Clin. exp. Immunol. (1991) 83, 169-174
ADONIS 0009910491000327
Immunoglobulin and cytokine production by neonatal lymphocytes W. WATSON*, K. OEN*t, R. RAMDAHIN*+ & C. HARMANt Departments of *Paediatrics and Child Health and
tObstetrics and Gynaecology, and tRheumatic Disease Unit Research Laboratory, University of Manitoba, Winnipeg, Manitoba, Canada
(Acceptedfor publication 10 July 1990)
SUMMARY Growth and differentiation of cord blood B cells were studied using T cell-depleted populations. In the absence of in vitro activation, cord blood B cells proliferated in response to cytokines including interleukin-2 (IL-2) and interleukin-4 (IL-4); anti-,u-stimulated cord B cells had a lesser response to IL-2 than adult cells. IgM synthesis by cord blood B cells was enhanced by interleukin-6 (IL-6) and decreased by IL-2. In cultures activated by Staphylococcus aureus Cowan I (SAC), cord blood B cells produced lesser increases in IgM than adult B cells regardless of the cytokine added. Cord blood B cells produced no IgG or IgA with any cytokine preparation with or without SAC activation. Supernatants of cord blood T cells pulse-stimulated with phytohaemagglutinin and phorbol myristate acetate contained less IL-2 and IL-6 and had less growth and differentiation activity than adult T cell supernatants. The results confirm a limited cord blood B cell response and also suggest a limitation in production of B cell stimulatory lymphokines by cord blood T cells. Keywords Cord B and T lymphocytes immunoglobulin synthesis cytokine production
INTRODUCTION
IgG and IgA in addition to IgM. Conversely we have investigated the ability of neonatal T cells to produce lymphokines which influence B cell maturation and differentiation.
The neonatal humoral response consists primarily of IgM antibody (Gathings, Kubagawa & Cooper, 1981; Cooper, 1987). Similarly, in vitro responses stimulated by pokeweed mitogen (PWM), Staphylococcus aureus Cowan I (SAC), or Epstein-Barr virus (EBV) result in immunoglobulin secretion, largely limited to the IgM isotype (Gathings et al., 1981; Ruuskanen et al., 1980; Pereira, Webster & Platts-Mills, 1982). Phenotypically neonatal B cells express surface (s) IgM as well as sIgG and sIgA but the latter are expressed on cells with sIgM and sIgD, a phenotype that has been referred to as 'immature' (Gathings, Lawton & Cooper, 1977; Conley & Cooper, 1981; Gathings et al., 1981). It has been debated whether the distinct B cell response of the neonate relates to functional differences in B lymphocytes or whether T cell influences such as suppression may account for decreased B cell responses (Hayward & Lawton, 1977; Morito, Bankhurst & Williams, 1979; Gathings et al., 1981). To these considerations one can add decreased lymphokine production by neonatal T cells. In order to assess the capacity of neonatal B and T lymphocytes separately we have studied proliferation and immunoglobulin synthesis by T-depleted populations. We hypothesized that given adequate activation signals as well as stimulation by cytokines, neonatal B lymphocytes may produce
MATERIALS AND METHODS Subjects Cord blood was obtained from placentas following routine repeat Caesarean section deliveries of term infants. Healthy adult volunteers, aged 25-45 years served as the comparison group. Tonsils were obtained from tonsillectomies performed for clinical indications. The study was approved by the University of Manitoba Faculty Committee on the Use of Human Subjects in Research.
Preparation of cells Tonsil and blood lymphocytes were separated into T and non-T cell fractions as previously described (Oen, Wilkins & Krzekotowska, 1985). For adult specimens B enriched cells were prepared from non-T cells by partial removal of adherent cells through incubation on plastic (Oen & Krzekotowska, 1987). Analysis of T cells, surface immunoglobulin-positive cells and esterase-positive cells were performed as previously described (Oen et al., 1985; Oen & Krzekotowska, 1987). Cord blood T cells contained 88 + 4% rosette-forming cells (RFC) and 0% esterase-positive cells; and non-T cells were 0-7 + 0-6% RFC, 42 + 10% surface immunoglobulin-positive, and 15 + 6% esterase-positive. Adult T cells were 91+2% RFC and 0-3 + 0-7% esterase-positive; and B-enriched cells were
Correspondence: Dr K. Oen, RR149 Rehabilitation Centre, 800 Sherbrook Street, Winnipeg, Manitoba, Canada, R3A 1M4.
169
170
W. Watson et al.
0-4 + 0 4% RFC, 44 +± 0% surface immunoglobulin-positive, and 21 +4% esterase-positive. Tonsil non-T cells contained 03+002% RFC, 83+80±% surface immunoglobulin- and 0% esterase-positive cells.
*
0
c
Cytokine preparations Recombinant interleukin-2 (IL-2), interleukin-4 (IL-4), and interleukin-6 (IL-6) were purchased from Genzyme Corporation (Boston, MA). T cells were pulsed with 1/1000 dilution of phytohaemaglutinin-M (PHA) (Difco Laboratories, Detroit, MI), or PHA 1/1000 and 20 ng/ml phorbol myristate acetate (PMA) (Sigma) for 2 h, according to methods based on Sakane et al. (1985). After washing, 2-5 x 106 viable cells/ml were cultured in RPMI 1640 medium (GIBCO, Grand Island, NY) and 10% fetal calf serum (FCS) (GIBCO) for 2 days. Supernatants were harvested and frozen at -20'C until used. T cell supernatants prepared as above from seven adult controls were pooled and used for stimulation of cultures. The supernatant pool contained 2176 units of IL-2, 16384 units of interferon, and 6-4 units of IL-6. Interferon was measured by inhibition of murine encephalomyocarditis virus-induced cytopathic effect on a human bronchial carcinoma cell line A549. Titres were compared with a laboratory standard previously measured against a National Institutes of Health standard. Cultures Optimal cell preparations for cord blood B cells consisted of I05 non-T cells and for adult controls, 5 x 104 B enriched cells per culture. B cell growth was assessed by stimulation with anti-p coated acrylamide beads (BioRad, Richmond, CA) at an optimal concentration of 5 pg/ml antibody and 3 or 12 U/ml IL2,3 or 12 U/ml IL-4, or 12-5% supernatant in 0-2 ml RPMI 1640 supplemented with 10% FCS and 5 x 10 -M 2-mercaptoethanol (2-ME) (Sigma). Cultures were also set up with medium or the cytokine preparations only. 3H-thymidine (0 4 pCi) was added 6 h prior to harvesting on day 3. Cells were harvested and ct/min were counted as previously described (Oen et al., 1988). Immunoglobulin synthesis was determined by stimulation with 0 001% SAC (Pansorbin, Calbiochem-Behring, San Diego, CA) and 30 or 60 U/ml IL-2; 10 or 50 U/ml IL-6; 25, 50, or 100 U/ml IL-4; combinations of 30 U IL-2 with 50 U/ml IL-4 or IL-6, and 50 U of IL-4 with 50 U IL-6; or 3% or 6% supernatant in 0 2 ml RPMI 16940 with 10% FCS and 10-5 M 2-ME. Cultures were incubated for 9 days and IgG, IgA and IgM were measured in supernatants by class specific ELISA assays as previously described (Oen et al., 1985). In certain experiments, 4 x 106 cord non-T cells were preincubated with medium alone or with 10 or 50 U/ml of IL-4 in 2 ml of RPMI 1640 with 10% FCS for 1 to 2 days. Cells were washed and re-incubated at a concentration of 105 cells/0 2 ml with 0 001% SAC and 30 U/ml IL-2 for 8 or 7 days. Alternatively, 10 or 50 U/ml IL-4 were added to I05 non-T cells at the start of culture and SAC with 30 U/ml IL-2 were added on days 1 or 2.
Analysis of cytokine production IL-2 was determined by proliferation of CTLL-2 cells (Gillis et al., 1978). A commercially obtained purified human IL-2 preparation of known activity (Electronucleonics, Silver Spring, MD) was used as a standard. IL-6 was measured by prolifer-
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ADONIS 0009910491000327
Immunoglobulin and cytokine production by neonatal lymphocytes W. WATSON*, K. OEN*t, R. RAMDAHIN*+ & C. HARMANt Departments of *Paediatrics and Child Health and
tObstetrics and Gynaecology, and tRheumatic Disease Unit Research Laboratory, University of Manitoba, Winnipeg, Manitoba, Canada
(Acceptedfor publication 10 July 1990)
SUMMARY Growth and differentiation of cord blood B cells were studied using T cell-depleted populations. In the absence of in vitro activation, cord blood B cells proliferated in response to cytokines including interleukin-2 (IL-2) and interleukin-4 (IL-4); anti-,u-stimulated cord B cells had a lesser response to IL-2 than adult cells. IgM synthesis by cord blood B cells was enhanced by interleukin-6 (IL-6) and decreased by IL-2. In cultures activated by Staphylococcus aureus Cowan I (SAC), cord blood B cells produced lesser increases in IgM than adult B cells regardless of the cytokine added. Cord blood B cells produced no IgG or IgA with any cytokine preparation with or without SAC activation. Supernatants of cord blood T cells pulse-stimulated with phytohaemagglutinin and phorbol myristate acetate contained less IL-2 and IL-6 and had less growth and differentiation activity than adult T cell supernatants. The results confirm a limited cord blood B cell response and also suggest a limitation in production of B cell stimulatory lymphokines by cord blood T cells. Keywords Cord B and T lymphocytes immunoglobulin synthesis cytokine production
INTRODUCTION
IgG and IgA in addition to IgM. Conversely we have investigated the ability of neonatal T cells to produce lymphokines which influence B cell maturation and differentiation.
The neonatal humoral response consists primarily of IgM antibody (Gathings, Kubagawa & Cooper, 1981; Cooper, 1987). Similarly, in vitro responses stimulated by pokeweed mitogen (PWM), Staphylococcus aureus Cowan I (SAC), or Epstein-Barr virus (EBV) result in immunoglobulin secretion, largely limited to the IgM isotype (Gathings et al., 1981; Ruuskanen et al., 1980; Pereira, Webster & Platts-Mills, 1982). Phenotypically neonatal B cells express surface (s) IgM as well as sIgG and sIgA but the latter are expressed on cells with sIgM and sIgD, a phenotype that has been referred to as 'immature' (Gathings, Lawton & Cooper, 1977; Conley & Cooper, 1981; Gathings et al., 1981). It has been debated whether the distinct B cell response of the neonate relates to functional differences in B lymphocytes or whether T cell influences such as suppression may account for decreased B cell responses (Hayward & Lawton, 1977; Morito, Bankhurst & Williams, 1979; Gathings et al., 1981). To these considerations one can add decreased lymphokine production by neonatal T cells. In order to assess the capacity of neonatal B and T lymphocytes separately we have studied proliferation and immunoglobulin synthesis by T-depleted populations. We hypothesized that given adequate activation signals as well as stimulation by cytokines, neonatal B lymphocytes may produce
MATERIALS AND METHODS Subjects Cord blood was obtained from placentas following routine repeat Caesarean section deliveries of term infants. Healthy adult volunteers, aged 25-45 years served as the comparison group. Tonsils were obtained from tonsillectomies performed for clinical indications. The study was approved by the University of Manitoba Faculty Committee on the Use of Human Subjects in Research.
Preparation of cells Tonsil and blood lymphocytes were separated into T and non-T cell fractions as previously described (Oen, Wilkins & Krzekotowska, 1985). For adult specimens B enriched cells were prepared from non-T cells by partial removal of adherent cells through incubation on plastic (Oen & Krzekotowska, 1987). Analysis of T cells, surface immunoglobulin-positive cells and esterase-positive cells were performed as previously described (Oen et al., 1985; Oen & Krzekotowska, 1987). Cord blood T cells contained 88 + 4% rosette-forming cells (RFC) and 0% esterase-positive cells; and non-T cells were 0-7 + 0-6% RFC, 42 + 10% surface immunoglobulin-positive, and 15 + 6% esterase-positive. Adult T cells were 91+2% RFC and 0-3 + 0-7% esterase-positive; and B-enriched cells were
Correspondence: Dr K. Oen, RR149 Rehabilitation Centre, 800 Sherbrook Street, Winnipeg, Manitoba, Canada, R3A 1M4.
169
170
W. Watson et al.
0-4 + 0 4% RFC, 44 +± 0% surface immunoglobulin-positive, and 21 +4% esterase-positive. Tonsil non-T cells contained 03+002% RFC, 83+80±% surface immunoglobulin- and 0% esterase-positive cells.
*
0
c
Cytokine preparations Recombinant interleukin-2 (IL-2), interleukin-4 (IL-4), and interleukin-6 (IL-6) were purchased from Genzyme Corporation (Boston, MA). T cells were pulsed with 1/1000 dilution of phytohaemaglutinin-M (PHA) (Difco Laboratories, Detroit, MI), or PHA 1/1000 and 20 ng/ml phorbol myristate acetate (PMA) (Sigma) for 2 h, according to methods based on Sakane et al. (1985). After washing, 2-5 x 106 viable cells/ml were cultured in RPMI 1640 medium (GIBCO, Grand Island, NY) and 10% fetal calf serum (FCS) (GIBCO) for 2 days. Supernatants were harvested and frozen at -20'C until used. T cell supernatants prepared as above from seven adult controls were pooled and used for stimulation of cultures. The supernatant pool contained 2176 units of IL-2, 16384 units of interferon, and 6-4 units of IL-6. Interferon was measured by inhibition of murine encephalomyocarditis virus-induced cytopathic effect on a human bronchial carcinoma cell line A549. Titres were compared with a laboratory standard previously measured against a National Institutes of Health standard. Cultures Optimal cell preparations for cord blood B cells consisted of I05 non-T cells and for adult controls, 5 x 104 B enriched cells per culture. B cell growth was assessed by stimulation with anti-p coated acrylamide beads (BioRad, Richmond, CA) at an optimal concentration of 5 pg/ml antibody and 3 or 12 U/ml IL2,3 or 12 U/ml IL-4, or 12-5% supernatant in 0-2 ml RPMI 1640 supplemented with 10% FCS and 5 x 10 -M 2-mercaptoethanol (2-ME) (Sigma). Cultures were also set up with medium or the cytokine preparations only. 3H-thymidine (0 4 pCi) was added 6 h prior to harvesting on day 3. Cells were harvested and ct/min were counted as previously described (Oen et al., 1988). Immunoglobulin synthesis was determined by stimulation with 0 001% SAC (Pansorbin, Calbiochem-Behring, San Diego, CA) and 30 or 60 U/ml IL-2; 10 or 50 U/ml IL-6; 25, 50, or 100 U/ml IL-4; combinations of 30 U IL-2 with 50 U/ml IL-4 or IL-6, and 50 U of IL-4 with 50 U IL-6; or 3% or 6% supernatant in 0 2 ml RPMI 16940 with 10% FCS and 10-5 M 2-ME. Cultures were incubated for 9 days and IgG, IgA and IgM were measured in supernatants by class specific ELISA assays as previously described (Oen et al., 1985). In certain experiments, 4 x 106 cord non-T cells were preincubated with medium alone or with 10 or 50 U/ml of IL-4 in 2 ml of RPMI 1640 with 10% FCS for 1 to 2 days. Cells were washed and re-incubated at a concentration of 105 cells/0 2 ml with 0 001% SAC and 30 U/ml IL-2 for 8 or 7 days. Alternatively, 10 or 50 U/ml IL-4 were added to I05 non-T cells at the start of culture and SAC with 30 U/ml IL-2 were added on days 1 or 2.
Analysis of cytokine production IL-2 was determined by proliferation of CTLL-2 cells (Gillis et al., 1978). A commercially obtained purified human IL-2 preparation of known activity (Electronucleonics, Silver Spring, MD) was used as a standard. IL-6 was measured by prolifer-
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