- Email: [email protected]
Comparative chemiluminescence of neonatal and adult ovine polymorphonuclear leukocytes
Veterinary Immunology and Immunopathology 134 (2010) 265–268
Contents lists available at ScienceDirect
Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm
Short communication
Comparative chemiluminescence of neonatal and adult ovine polymorphonuclear leukocytes Eugene H. Johnson *, Khalid Al-Habsi, Rashid Al-Busaidy Department of Animal and Veterinary Sciences, College of Agricultural and Marine Sciences, Sultan Qaboos University, P.O. Box 34, Al-Khod 123, Oman
A R T I C L E I N F O
A B S T R A C T
Article history: Received 15 March 2009 Received in revised form 5 September 2009 Accepted 16 September 2009
Polymorphonuclear leukoctyes (PMN) serve on the first line of the immunological defense of ruminants. However, despite the high morbidity and mortality of neonatal lambs to a wide range of infections there have been no definitive studies undertaken to ascertain whether there might be functional differences in PMN from neonatal lambs when compared to those from adult sheep. To determine whether there were differences in the oxidative respiratory burst of PMN, luminol-dependent chemiluminescence (CL) measurements were made of PMN from lambs at 1 week, 1, 2 and 3 months of age and at the same time from their respective dams. PMN isolated from lambs exhibited significantly lower levels of CL until 2 months of age. At 3 months of age the PMN produced levels of CL equal to that of their dams. As CL mirrors the ability of PMN to efficiently phagocytize and kill pathogens the present findings would suggest that PMN of neonatal lambs during the first 2 months are likely less capable of defending them from pathogenic organisms. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Lambs Chemiluminescence Respiratory burst Phagocytosis
1. Introduction Many researchers have focused their attention on the contributory role of failure of passive transfer of immunoglobulins or on the impairment of humoral and cellular immunity in neonatal ruminants as the principle immunological factors responsible for the proclivity of the high incidence of neonatal infections in ruminants (Senogles et al., 1987; Higuchi et al., 1997). Surprisingly, the functionality of polymorphonuclear leukocytes (PMN) from newborn ruminants has received little attention, although these cells are the first to arrive at a site of infection and play a central role in the defense as phagocytes, and in the killing of pathogenic microorganisms by releasing reactive oxygen species, degradative enzymes and cationic peptides (McCaffrey and Allen, 2006).
Abbreviations: PMN, polymorphonuclear leukocytes; CL, chemiluminescence; AUC, area under the curve. * Corresponding author. Tel.: +968 24143679; fax: +968 24413481. E-mail address: [email protected] (E.H. Johnson). 0165-2427/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2009.09.004
In humans there are a number of studies with conflicting results relative to the comparative phagocytic efficiency of PMN from newborns and adults. Whereas some investigators have reported that PMN from neonates and adults exhibit no difference in their phagocytic activity (Shigeoka et al., 1979; Wilson, 1990) others have demonstrated lower levels of phagocytic activity (Van Epps et al., 1978; Strauss et al., 1980). During the phagocytic process PMN generate an oxidative respiratory burst that results in increased oxygen consumption, hexose monophosphate shunt activity and production of reactive oxygen metabolites, including superoxide anions (O2 ) hydrogen peroxide (H2O2), singlet oxygen among others (Hampton et al., 1998; Babior, 1999). Actively, phagocytizing PMN emit light or chemiluminescence (CL) which is enhanced by luminol (5-amino2,3-dihydro-1,4-phthalazinedione) and is linked to the oxidative activity of the PMN (Stevens et al., 1978). These reactive oxygen species have microbicidal properties and the measurement of CL has gained widespread acceptance as an assay for phagocytic and oxidative metabolic function (Allen et al., 1972; Leino and Paape, 1993; Mehrzad et al., 2005).
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E.H. Johnson et al. / Veterinary Immunology and Immunopathology 134 (2010) 265–268
In the present study we compared CL of PMN from ovine neonates and their respective dams to ascertain whether there are differences in the oxidative respiratory burst of these cells which might help explain the increased susceptibility of newborns to infections. 2. Materials and methods 2.1. Animals and blood sampling Clinically healthy Omani sheep bred and maintained at the Sultan Qaboos University Agriculture Experimental Station were used for this study. Blood samples were collected by jugular venipuncture into heparinized tubes for PMN isolation from five full term Omani lambs at 1 week of age and subsequently at 1, 2 and 3 months of age, as well as from their respective dams. This study was approved in advance by the institutional animal care and use committee. 2.2. Isolation of PMN PMN were isolated from heparinized whole blood by density centrifugation over Ficol-hypague, as previously described (Johnson et al., 1991). Briefly, 7 ml of whole blood were layered onto 3 ml of Histopaque 1077 (Sigma Chemical Co., St. Louis, MO) and centrifuged at 1500 g for 30 min. The red blood cell layer was removed from the granulocyte containing pellets by hypotonic lysis and the PMN were washed three times in PBS and re-suspended to a concentration of 5 106 PMN/ml in RPMI. Cell counts were performed in a routine haemocytometer and differentials were determined using Wright–Giemsa stain. Cell viability was assessed by trypan blue exclusion. Only samples containing >95% neutrophils with >95% viability were used. Accompanying cells were predominately eosinophils. After isolation, PMN were immediately used for CL assays. 2.3. Opsonization of zymosan 100 mg of zymosan A particles (Sigma Chemical Co., St. Louis, MO) were suspended in 2 ml of PBS and boiled in a water bath for 1 h with frequent mixing. The zymosan was washed three times in PBS and re-suspended to a final concentration of 50 mg/ml. One part of the zymosan (0.1 ml) solution was mixed with nine parts of pooled adult ovine serum (0.9 ml) and incubated at 37 8C for 15 min with continuous mixing. The mixture was then washed and centrifuged at 850 g for 20 min. The zymosan pellet was re-suspended in 4 ml of RPMI.
plates at 37 8C with intermittent shaking between readings and measurements were made in an Ascent Luminoskan luminometer (Thermo Electron Corp., Vantaa, Finland). Prior to the commencement of the studies it was demonstrated that background CL of PMN was negligible. Based on trypan blue exclusion it was determined that there were no relevant differences in PMN viability at the beginning and end of the CL assays. The area under the curve (AUC), defined by relative light units and time, was calculated over a 60 min recording period. CL was recorded as mean relative light units (RLU). 2.5. Statistical analysis Data are presented as means standard error of the mean. For CL measurements over a 60 min recording period the AUC was calculated from plotted data points using Graph Pad Prism, version 4.00 for Windows (Graph Pad Software, Inc., San Diego, CA). For comparisons between means of adult and newborn PMN samples an unpaired t-test was applied. A p-value <0.05 was considered significant. 3. Results and discussion A comparison of the CL response of the PMN from the dams and their 1-week-old lambs is shown in Fig. 1. In both groups the CL response showed a similar kinetic response. However, there was a significant difference in the average counts per minute, peak CL response and the AUC. These differences continued for the readings of the PMN of lambs at 1 and 2 months of age. At month 3 the counts per minute of the PMN of the lambs had reached the level of the dams (Table 1). These results are in accordance with those of human studies which have shown that PMN from normal term neonates exhibited depressed oxidative metabolic responsiveness as measured by peak CL responses (Van Epps et al., 1978; Mills et al., 1979; Strauss et al., 1980). It is important however to note that many human studies have not been able to demonstrate a difference in CL response, phagocytic or bactericidal activity of PMN from healthy full term neonates when compared to adults (McCracken and Eichenwald, 1971; Shigeoka et al., 1979; Gahr et al.,
Table 1 Comparison of chemiluminescence (CL) response of polymorphonuclear leukocytes stimulated with opsonized zymosan from lambs and their respective dams. Age group
Mean RLU
Peak CL
Lambs (1 week) Dams
129 8.4 445.2 37.8*
187 15.1 574.2 51.7*
AUC 7632.1 686.7 26082 2832.4*
2.4. Chemiluminescence (CL)
Lambs 1 month Dams
130.2 7.7 450.4 39.6*
195.2 11.7 583.6 52.5*
8016.7 721.4 27503.4 2200.2*
The procedure for CL closely followed that described by Saeed and Castle (1998) with minor modifications. The reaction mixtures consisted of 100 ml of opsonized zymosan, 100 ml of PMN (5 106/ml) and 100 ml of RPMI containing 10 5 luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, Sigma Chemical Co., St. Louis, MO, USA). Assays were performed in triplicates in 96 well microtiter
Lambs 2 months Dams
73.2 6.9 452.4 44.8*
103.8 6.6 574.6 54.5*
4495.7 494.5 27337.8 1913.6*
Lambs 3 months Dams
451.7 31.2 459.4 38.1
582.1 40.7 590.3 38.4
28516.3 1996.1 28732.2 2585.9
Values represent mean SEM. CPM = counts per minute; AUC = area under the curve; RLU = relative light units. * P-values 0.05.
E.H. Johnson et al. / Veterinary Immunology and Immunopathology 134 (2010) 265–268
267
Fig. 1. Comparison of CL values of PMN induced by opsonized zymosan from 1-week-old lambs and their respective dams measured at 2 min intervals.
1985). It has also been reported that the resting levels of oxygen consumption, hexose monophosphate shunt activity, O2 , and nitro blue tetrazolium reduction is higher in neonatal PMN than in adult cells (Humbert et al., 1970; Park et al., 1970; Anderson et al., 1974; Strauss and Seifert, 1978; Ambruso et al., 1984). Some authors have only been able to demonstrate a difference in CL response of preterm but not in normal term infants (Shigeoka et al., 1979; Bektas et al., 1990; Kallman et al., 1998). Of interest is that some studies have not been able to demonstrate a difference in peak CL response of PMN from adult and neonates. However the CL activity of the PMN from newborns has been reported to decline more rapidly (Strauss and Seifert, 1978) suggesting that PMN from the neonates were unable to maintain the same level of oxygen dependent metabolic activity as those from adult animals. This difference in oxidative metabolism has also been observed in cord blood PMN from normal term human infants (Strauss and Seifert, 1978). These authors concluded that this aberration of leukocyte function might indicate a deficiency of metabolic reserve that could be related to the increased susceptibility of newborn humans to bacterial infections. Although the present study presents preliminary findings relative to the oxidative metabolism of ovine neutrophils they are of particular interest as there is mounting evidence, at least in human newborns, that the neutrophil might function at less than optimal levels also in oxygen-independent bacterial systems. Levy et al. (1999) demonstrated that neutrophils from newborns have less bactericidal/permeability increasing protein, a 55-kDa protein that binds with high affinity to bacterial lipopolysaccharides and kills Gram-negative bacteria.
Neutrophils from newborns have also been shown to have lesser amounts of lactoferrin and lysozyme in their specific granules (Ambruso et al., 1984) and are less efficiently primed by lipopolysaccharide (Qing et al., 1996) and tumor necrosis factor (Bortloussi et al., 1993). Neonatal neutrophils have also been reported to exhibit reduced expression of L-selectin and lower up regulation of CD11b (Kim et al., 2003). These findings, coupled with likely low bone marrow reserves (Bracho et al., 1998) and impaired neutrophil migration, chemotaxis and adherence (Krause et al., 1986) might help explain why newborns are especially susceptible to bacterial infections. Although it is widely accepted that failure of passive transfer and resulting low levels of circulating antibodies in newborn ruminants is associated with an increased susceptibility to infectious diseases (Besser and Gay, 1994; Rogers and Capucille, 2000; Weaver et al., 2000) the possibility of impaired neutrophil function in neonates should also be considered. This is highlighted in the findings of the present study which gives the first evidence of an impaired oxygen respiratory burst of PMN from neonatal lambs that persists for the first 2 months of their lives when compared to that from the PMN of adults. This might help to explain why some authors have been unable to establish a relationship between serum immunoglobulin levels and the subsequent incidence of infections in newborn calves (Adams et al., 1992; Bradley et al., 1979).
References Adams, R., Garry, F.B., Aldridge, B.M., Holland, M.D., Odde, K.G., 1992. Hematologic values in newborn beef calves. Am. J. Vet. Res. 53, 944– 950.
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Allen, R.C., Stjernholm, R.L., Steele, R.H., 1972. Evidence for the generation of an electronic excitation state(s) in human polymorphonuclear leukocytes and its participation in bactericidal activity. Biochem. Biophys. Res. Commun. 47, 679–684. Anderson, D.C., Pickering, L.K., Feigin, R.D., 1974. Leukocyte function in normal and infected neonates. J. Pediatr. 85, 420–425. Ambruso, D.R., Bentwood, B., Hanson, P.M., Johnston Jr., R.B., 1984. Oxidative metabolism of cord blood neutrophils: relationship to content and degranulation of cytoplasmic granules. Pediatr. Res. 18, 1148–1153. Babior, B.M., 1999. NADH oxidase: an update. Blood 93, 1464–1476. Bektas, S., Goetze, B., Speer, C.P., 1990. Decreased adherence, chemotaxis and phagocytic activities of neutrophils from preterm neonates. Acta Paediatr. Scand. 79, 1031–1038. Besser, T.E., Gay, C.C., 1994. The importance of colostrum to the health of the neonatal calf. Vet. Clin. N. Am. Food Anim. 10, 107–117. Bortloussi, R., Howlett, K., Rajaraman, K., Halperin, S., 1993. Deficient priming activity of newborn cord blood derived polymorphonuclear neutrophilic granulocytes with lipopolysaccharide and tumor necrosis factor-a triggered with formyl-methionyly-leucyl-phenylalanine. Pediatr. Res. 34, 243–248. Bracho, F., Goldman, S., Cairo, M., 1998. Potential use of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor in neonates. Curr. Opin. Hematol. 5, 215–220. Bradley, J.A., Niilo, L., Dorward, W.J., 1979. Some observations on serum gammaglobulin concentrations in suckled beef calves. Can. Vet. J. 20, 227–232. Gahr, M., Blanke, R., Speer, C.P., 1985. Polymorphonuclear leukocyte function in term and preterm newborn infants. Biol. Neonate 48, 15–20. Hampton, M.B., Kettle, A.J., Winterbourn, C.C., 1998. Inside the neutrophil phagosome. Oxidants, myeloperoxidase, and bacterial killing. Blood 92, 3007–3017. Higuchi, K., Nagahata, H., Kiroki, M., Noda, H., 1997. Relationship between age-dependent changes of bovine neutrophil functions and their intracellular Ca2+ concentrations. J. Vet. Med. Sci. 59, 277–279. Humbert, J.R., Kurtz, M.L., Hathaway, W.E., 1970. Increased reduction of nitroblue tetrazolium by neutrophils of newborn infants. Pediatrics 45, 125–128. Johnson, E.H., Lustigman, S., Brotman, B., Browne, J., Prince, A.M., 1991. Onchocerca volvulus: In vitro killing of microfilaria by neutrophils and eosinophils from experimentally infected chimpanzees. Trop. Med. Parasitol. 42, 351–355. Kallman, J., Schollin, J., Schalen, C., Erlandsson, A., Kihlstrom, E., 1998. Impaired phagocytosis and opsonisation towards group B streptococci in preterm neonates. Arch. Dis. Child Fetal Neonatal Ed. 78, 46– 50. Kim, S.K., Keeney, S.E., Alpard, S.K., Schmalstieg, F.C., 2003. Comparison of L-selectin and CD11b on neutrophils of adults and neonates during the first month of life. Pediatr. Res. 53, 132–136. Krause, P.J., Herson, V.C., Boutin-Lebowitz, J., Eisenfeld, L., Block, C., LoBello, T., Maderazo, E.G., 1986. Polymorphonuclear leukocyte adherence and chemotaxis in stressed and healthy neonates. Pediatr. Res. 20, 296–300.
Leino, L., Paape, M.J., 1993. Comparison of the chemiluminescence responses of bovine neutrophils to differently opsonized zymosan particles. Am. J. Vet. Res. 54, 1055–1059. Levy, O., Martin, S., Eichenwald, E., Ganz, T., Valore, E., Carroll, S.F., Lee, K., Goldmann, D., Thorne, G.M., 1999. Impaired innate immunity in the newborn: newborn neutrophils are deficient in bactericidal/permeability-increasing protein. Pediatrics 106, 1327–1333. McCaffrey, R.L., Allen, L-A.H., 2006. Pivotal advance: francisella tularensis LVS evades killing by human neutrophils via inhibition of the respiratory burst and phagosome escape. J. Leukoc. Biol. 80, 1224–1230. McCracken Jr., G.H., Eichenwald, H.F., 1971. Leukocyte function and the development of opsonic and complement activity in the neonate. Am. J. Dis. Child. 121, 120–126. Mehrzad, J., Duchateau, L., Burvenich, C., 2005. High milk neutrophil chemiluminescence limits the severity of bovine coliform mastitis. Vet. Res. 36, 101–116. Mills, E.I., Thompson, T., Bjorksten, B., Filipovich, D., Quie, P.G., 1979. The chemiluminescence response and bactericidal activity of polymorphonuclear neutrophils from newborns and their mothers. Pediatrics 63, 429–434. Park, B.H., Holmes, B., Good, R.A., 1970. Metabolic activities in leukocytes of newborn infants. J. Pediatr. 76, 237–241. Qing, G., Howlett, S., Bortolussi, R., 1996. Lipopolysaccharide binding proteins on polymorphonuclear leukocytes: comparison of adult and neonatal cells. Infect. Immun. 64, 4638–4642. Rogers, G.M., Capucille, D.J., 2000. Colostrum management: keeping beef calves alive and performing. Compend. Contin. Educ. Vet. 22, 6–13. Saeed, F.A., Castle, G.E., 1998. Neutrophil chemiluminescence during phagocytosis is inhibited by abnormally elevated levels of acetoacetate: implications for diabetic susceptibility to infections. Clin. Diagn. Lab. Immunol. 5, 740–743. Senogles, D.R., Muscoplat, C.C., Paul, P.S., Johnson, D.W., 1987. Ontogeny of circulating B lymphocytes in neonatal calves. Res. Vet. Sci. 25, 34–36. Shigeoka, A.O., Santos, J.I., Hill, H.R., 1979. Functional analysis of neutrophil granulocytes from healthy, infected, and stressed neonates. J. Pediatr. 95, 454–460. Stevens, P., Winston, D.J., Van Dyke, K., 1978. In vitro evaluation of opsonic and cellular granulocyte function by luminol-dependent chemiluminescence: utility in patients with severe neutropenia and cellular deficiency states. Infect. Immun. 22, 41–51. Strauss, R.G., Seifert, M.J., 1978. Oxidative metabolism in cord-blood polymorphonuclear leucocytes. Arch. Dis. Child. 53, 78–80. Strauss, R.G., Rosenberger, T.G., Wallace, P.D., 1980. Neutrophil chemiluminescence during the first month of life. Acta Haematol. 63, 326–329. Van Epps, D.E., Goodwin, J.S., Murphy, S., 1978. Age-dependent variations in polymorphonuclear leukocyte chemiluminescence. Infect. Immun. 22, 57–61. Weaver, D.M., Tyler, J.W., VanMetre, D.C., Hostetler, D.E., Barrington, G.M., 2000. Passive transfer of colostral immunoglobulins in calves. J. Vet. Intern. Med. 14, 569–577. Wilson, C.B., 1990. Developmental immunology and role of host defenses in neonatal susceptibility. In: Remington, J.S., Klein, O.J. (Eds.), Infectious Diseases of the Fetus and Newborn Infant. W.B. Saunders Co., Philadelphia, pp. 17–67.
Contents lists available at ScienceDirect
Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm
Short communication
Comparative chemiluminescence of neonatal and adult ovine polymorphonuclear leukocytes Eugene H. Johnson *, Khalid Al-Habsi, Rashid Al-Busaidy Department of Animal and Veterinary Sciences, College of Agricultural and Marine Sciences, Sultan Qaboos University, P.O. Box 34, Al-Khod 123, Oman
A R T I C L E I N F O
A B S T R A C T
Article history: Received 15 March 2009 Received in revised form 5 September 2009 Accepted 16 September 2009
Polymorphonuclear leukoctyes (PMN) serve on the first line of the immunological defense of ruminants. However, despite the high morbidity and mortality of neonatal lambs to a wide range of infections there have been no definitive studies undertaken to ascertain whether there might be functional differences in PMN from neonatal lambs when compared to those from adult sheep. To determine whether there were differences in the oxidative respiratory burst of PMN, luminol-dependent chemiluminescence (CL) measurements were made of PMN from lambs at 1 week, 1, 2 and 3 months of age and at the same time from their respective dams. PMN isolated from lambs exhibited significantly lower levels of CL until 2 months of age. At 3 months of age the PMN produced levels of CL equal to that of their dams. As CL mirrors the ability of PMN to efficiently phagocytize and kill pathogens the present findings would suggest that PMN of neonatal lambs during the first 2 months are likely less capable of defending them from pathogenic organisms. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Lambs Chemiluminescence Respiratory burst Phagocytosis
1. Introduction Many researchers have focused their attention on the contributory role of failure of passive transfer of immunoglobulins or on the impairment of humoral and cellular immunity in neonatal ruminants as the principle immunological factors responsible for the proclivity of the high incidence of neonatal infections in ruminants (Senogles et al., 1987; Higuchi et al., 1997). Surprisingly, the functionality of polymorphonuclear leukocytes (PMN) from newborn ruminants has received little attention, although these cells are the first to arrive at a site of infection and play a central role in the defense as phagocytes, and in the killing of pathogenic microorganisms by releasing reactive oxygen species, degradative enzymes and cationic peptides (McCaffrey and Allen, 2006).
Abbreviations: PMN, polymorphonuclear leukocytes; CL, chemiluminescence; AUC, area under the curve. * Corresponding author. Tel.: +968 24143679; fax: +968 24413481. E-mail address: [email protected] (E.H. Johnson). 0165-2427/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2009.09.004
In humans there are a number of studies with conflicting results relative to the comparative phagocytic efficiency of PMN from newborns and adults. Whereas some investigators have reported that PMN from neonates and adults exhibit no difference in their phagocytic activity (Shigeoka et al., 1979; Wilson, 1990) others have demonstrated lower levels of phagocytic activity (Van Epps et al., 1978; Strauss et al., 1980). During the phagocytic process PMN generate an oxidative respiratory burst that results in increased oxygen consumption, hexose monophosphate shunt activity and production of reactive oxygen metabolites, including superoxide anions (O2 ) hydrogen peroxide (H2O2), singlet oxygen among others (Hampton et al., 1998; Babior, 1999). Actively, phagocytizing PMN emit light or chemiluminescence (CL) which is enhanced by luminol (5-amino2,3-dihydro-1,4-phthalazinedione) and is linked to the oxidative activity of the PMN (Stevens et al., 1978). These reactive oxygen species have microbicidal properties and the measurement of CL has gained widespread acceptance as an assay for phagocytic and oxidative metabolic function (Allen et al., 1972; Leino and Paape, 1993; Mehrzad et al., 2005).
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E.H. Johnson et al. / Veterinary Immunology and Immunopathology 134 (2010) 265–268
In the present study we compared CL of PMN from ovine neonates and their respective dams to ascertain whether there are differences in the oxidative respiratory burst of these cells which might help explain the increased susceptibility of newborns to infections. 2. Materials and methods 2.1. Animals and blood sampling Clinically healthy Omani sheep bred and maintained at the Sultan Qaboos University Agriculture Experimental Station were used for this study. Blood samples were collected by jugular venipuncture into heparinized tubes for PMN isolation from five full term Omani lambs at 1 week of age and subsequently at 1, 2 and 3 months of age, as well as from their respective dams. This study was approved in advance by the institutional animal care and use committee. 2.2. Isolation of PMN PMN were isolated from heparinized whole blood by density centrifugation over Ficol-hypague, as previously described (Johnson et al., 1991). Briefly, 7 ml of whole blood were layered onto 3 ml of Histopaque 1077 (Sigma Chemical Co., St. Louis, MO) and centrifuged at 1500 g for 30 min. The red blood cell layer was removed from the granulocyte containing pellets by hypotonic lysis and the PMN were washed three times in PBS and re-suspended to a concentration of 5 106 PMN/ml in RPMI. Cell counts were performed in a routine haemocytometer and differentials were determined using Wright–Giemsa stain. Cell viability was assessed by trypan blue exclusion. Only samples containing >95% neutrophils with >95% viability were used. Accompanying cells were predominately eosinophils. After isolation, PMN were immediately used for CL assays. 2.3. Opsonization of zymosan 100 mg of zymosan A particles (Sigma Chemical Co., St. Louis, MO) were suspended in 2 ml of PBS and boiled in a water bath for 1 h with frequent mixing. The zymosan was washed three times in PBS and re-suspended to a final concentration of 50 mg/ml. One part of the zymosan (0.1 ml) solution was mixed with nine parts of pooled adult ovine serum (0.9 ml) and incubated at 37 8C for 15 min with continuous mixing. The mixture was then washed and centrifuged at 850 g for 20 min. The zymosan pellet was re-suspended in 4 ml of RPMI.
plates at 37 8C with intermittent shaking between readings and measurements were made in an Ascent Luminoskan luminometer (Thermo Electron Corp., Vantaa, Finland). Prior to the commencement of the studies it was demonstrated that background CL of PMN was negligible. Based on trypan blue exclusion it was determined that there were no relevant differences in PMN viability at the beginning and end of the CL assays. The area under the curve (AUC), defined by relative light units and time, was calculated over a 60 min recording period. CL was recorded as mean relative light units (RLU). 2.5. Statistical analysis Data are presented as means standard error of the mean. For CL measurements over a 60 min recording period the AUC was calculated from plotted data points using Graph Pad Prism, version 4.00 for Windows (Graph Pad Software, Inc., San Diego, CA). For comparisons between means of adult and newborn PMN samples an unpaired t-test was applied. A p-value <0.05 was considered significant. 3. Results and discussion A comparison of the CL response of the PMN from the dams and their 1-week-old lambs is shown in Fig. 1. In both groups the CL response showed a similar kinetic response. However, there was a significant difference in the average counts per minute, peak CL response and the AUC. These differences continued for the readings of the PMN of lambs at 1 and 2 months of age. At month 3 the counts per minute of the PMN of the lambs had reached the level of the dams (Table 1). These results are in accordance with those of human studies which have shown that PMN from normal term neonates exhibited depressed oxidative metabolic responsiveness as measured by peak CL responses (Van Epps et al., 1978; Mills et al., 1979; Strauss et al., 1980). It is important however to note that many human studies have not been able to demonstrate a difference in CL response, phagocytic or bactericidal activity of PMN from healthy full term neonates when compared to adults (McCracken and Eichenwald, 1971; Shigeoka et al., 1979; Gahr et al.,
Table 1 Comparison of chemiluminescence (CL) response of polymorphonuclear leukocytes stimulated with opsonized zymosan from lambs and their respective dams. Age group
Mean RLU
Peak CL
Lambs (1 week) Dams
129 8.4 445.2 37.8*
187 15.1 574.2 51.7*
AUC 7632.1 686.7 26082 2832.4*
2.4. Chemiluminescence (CL)
Lambs 1 month Dams
130.2 7.7 450.4 39.6*
195.2 11.7 583.6 52.5*
8016.7 721.4 27503.4 2200.2*
The procedure for CL closely followed that described by Saeed and Castle (1998) with minor modifications. The reaction mixtures consisted of 100 ml of opsonized zymosan, 100 ml of PMN (5 106/ml) and 100 ml of RPMI containing 10 5 luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, Sigma Chemical Co., St. Louis, MO, USA). Assays were performed in triplicates in 96 well microtiter
Lambs 2 months Dams
73.2 6.9 452.4 44.8*
103.8 6.6 574.6 54.5*
4495.7 494.5 27337.8 1913.6*
Lambs 3 months Dams
451.7 31.2 459.4 38.1
582.1 40.7 590.3 38.4
28516.3 1996.1 28732.2 2585.9
Values represent mean SEM. CPM = counts per minute; AUC = area under the curve; RLU = relative light units. * P-values 0.05.
E.H. Johnson et al. / Veterinary Immunology and Immunopathology 134 (2010) 265–268
267
Fig. 1. Comparison of CL values of PMN induced by opsonized zymosan from 1-week-old lambs and their respective dams measured at 2 min intervals.
1985). It has also been reported that the resting levels of oxygen consumption, hexose monophosphate shunt activity, O2 , and nitro blue tetrazolium reduction is higher in neonatal PMN than in adult cells (Humbert et al., 1970; Park et al., 1970; Anderson et al., 1974; Strauss and Seifert, 1978; Ambruso et al., 1984). Some authors have only been able to demonstrate a difference in CL response of preterm but not in normal term infants (Shigeoka et al., 1979; Bektas et al., 1990; Kallman et al., 1998). Of interest is that some studies have not been able to demonstrate a difference in peak CL response of PMN from adult and neonates. However the CL activity of the PMN from newborns has been reported to decline more rapidly (Strauss and Seifert, 1978) suggesting that PMN from the neonates were unable to maintain the same level of oxygen dependent metabolic activity as those from adult animals. This difference in oxidative metabolism has also been observed in cord blood PMN from normal term human infants (Strauss and Seifert, 1978). These authors concluded that this aberration of leukocyte function might indicate a deficiency of metabolic reserve that could be related to the increased susceptibility of newborn humans to bacterial infections. Although the present study presents preliminary findings relative to the oxidative metabolism of ovine neutrophils they are of particular interest as there is mounting evidence, at least in human newborns, that the neutrophil might function at less than optimal levels also in oxygen-independent bacterial systems. Levy et al. (1999) demonstrated that neutrophils from newborns have less bactericidal/permeability increasing protein, a 55-kDa protein that binds with high affinity to bacterial lipopolysaccharides and kills Gram-negative bacteria.
Neutrophils from newborns have also been shown to have lesser amounts of lactoferrin and lysozyme in their specific granules (Ambruso et al., 1984) and are less efficiently primed by lipopolysaccharide (Qing et al., 1996) and tumor necrosis factor (Bortloussi et al., 1993). Neonatal neutrophils have also been reported to exhibit reduced expression of L-selectin and lower up regulation of CD11b (Kim et al., 2003). These findings, coupled with likely low bone marrow reserves (Bracho et al., 1998) and impaired neutrophil migration, chemotaxis and adherence (Krause et al., 1986) might help explain why newborns are especially susceptible to bacterial infections. Although it is widely accepted that failure of passive transfer and resulting low levels of circulating antibodies in newborn ruminants is associated with an increased susceptibility to infectious diseases (Besser and Gay, 1994; Rogers and Capucille, 2000; Weaver et al., 2000) the possibility of impaired neutrophil function in neonates should also be considered. This is highlighted in the findings of the present study which gives the first evidence of an impaired oxygen respiratory burst of PMN from neonatal lambs that persists for the first 2 months of their lives when compared to that from the PMN of adults. This might help to explain why some authors have been unable to establish a relationship between serum immunoglobulin levels and the subsequent incidence of infections in newborn calves (Adams et al., 1992; Bradley et al., 1979).
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