Gerald Rothstein

Defective Production of Interleukin-6 by Monocytes: A Possible Mechanism Underlying Several Host Defense Deficiencies of Neonates

ABSTRACT: Several deficiencies in antibacterial defense have been described in neonates. Among those best characterized are delayed maturation of B cells into antibody producing cells, deficient T-cell maturation, and delayed cycling of hematopoietic progenitor cells after an infectious challenge. No unifying theory has been forwarded, however, to explain the concomitance of these three developmental deficiencies. IL-6, a cytokine produced primarily by monocytes and macrophages in response to stimulation by IL-1, is involved in the regulation of these three processes. Thus, we postulated that defective production of IL-6 could be a mechanism underlying these immune deficiencies of neonates. Indeed, we observed that at peak production, cells of five term neonates produced only one half as much IL-6 (14 120 ± 2590 pg IL-6/108 monocytes) as those of five adults (28 940 ± 1680 pg, p < 0.001). Peak production was lower still by monocytes of six preterm neonates (7190 ± 1400 pg, p < 0.001 versus term). Production of IL-6 protein was inhibited by actinomycin D and the IL-6 mRNA content of monocytes from neonates, as assessed by competitive polymerase chain reaction, was less than that of adult monocytes. We speculate that defective IL-6 transcription might underlie some of the defects in immune regulation observed in neonates.

Blood and marrow neutrophils during experimental group B streptococcal infection: quantification of the stem cell, proliferative, storage and circulating pools.

Summary: In an attempt to explain the profound neutropenia and depletion of marrow neutrophil reserves observed in human and experimental group B streptococcal infection, we have studied the stem cell, proliferative, storage, and circulating neutrophil pools in groups of adult and neonatal rats inoculated with 3 × 106 type II group B streptococci/g body weight. At intervals after inoculation, animals were sacrificed and the unipotent granulocytic stem cells, (Colony Forming Units in culture, CFUC) from the femoral marrow were quantified. In addition, the total body neutrophil proliferative pool, neutrophil storage pool, circulating blood neutrophil concentration and blood immature to total neutrophil ratio were measured. Adult animals developed neutrophilia with a peak blood neutrophil concentration of 3.4 times control by 24 h after inoculation (P < 0.001). A left shift was seen in these mature animals as early as 2 h but by 24 h was no longer present. The neutrophil storage pool diminished to 65% of control by 15 h (P < 0.001) but then increased to exceed the control in all animals by 48 h (P < 0.01). The neutrophil proliferative pool did not change during infection in the adult animals, but CFUC increased to three times control 48 h after inoculation (P < 0.001) and returned to baseline by 72 h.The myeloid response of the neonatal rats was markedly different from that of the adults. Neonates developed profound neutropenia with an extreme left shift, exhaustion of the neutrophil storage pool (less than 10% of control by 15 h, P < 0.001), and diminution of the proliferative pool to 58% of control by 10 h (P < 0.05). In contrast to the adult animals, neonatal rats infected with group B streptococci failed to increase CFUC, and in fact a diminution of CFUC to 48% of control was seen by 15 h (P < 0.05).Speculation: When adults develop bacterial infection, they often demonstrate neutrophilia and a leukocyte left shift. We observed these findings in infected adult rats accompanied by a moderate, temporary diminution of storage neutrophils and by an elevation in granulocytic stem cells. The adult response enables the animal to deliver large numbers of neutrophils to the infected tissues. In contrast, infected neonates developed profound neutropenia, exhaustion of neutrophil reserves and decreased neutrophii production. The neonatal response results in diminished delivery of neutrophils to infected tissues and a marked increase in mortality. The decreased production of neutrophils accompanying neutrophil storage pool depletion and neutropenia in neonates may constitute a condition for which neutrophil transfusion may represent a reasonable therapeutic approach.

Granulocyte-macrophage progenitor cells in term and preterm neonates

In groups of adults, and term and preterm neonates, we determined: the blood concentration, the proliferative rate, and the variety of progeny of committed granulocyte-macrophage progenitor cells (CFU-GM). In five of eight term neonates and in all premature infants, a potentially significant limitation of neutrophil production was detected. Unlike the slowly proliferating CFU-GM present in the blood of healthy adult subjects (7% thymidine suicide, range 0% to 32%), the circulating CFU-GM in the premature subjects were proliferating at a near maximal rate (55%, range 40% to 75%, P less than 0.001). Because CFU-GM proliferation is nearly maximal in the baseline, noninfected state, neonates may have restricted ability to increase neutrophil production from CFU-GM during times of increased neutrophil need, such as during bacterial infection. Such inability may predispose neonates to exhaustion of the neutrophil supply during bacterial infection.

Neutropenia and Thrombocytopenia in Renal Allograft Recipients Treated with Trimethoprim-Sulfamethoxazole

Hematologic toxicity occurred when trimethoprim-sulfamethoxazole was given for prolonged periods to renal allograft subjects also treated with azathioprine. In six such patients, the incidences and duration of neutropenia and thrombocytopenia were greater than in 25 similar allograft recipients treated with azathioprine alone. In bone marrow culture, the antifolate action of trimethoprim-sulfamethoxazole enhanced the marrow-suppressive effect of 6-mercaptopurine, the active moiety cleaved from azathioprine. These studies show that in renal allograft recipients treated with azathioprine, the prolonged use of trimethoprim-sulfamethoxazole may result in life-threatening hematotoxicity.

Hematopoiesis is not clonal in healthy elderly women.

Clonality assays, based on X-chromosome inactivation, discriminate active from inactive alleles. Skewing of X-chromosome allelic usage, based on preferential methylation of one of the HUMARA alleles, was reported as evidence of clonal hematopoiesis in approximately 30% of elderly women. Using a quantitative, transcriptionally based clonality assay, we reported X-chromosome-transcribed allelic ratio in blood cells of healthy women consistent with random X-inactivation of 8 embryonic hematopoietic stem cells. Furthermore, we did not detect clonal hematopoiesis in more than 200 healthy nonelderly women. In view of the susceptibility of aging hematopoietic stem cells to epigenetic dysregulation, we reinvestigated the issue of clonality in elderly women. Forty healthy women (ages 65-92 years; mean, 81.3 years) were tested by a novel, quantitative polymerase chain reaction (qPCR) transcriptional clonality assay. We did not detect clonal hematopoiesis in any of the tested subjects. We also tested DNA from the same granulocyte samples using the methylation-based HUMARA assay, and confirmed previous reports of approximately 30% extensively skewed or monoallelic methylation, in agreement with likely age-related deregulated methylation of the HUMARA gene locus. We conclude that the transcriptionally based X-chromosome clonality assays are suitable for evaluation of clonal hematopoiesis in elderly women.

Disordered Hematopoiesis and Myelodysplasia in the Elderly

Normal hematopoiesis constitutes the process of producing diverse, differentiated blood cell types in a manner related to physiological requirement. During aging, modulation of hematopoiesis becomes disordered, impairing the ability of older people to respond appropriately to the physiological demand for blood cell replacement triggered by stimuli such as blood loss or cytoreductive chemotherapy. This may contribute to the increase in the prevalence of anemia that is observed during aging. In addition, various age‐related events, such as genomic mutations secondary to oxidative stress and impaired regulation of cytokine production, may contribute to or cause the emergence of abnormal clones of hematopoietic cells. Therefore, normal hematopoiesis is disrupted, and the hematopoietic system is populated with cells that are quantitatively and functionally deficient and are also subject to leukemic transformation. These defects in the production and maturation of the various differentiated blood cells are referred to as myelodysplastic syndromes. These syndromes are so tightly associated with aging that they are considered to be geriatric disorders; they can lead to anemia, neutropenia, and thrombocytopenia and to the development of acute nonlymphoblastic leukemia. Dysregulation of mechanisms controlling hematopoiesis is therefore an important characteristic of the hematopoietic system in the elderly, but the response of progenitor cells to humoral stimulators is preserved and accounts for the effectiveness of recombinant hematopoietic growth factors used as emerging treatment modalities for hematopoietic disorders in the elderly.

Diminished transcription of interleukin-8 by monocytes from preterm neonates

Developmental delays, which impair antibacterial host defense, are present in the neutrophil system of human preterm neonates. We hypothesized that diminished production of interleukin‐8 (IL‐8), a neutrophil chemotactic peptide, might in part be responsible for these defects. To test this, monocytes from the blood of preterm neonates, term neonates, and adults were isolated immunologically by “negative panning” and subsequently stimulated with interleukin‐1α (IL‐lα), tumor necrosis factor a (TNF‐α), or lipopolysaccharide (LPS), after which IL‐8 levels in the supernatants were measured by ELISA. Total cellular RNA was extracted and IL‐8 mRNA was assessed by Northern blotting and by competitive polymerase chain reaction (PCR) analyses. After stimulation with IL‐lα, IL‐8 accumulation was lowest in supernatants of monocytes from preterm neonates, intermediate in supernatants of monocytes from term neonates and greatest from monocytes of adults. Similarly, when stimulated with TNF‐α or LPS, monocytes from preterm neonates produced less IL‐8 than cells from term neonates and adults. These differences in IL‐8 concentrations paralleled differences in IL‐8 mRNA expression.

Fatal early onset group B streptococcal sepsis with normal leukocyte counts.

In contrast to the attitude prevalent a decade ago, clinicians entertaining the diagnosis of neonatal bacterial sepsis now often place considerable reliance on the blood neutrophil count and degree of left shift. In this report we present four cases which illustrate that in some patients, no derangement of the complete blood count (CBC) is present early in the course of bacterial sepsis. In order to determine the length of time between bacterial inoculation and the appearance of changes in the CBC, we used an animal model of early onset Group B streptococcal sepsis. In adult animals we observed characteristic changes in the CBC within 1 hour of bacterial inoculation, but in neonates this latent period was considerably longer, lasting 4 hours. Thus a normal CBC might actually be expected during the first several hours of early onset neonatal sepsis. This delay in appearance of CBC changes constitutes a previously uninvestigated feature of neonatal neutrophil kinetics, the “latent period.”

Circulating and Storage Neutrophil Changes in Experimental Type II Group B Streptococcal Sepsis

Summary: The availibility of neutrophils is an important factor in host resistance to bacterial infection. Therefore, circulating and storage neutrophil quantification was carried out on groups of neonatal rats intranasally inoculated with type II group B streptococci. The dose of type II group B streptococci used produced 56% mortality in 48 neonatal rats with death being due to pneumonia and sepsis. Neutropenia (1300 ± 150/mm3 versus 2300 ± 170/mm3; mean ± S.D.; P < 0.01) and an elevation in band/polymorphonuclear ratio (0.98 ± 0.04 versus 0.30 ± 0.04: P < 0.01) were observed in infected neonatal rats 24 hr following inoculation. Femoral marrow as well as splenic and hepatic neutrophil storage compartment quantification revealed dimunition of postmitotic (polymorphonuclear, band, and metamyelocyte) neutrophils in the infected group (P < 0.01) with sparing of the proliferative neutrophils (myeloblasts, promyelocytes, and myelocytes). Repletion of the myeloid but not the splenic or hepatic neutrophil storage compartments with normalization of the neutrophil count and band/polymorphonuclear ratio occurred in animals surviving 72 hr. These studies establish that neutropenia and neutrophil storage pool depletion are prominent features of experimental type II group B streptococci infection in neonates.Speculation: Although neutrophil storage pool depletion is rare in infected adult animals or humans, its presence can be correlated with an increased mortality. It appears that neutrophil storage pool depletion is a prominent feature of experimental neonatal type II group B streptococci infection. The resulting deficiency of phagocytic cell supply probably represents diminution in the already compromised host defense mechanism of the neonate. Thus, it is postulated that neutrophil storage pool depletion represents a pathophysiologic factor contributing to the high mortality rate observed in neonatal type II group B streptococci infection.

Pre- and postnatal development of granulocytic stem cells in the rat

Summary: Bacterial sepsis is a relatively common problem in the neonatal period, particularly among prematurely delivered infants. The newborn rat has been widely used as a model for sepsis neonatorum, and in that model incomplete development of the neutrophil system has been postulated to be an important factor predisposing neonates to death from bacterial infection. In this study, that hypothesis was further tested by assessing neutrophil development in rats of various pre-and postnatal ages. Using standard soft agar colony techniques for detecting granulocyte-macrophage progenitor cells [CFU(c)], the number of CFU(c)/g of body weight was seen to increase from 0.5 + 0.1 × 103 at 19-20 days gestation to 10.5 ± 0.2 × 103 at 4 weeks. The anatomic location of CFU(c) changed from totally hepatic at 16 days gestation to almost totally myeloid at 4 weeks. Lastly, the proportion of mature, stored neutrophils/CFU(c) decreased from 2440 ± 40 at 19-20 days gestation to 430 ± 75 at 4 weeks.