Summary: Lymphokine production by newborn lymphocytes was assessed by measuring migration inhibition factor (MIF) and leukocyte inhibition factor (LIF) of isolated mononuclear cells from cord blood, 1–7-days-old newborns, and adult controls. Ficoll-Hypaque separated mononuclear cells were stimulated with phytohemagglutinin (PHA) or allogeneic lymphocytes in a mixed leukocyte culture (MLC), and the supernatants were harvested at optimal times for lymphokine assays. Thymidine incorporation into DNA was also assayed to calculate a proliferative index. MIF was assessed by the inhibition of adult mononuclear phagocyte cell migration under agarose; LIF was assessed by polymorphonuclear cell migration under agarose. Although the proliferative responses of cord and newborn cells are equivalent or greater than those of adult controls, the PHA-induced MIF production in cord blood and newborn lymphocytes was only 46% and 12.5% respectively of mean adult levels; MLC-induced MIF production was 44% and 7%, respectively of mean adult levels. PHA-induced LIF production in cord blood was 27% of adult levels. These differences are only appreciated if dilutions of the supernatants are assayed. Simultaneous assay of MIF and LIF production in dilution of supernatants from adult lymphocytes showed higher LIF activity, whereas in cord lymphocytes MIF activity was greater than LIF activity. This further emphasizes the non-identity of MIF and LIF. These results indicate another abnormality of T cellular immunity in newborns not detected by T-cell enumeration or proliferative responses and parallels other defects in specialized T cell function such as cytotoxicity and immune interferon production.

Two cytotoxic assays, lectin-dependent cytotoxicity and natural killer (NK) cytotoxicity, were used to assess the competence of cord blood and neonatal peripheral blood mononuclear cell (PBMC) and T-cell cytotoxic reactions. The effect of exogenous interferon was also studied. Results were compared with cytotoxic capabilities of adult cells and cells from patients with primary immunodeficiency syndromes. Lectin-dependent cytotoxicity (LDCC), a property of both T and non-T cells, was assessed by lysis of chromium-labeled EL4 tumor target cells in the presence or absence of exogenous fibroblast interferon (IFN-β). Natural killer cytotoxicity was assessed by lysis of two different chromium-labeled tumor target cells, Molt 4f and K562 in the presence or absence of IFN-β. Lectin-dependent cytotoxicity (LDCC) of PBMC of cord blood (32 ± 4% SEM) and adult cells (36 ± 2% SEM) were equivalent but neonatal cells had slightly decreased LDCC (22 ± 3% lysis). T-depleted cells from cord or neonatal blood had increased LDCC but T-enriched (>95% sheep erythrocyte rosette-forming cells) from both cord (22 ± 3%) and neonatal blood (18 ± 5%) had significantly reduced LDCC compared to 55 ± 2% for adult T cells. This deficiency corrects with age and is near normal after age 2. Preincubation with IFN-β did not enhance LDCC of newborn or adult cells. The LDCC of some cord T cells was markedly reduced and was in the same low range as patients with severe combined immunodeficiency. Natural killer (NK) cytotoxicity of PBMC from cord and adult cells was equivalent at three effector:target ratios against the Molt 4f target but against the K562 target, cord PBMC had significantly less NK activity (22 ± 11 SD) compared to adult NK activity (50.5 ± 22.2 SD) at a 50:1 effector:target ratio. Similar differences were noted at 25:1 and 10:1 target:effector ratios. NK cytotoxicity against Molt 4f targets of adult cells was significantly enhanced by preincubation with IFN-β but NK of cord cells was only variably enhanced. By contrast, IFN-β enhanced NK against K562 targets of both adult and cord cells, adult greater (67.7 ± 20) than cord cells (37.8 ± 2.0). These T-cell effector deficiencies are in marked contrast to the vigorous proliferative responses of newborn T cells, and parallel deficiencies of certain neonatal lymphokines. These defects may explain the newborns' enhanced susceptibility to intracellular viruses and to congenital viral infections.

Host defense mechanisms were studied in six patients with pemphigus vulgaris (PV) and six patients with bullous pemphigoid (BP). Polymorphonuclear (PMN) leukocyte killing of Staphylococcus organisms was evaluated, and chemotaxis of PMN and mononuclear (MN) leukocytes in patients was compared with that in twenty age- and sex-matched controls. All patients had extensive widespread disease with the clinical diagnosis confirmed by immunopathologic studies. No statistically significant differences were observed in the PMN leukocyte bactericidal activity between PV patients and controls. In BP patients, PMN leukocyte bactericidal activity was very slightly reduced when normal cells and patient serum were used, but activity was normal when patient cells and patient serum were used. PMN leukocyte chemotaxis was normal in PV and BP patients. MN leukocyte chemotaxis was normal in PV patients and increased in BP patients when compared with that in controls. This study indicated that in spite of very severe and extensive disease, patients with PV and BP have intact neutrophil and monocyte functions. Drugs that compromise the patient's ability to fight infections should be used cautiously and judiciously.

Cellular (T-cell) immunity in infants and young children is suspect because of diminished delayed skin tests and increased susceptibility to infection, engraftment, and malignancy. However, T cell numbers and proliferative responses are normal, and lymphokine production is variably reduced. We therefore studied two cytotoxic functions of T cells, natural killer (NK) and lect-in-dependent (LD) cytotoxicity in newborns, young children less than 24 months, and adults. In the PHA-enhanced LD cytotoxicity assay, whole mononuclear (MN), T enriched (>95% T cells) and T depleted (<1% T cells) fractions were tested against 51Cr-labeled EL-4 target cells in a 4 hour incubation at an effector target ratio of 40:1. In the NK assay, MN cells were used against 51Cr labeled Molt-4f tumor cells with and without the addition of exogenous interferon (100 units) at effector:target ratios of 50:1, 25:1, and 10:1. Cyclic AMP levels were also assessed in the cell fractions. In the LD assay, the MN cord blood cells had equivalent specific cytotoxicity (34 vs 32%, N=14) to adult cells but the T-enriched fractions had a strikingly decreased cytotoxicity (22 vs 51%) compared to adults. This normalizes after age 2. By contrast NK activity and interferon enhancement was equivalent in newborn and adult cells (53 vs 55%, N=10). Cyclic AMP levels of newborn cells were markedly reduced. These results indicate that NK maturation occurs very early, that an important T-effector function is immature in newborns, and that their T cells resemble those of some patients with T cell immunodeficiency.

Summary: Using Ficoll-Hypaque-separated cells, monocyte chemotaxis was measured by an agarose technique in patients with increased susceptibility to infection, with atopic dermatitis, and in individuals taking aspirin. In vitro effects of aspirin, hydrocortisone, aminophylline, ephedrine, and diphenhydramine were also studied. Significantly decreased chemotaxis was found in one 9-year-boy with severe mucocutaneous candidiasis and three of 22 patients with atopic dermatitis. In the atopic group of patients greater than 10 years of age, mean monocyte chemotaxis was significantly decreased from the age-matched control group. This decrease did not correlate with serum IgE levels, absolute blood eosinophil counts, or clinical symptom scores. Following aspirin ingestion, mean monocyte chemotaxis significantly decreased whereas neutrophil chemotaxis was unaffected. Using therapeutic concentrations, drug levels of aspirin and aminophylline in vitro caused greater than 35% inhibition of monocyte movement.Speculation: Measurement of monocyte chemotaxis in immunodeficiency and atopic disease may uncover defects amenable to agents which stimulate cell movement.

The increased susceptibility of newborns to disseminated infection may result from defective or immature lymphokine production. Thus, we assessed monocyte migration inhibition factor (MIF) and interferon (IF) production of the lymphocytes of 20 newborns (cord blood), 20 1-7 day old neonates, and 20 normal adults. Ficoll-hypaque separated mononuclear cells were stimulated with phytohemagglutinin (PHA), allogeneic lymphocytes in a mixed leukocyte culture (MLC), or Newcastle Disease Virus (NDV), and supernatants were harvested at optimal times. MIF was assessed by the inhibition of adult mononuclear cell migration under agarose; IF was assessed by micro-dye uptake of human diploid cells after encephalomyocarditis virus challenge. Mean PHA-induced MIF production in cord and newborn lymphocytes was 30 and 10% respectively of adult levels, MLC-induced MIF production was 8 and 5% of adult levels. PHA-induced (immune) IF was produced in only 1 of 20 cord bloods and 3 of 18 newborns in small amounts whereas IF was produced by all adults (mean 225 units ±2 SD). No IF was produced by MLC in newborns, cords or adults. NDV-induced (classical) IF was produced in normal amounts by adult (168±2), cord (200±2), and newborn cells (228±3). These results indicate an abnormality of cellular immunity in newborns not detected by T-cell numbers or transformation indices and suggest a mechanism for viral infection in newborns with concomitant bacterial infections and in patients undergoing graft-versus-host reactions.

Monocyte chemotaxis was determined by an agarose technique in normal newborns, infants, older children, and adults and in selected patients. Monocytes were obtained from heparinized blood by Ficoll-Hypaque separation and exposed to zymosan treated serum as the chemotactic source. Newborn values (50 ± 33; ± 1 S.D.) were significantly different from adult values (216 ± 75, p<.005). An age related increase was noted (6 weeks to 2 yrs., 31 ± 25; 3 yrs. to 5 yrs., 71 ± 57; 6 yrs. to 10 yrs., 142 ± 64). Normal adult mean values were observed in the 11 to 16 year age group (214 ± 70). Monocyte chemotactic defects have previously been described in several conditions. We have noted marked defects (>2 S.D.) in one patient with chronic mucocutaneous candidiasis and 4/29 patients with atopic dermatitis. In 8 patients on salicylate therapy, a mean decrease of monocyte chemotaxis of 25% was seen with salicylate levels >15 mgs% compared to a mean increase of 8% when neutrophil chemotaxis was concurrently measured. We also noted decreased monocyte chemotaxis in several seemingly normal subjects with absent delayed hypersensitivity skin tests to tetanus toxoid but normal in vitro lymphocyte transformation to tetanus. These findings extend previous studies demonstrating maturational defects in human phagocytic cells, re-emphasize the possible role of defective monocyte chemotaxis in immunodeficient states, and suggest a possible mechanism for the anti-inflammatory effects of salicylates.

A new method, chemotaxis under agarose gel, was used to assess the directed motility of polymorphonuclear (PMN) and mononuclear (MN) phagocytes of 21 newborns, 71 infants and children, and 50 adults. This assay requires only small quantities of cells and is rapid, easy, reproducible, and provides a permanent record. The chemotactic substance was zymosan-activated human serum. Monocyte chemotaxis in the newborn was approximately 50% of adult control values, using the Boyden chamber (8.1 ± 2 cells per high-power field[HPF] [SE] in newborns compared to 17 ± 3 cells per HPF in adults), and 25% of adult control values, using the agarose method (50 ± 10 cells in newborns compared to 216 ± 15 cells in adults). Both are significant differences (P < .005). MN chemotaxis remains extremely low through age 5, and remains moderately reduced until age 10. PMN chemotaxis in the newborn was 82 ± 21 cells compared to adult controls of 300± 42 cells, also a significant difference (P < .05). PMN chemotaxis remains markedly depressed through age 2. Thereafter, PMN chemotaxis increases but remains significantly less than in adults until age 16. These chemotactic defects may play an important role in depressed delayed hypersensitivity skin tests, diminished inflammatory reactions, and increased susceptibility to infection present in the newborn and young infant.