and endotoxm together with the released pro-inflammatory cytokines indue a synergistic effect un the marphogenesls ofmflammatory lesmns in shigellasis with a probable mununomodulatory role of INF-y in the convaiesccnt stage.
Al82
Al85