Multiple Organ Failure Children with Nosocomial Sepsis and Depletion

Prolonged Lymphopenia, Lymphoid Depletion, and Hypoprolactinemia in Children with Nosocomial Sepsis and Multiple Organ Failure This information is current as of September 29, 2021. Kate A. Felmet, Mark W. Hall, Robert S. B. Clark, Ronald Jaffe and Joseph A. Carcillo J Immunol 2005; 174:3765-3772; ; doi: 10.4049/jimmunol.174.6.3765 http://www.jimmunol.org/content/174/6/3765 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Prolonged Lymphopenia, Lymphoid Depletion, and Hypoprolactinemia in Children with Nosocomial Sepsis and Multiple Organ Failure1

Kate A. Felmet,* Mark W. Hall,‡ Robert S. B. Clark,* Ronald Jaffe,† and Joseph A. Carcillo2*

Lymphopenia and lymphoid depletion occur in adults dying of sepsis. Prolactin increases Bcl-2 expression, suppresses stress- induced lymphocyte apoptosis, and improves survival from experimental sepsis. We hypothesized that prolonged lymphopenia, lymphoid depletion, and hypoprolactinemia occur in children dying with sepsis and multiple organ failure (MOF). Fifty-eight critically ill children with and 55 without MOF admitted to a university hospital pediatric intensive care unit were enrolled in a prospective, longitudinal, observational clinical study. Prolactin levels and absolute lymphocyte count were measured on days 1, 3, 7, 14, and 21. Lymph node, thymus, and spleen autopsy specimens were examined for lymphoid depletion, with immunohis- Downloaded from tochemical staining for CD4, CD20, and CD21 and for lymphoid apoptosis. Prolonged lymphopenia (absolute lymphocyte count < 1000 for >7 days) occurred only in children with MOF (29 vs 0%, p < 0.05) and was associated independently with nosocomial infection (odds ratio (OR), 5.5, 95% conﬁdence interval (CI), 1.7–17, p < 0.05), death (OR, 6.8, 95% CI, 1.3–34, p < 0.05), and splenic and lymph node hypocellularity (OR, 42, 95% CI, 3.7–473, p < 0.05). Lymphocyte apoptosis and ante/postmortem infection were observed only in children with lymphoid depletion. Prolonged hypoprolactinemia (>7 days) was more common in

children with MOF (17 vs 2%, p < 0.05) and was associated independently with prolonged lymphopenia (OR, 8.3, 95% CI, 2.1–33, http://www.jimmunol.org/ p < 0.05) and lymphoid depletion (OR, 12.2, 95% CI, 2.2–65, p < 0.05). Prolonged lymphopenia and apoptosis-associated depletion of lymphoid organs play a role in nosocomial sepsis-related death in critically ill children. Prolonged hypoprolactinemia is a previously unrecognized risk factor for this syndrome. The Journal of Immunology, 2005, 174: 3765–3772.

evere sepsis and multiple organ failure (MOF)3 remain the Stress-induced lymphocyte apoptosis is thought to be mediated, fifth leading cause of death in infants and the second lead- in part, through the adrenocorticotropic hormone-cortisol axis. S ing cause of death in children in the United States (1). The Prolactin is the counterregulatory stress hormone produced by the majority of children dying with severe sepsis and MOF do so with anterior pituitary and by lymphocytes and monocytes, which pre- uneradicated infection. There is good evidence that cellular com- vents cortisol/stress-induced lymphocyte apoptosis through in- by guest on September 29, 2021 ponents of the immune system are depleted profoundly in critically creased Bcl-2 production (9–11). Anterior pituitary dysfunction ill adult patients with MOF. Trauma and burn patients have been has been documented in adult critical illness, with respect to the shown to be anergic (2, 3), and critical illness and sepsis are as- growth hormone, the thyroid-stimulating hormone, and the gona- sociated with the production of anti-inflammatory cytokines and a dotropin-releasing hormone, but the prolactin axis has not been predisposition to nosocomial infections (4–6). Lymphocytes are investigated in detail (12). Chaudry and colleagues (13, 14) found important to host defense against infection. Hotchkiss et al. (7) that experimentally induced hemorrhagic shock caused decreased found lymphocyte depletion and apoptosis in adult patients dying lymphocyte proliferation, increasing susceptibility to death from of sepsis-induced MOF. This group of investigators demonstrated cecal ligation, and puncture-induced sepsis. Administration of ei- that administration of a caspase inhibitor prevents lymphocyte ap- ther prolactin or a prolactin secretalogue (metoclopramide) re- optosis, increasing lymphocyte counts, reducing systemic bacterial versed this process, preventing hemorrhage-induced suppression cell counts, and improving survival in a cecal ligation puncture of splenocyte proliferation and cytokine release capacity and im- rodent sepsis model (8). proving survival from subsequent cecal ligation and puncture-induced sepsis (13, 14). Although this phenomenon has not been examined in humans, it appears plausible biologically that a state of prolonged hypoprolactinemia could predispose critically ill pa- Departments of *Critical Care Medicine and †Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213; and ‡Department of Pediatrics, Ohio State tients to stress-induced lymphopenia and lymphoid depletion. University College of Medicine, Columbus, OH 43210 In the present study, we hypothesized that critically ill children Received for publication May 24, 2004. Accepted for publication January 5, 2005. with prolonged lymphopenia are more likely to develop nosoco- The costs of publication of this article were defrayed in part by the payment of page mial infection or apoptosis-associated lymphoid depletion or to charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. die. We also hypothesized that prolonged hypoprolactinemia pre- 1 This work was supported by Children’s Hospital of Pittsburgh (to R.S.B.C.) and disposes critically ill children to the development of prolonged 3M01RR0056GCRC (to J.A.C.). lymphopenia and apoptosis-associated lymphoid depletion. 2 Address correspondence and reprint requests to Dr. Joseph A. Carcillo, Division of Critical Care Medicine, 6th Floor, Children’s Hospital of Pittsburgh, 3705 5th Ave- nue, Pittsburgh PA, 15213. E-mail address: [email protected] Materials and Methods 3 Abbreviations used in this paper: MOF, multiple organ failure; OFI, organ failure index; PRISM, pediatric risk of mortality; ALC, absolute lymphocyte count; OR, odds The local institutional review board approved the study. Informed consent ratio; CI, confidence interval; PICU, pediatric intensive care unit; IVIG, i.v. Ig. was obtained from the parents of children participating in the study.

FIGURE 1. Lymphocyte counts (ALC) over time in patients with and without MOF. MOF patients had signiﬁcantly lower lymphocyte counts and remained lymphopenic, whereas the lymphocyte counts in non-MOF patients increased (two-factor ANOVA, p Ͻ 0.05). FIGURE 2. Neutrophil counts (absolute neutrophil count) and lymphocyte counts (ALC) in MOF patients. Prolonged lymphopenia, which only Patient selection occurred in patients with MOF, was not associated with neutropenia.

Two patient cohorts were collected. The first cohort, collected between by guest on September 29, 2021 March 1999 and June 2000, enrolled consecutive patients with MOF (defined as an organ failure index (OFI) of two or more; Ref. 15) who required normal lymphocyte population in each tissue was seen. Paraffin-embedded an arterial line within 24 h of admission. The second cohort, collected sections of lymphocyte, thymus, and spleen were evaluated for the pres- between March 2001 and June 2001, enrolled consecutive critically ill ence of lymphocyte apoptosis by TUNEL staining with a concomitant nu- children who required an arterial line within 24 h of pediatric intensive care clear stain. Cell populations were identified using immunohistochemical unit (PICU) admission. Blood samples were collected on days 1, 3, 7, 14, staining for CD4 (T lymphocytes), CD20 (B lymphocytes), and CD21 and 21. The sample collection was terminated at the time the invasive (dendritic cells). access was discontinued, the patient died, or the patient was discharged TUNEL staining was performed as follows. Slides were deparaffinized from the intensive care unit. and rehydrated, then treated with proteinase for 30 min (Boehringer Mann- heim) to increase membrane permeability. Enzyme activity was quenched Data collection with a solution of 3% hydrogen peroxide in 30% methanol for 30 min. Sections were incubated with a mixture of recombinant TdT (Invitrogen In all patients, a morning blood sample was drawn in a heparinized tube Life Technologies) and biotin-16-2-deoxy-uridine-5-triphosphate (Roche) and immediately spun. The serum was decanted and frozen at Ϫ20°C. At for 2 h. Sections were then incubated for 1 h with a streptavidin-conjugated the end of the study, prolactin levels were measured in all samples by an fluorescent dye (Alexa Fluor 488; Molecular Probes) and the nuclear dye automated chemiluminescent assay (Immulite; Diagnostic Products Cor- bis-benzamide for 1 min (Sigma-Aldrich). TUNEL-positive cells were poration) Hypoprolactinemia was defined as Ͻ2.5 ng/ml in children Ͼ6mo identified by the presence of fluorescence in the spectrum of bis-benzimide of age and Ͻ20 ng/ml in children Ͻ6 mo of age (16, 17). Prolonged and streptavidin colocalized to the same cell. Tissues were defined as pos- hypoprolactinemia was defined as low prolactin levels for Ͼ1 wk or until itive for apoptosis when more than three TUNEL-positive cells were seen death. per high-powered field. On the day of admission, a pediatric risk of mortality (PRISM) score Immunohistochemical staining for cluster of differentiation markers was was calculated for all patients. At the time of collection, OFI, pressor type performed as follows. Slides were deparaffinized and rehydrated. Endog- and dose, use of metoclopramide, use of steroids, and other immune sup- enous peroxidase activity was blocked in 0.3% hydrogen peroxide for 30 pressive agents were recorded. Absolute lymphocyte counts (ALC) were min. Slides were microwaved for 10 min in the buffer recommended by the recorded as measured by a routine hospital laboratory complete blood manufacturer for each primary Ab. After the application of a blocking count. Lymphopenia was defined as ALC Ͻ 1000 (18–21). Lymphopenia agent, slides were incubated with mAbs against human CD4, CD20, and was defined as prolonged when lasting Ͼ1 wk or until death. Nosocomial CD21 for1hatroom temperature (Vector Laboratories). Slides were in- infection was defined by a positive culture from blood, urine, or endotra- cubated with a biotinylated secondary Ab for 30 min, then developed using cheal tube with increased white blood cells (Ͼ12,000) occurring between an avidin-biotinylated enzyme complex with a vasoactive intestinal peptide days 5 and 25 (mean, 11 days of PICU admission) and requiring antibiotics substrate (Vector Laboratories). per the attending physician. Statistical analysis Screening for lymphocyte depletion and apoptosis Data were analyzed using Sigmastat and Stata. Data involving two groups In study patients who died and underwent autopsy, samples of lymph node, were compared using the Mann-Whitney rank-sum test, the ␹2 analysis, thymus, and spleen were examined for evidence of lymphocyte depletion and the Fisher’s exact test when appropriate. Data involving comparison of and lymphocyte apoptosis. Lymphocyte depletion in each tissue was two groups over time were analyzed using two-factor ANOVA for ranks. graded by the blinded pathologist and defined as severe when Ͻ75% of Significance was accepted at p Ͻ 0.05. To determine which factors were The Journal of Immunology 3767 associated with lymphopenia, lymphoid depletion, nosocomial infection, Transient lymphopenia for 3–7 days was associated with an in- and death, we performed a univariate analysis with the following variables: creased likelihood of nosocomial infection (OR, 4.4, 95% confi- age, presence of steroids, presence of immune suppression, prolonged hy- dence interval (CI), 1.2–15.4, p Ͻ 0.02) independent of immune poprolactinemia, PRISM score, and dopamine exposure. Odds ratios (OR) were determined using all factors with p Ͻ 0.1 in a logistic regression suppression or steroid use but not an increased likelihood of MOF analysis. or death. Prolonged lymphopenia was associated with secondary infection (OR, 5.5, 95% CI, 1.7–17, p Ͻ 0.05), MOF ( p Ͻ 0.001, Results Fisher’s exact test), and death (OR, 6.8, 95% CI, 1.3–34, p Ͻ 0.05) Patient demographics independent of steroid use, immune suppression, and risk of mor- A total of 113 patients was enrolled. Primary admission diagnoses tality (PRISM score). Prolonged peripheral lymphopenia was an included sepsis (n ϭ 35), respiratory failure (n ϭ 8), postoperative independent predictor of lymphocyte depletion at autopsy with an ϭ (n ϭ 14), trauma (n ϭ 7), status postorgan transplantation (n ϭ OR of 42.2 (95% CI, 3.7–473, p 0.001). Patients with prolonged 28), fulminant hepatic failure (n ϭ 7), and other (n ϭ 17). There lymphopenia had normal neutrophil counts (Fig. 2). were 62 males and 51 females with an average age of 7.5 years (range, 2 wk to 23 years) and a mean PRISM score of 10 (range, Lymphoid depletion and autopsy findings 0–26). There were 58 patients with MOF (135 patient days) and 55 Sixteen patients died and 11 underwent autopsy. Median time from patients without MOF (120 patient days). Among patients with death to autopsy was 13 h (range, 3–39 h). A summary of autopsy MOF, the mean OFI at entry into the study was 2.9 compared with findings can be found in Table I. Severe lymphocyte depletion a mean OFI of 0.65 among patients enrolled without MOF. Over- (Fig. 3) was seen in eight of nine (89%) patients dying with MOF. Downloaded from all, there were 16 patient deaths, 14 among patients with MOF The one patient who died with MOF without lymphocyte depletion (24%) and 2 among patients without MOF (4%). had a rapid course with fulminant hepatic failure. Neither of the two patients who died without MOF showed evidence of lympho- Prolonged lymphopenia cyte depletion ( p ϭ 0.05, Fisher’s exact test). Lymph nodes and Forty-nine percent of the patients had lymphopenia for Ն1 days spleens of patients with MOF were hypocellular with sparse and and 15% had prolonged lymphopenia. Twenty-nine percent (17 of atrophic lymphoid nodules. The thymuses in patients with MOF 58) of patients with MOF had prolonged lymphopenia vs 0 of 55 were hypocellular. The same tissues in the two patients who died http://www.jimmunol.org/ patients without MOF ( p Ͻ 0.001, ␹2 test). Lymphocyte counts without MOF showed normal cell populations. In the lymphocyte- were lower in patients with MOF than those without (median, 864 depleted patients, seven of eight or 88% had autopsy evidence of vs 1787, p Ͻ 0.001, Mann-Whitney rank-sum test). MOF patients unresolved nosocomial infection. Four of these patients grew mul- remained lymphopenic over time, while the counts of patients tiple organisms from autopsy specimens. None of the three patients without MOF increased (two-factor ANOVA for ranks; time ϫ without lymphocyte depletion had infection before death or in au- group, p Ͻ 0.05; Fig. 1). topsy cultures ( p ϭ 0.02, Fisher’s exact test). by guest on September 29, 2021

Table I. Autopsy ﬁndings

Days from Onset of Prolonged Patient Known Nosocomial Unrecognized Infection at MOF to Lymphocyte Low Immune No. Cause of Death Infection Autopsy Death Depletion Prolactin? Suppression

Patients with MOF 24 Pulmonary hemorrhage Micrococcus, Candida Disseminated C. albicans 16 days Yes No Cyclosporin albicans, and CNS (blood) 29 Refractory hypotension None None 13 days Yes Yes Atgam after heart transplant 35 Acute intracranial Enterobacter cloacae Pseudomonas, Citrobacter, 16 days Yes No Tacrolimus, hemorrhage (blood) and Serratia (lung high-dose tissue) steroids 38 Support limited- Enterococcus (blood) Enterococcus fecalis and 49 days Yes Yes None refractory and parainﬂuenza CNS (blood) hypotension 43 Withdrawal of support C. albicans (lungs) E. fecalis (blood) 16 days Yes Yes Cyclosporin 44 Brain herniation C. albicans (urine) Disseminated C. albicans 9 days Yes Yes None 46 Fulminant hepatic None None 2 days No No None failure 53 Refractory hypotension None E. fecalis, Enterobacter 34 days Yes No None aerogenes, and Staphylococcus epidermidis (blood), pseudomembranous colitis 57 Intracranial hemorrhage None Severe pseudomembranous 6 days Yes Yes None colitis and C. albicans (mediastinum) Patients without MOF 12 Pulmonary hypertensive None None 2 days No No No crisis 32 Withdrawal of support None None 3 days No No No 3768 LYMPHOPENIA AND LYMPHOCYTE APOPTOSIS AND HYPOPROLACTINEMIA Downloaded from

FIGURE 4. Immunohistochemical staining for B cells. Unmagniﬁed image of slides of a spleen stained for B cells (CD20) from patients with and without lymphocyte depletion. The number and size of lymphoid fol-

licles (dark spots) are decreased dramatically in the patients with lympho- http://www.jimmunol.org/ cyte depletion. A, B, and C are patients 12, 32, and 46 from Table I. Patients D, E, and F are patients 29, 35, and 38 from Table I.

FIGURE 3. H&E-stained sections of lymph node, thymus, and spleen. H&E-stained sections of lymph node, thymus, and spleen from two age- hypoprolactinemia had lower peripheral lymphocyte counts (two- matched patients, one with MOF and one without. Images are at ϫ200 factor ANOVA for ranks, group, p Ͻ 0.05; Fig. 7). magniﬁcation. The patient with MOF has a decrease in the number and size In univariate analysis, age, steroid use, dopamine use, immune of inactive lymphoid nodules (arrows) in the spleen and lymph nodes com- suppression, and prolonged hypoprolactinemia were associated

pared with the patient without MOF. The patient with MOF has a marked by guest on September 29, 2021 with prolonged lymphopenia ( p Ͻ 0.1). In multivariate analysis, decrease in the cellularity of the thymus. prolonged hypoprolactinemia was associated independently with prolonged peripheral lymphopenia (OR, 8.3, 95% CI, 2.1–33, p ϭ 0.003). Dopamine use was a risk factor for prolonged hypopro- Immunohistochemical staining revealed a reduction in numbers lactinemia with an OR of 7.3 (95% CI, 1.6–32.3), but it was not an of lymphocyte cell lines in patients with lymphocyte depletion independent risk factor for prolonged lymphopenia. Three patients (Figs. 4 and 5). A decrease in the number and size of CD20-stained received metoclopramide, each for 1 day only, and all three had lymphoid follicles (zones of active B cell replication) in the normal prolactin levels and ALC. spleens and lymph nodes of patients with lymphoid depletion was In univariate analysis, age, steroid use, immune suppression, and found in most patients with MOF. Individual MOF patients with prolonged hypoprolactinemia were associated with lymphoid deple- lymphoid depletion also had a near absence of mature dendritic tion ( p Ͻ 0.1). In multivariate analysis, prolonged hypoprolactinemia cells as demonstrated by CD21 staining. Individual patients with was associated independently with lymphoid depletion (OR, 12.2, lymphoid depletion had a decrease in CD4-positive T cell staining 95% CI, 2.2–65, p ϭ 0.01). Five of the eight patients with lymphoid as well. Patients dying without MOF had normal populations of all depletion had prolonged hypoprolactinemia (Table I). Four of the three cell lines. eight patients with lymphoid depletion were treated with drugs that TUNEL-positive apoptotic cells were seen in patients with MOF are known to contribute to lymphocyte loss: one patient received anti- but not in those without MOF (Fig. 6). Fifty percent of thymuses, thymocyte globulin (a lympholytic agent), one patient received ta- 57% of lymph nodes, and 89% of spleens were TUNEL positive in crolimus, and two patients received cyclosporine A (a prolactin re- patients dying with MOF ( p ϭ 0.05, Fisher’s exact test). The ceptor antagonist). Only one of the eight patients with lymphoid median delay between death and tissue fixation was similar for depletion neither received a drug known to cause lymphopenia, nor patients with TUNEL-positive tissues compared with those experienced prolonged hypoprolactinemia. without. Discussion Prolonged hypoprolactinemia, prolonged lymphopenia, and Three novel findings are presented in this study. First, a simple lymphoid depletion clinical laboratory test, an ALC Ͻ 1000, not only identified crit- Fifty percent of PICU patients had one or more episodes of hy- ically ill children at risk for nosocomial infection, but an ALC poprolactinemia and 11% had prolonged hypoprolactinemia. Pro- Ͻ 1000 for Ͼ7 days identified those at risk for death from un- longed hypoprolactinemia occurred more commonly in patients eradicated nosocomial infection. Second, apoptosis-associated B with MOF. Seventeen percent (10 of 58) of patients with MOF had cell, T cell, and dendritic cell depletion was found in the lymphoid prolonged hypoprolactinemia compared with 2% (1 of 55) of pa- organs from children who died from MOF and uneradicated in- tients without MOF ( p Ͻ 0.01, ␹2 test). Patients with prolonged fection. Similar to adults, children dying from sepsis-induced The Journal of Immunology 3769

MOF may do so, in part, because of lymphoid depletion. Appar- ently, lymphocyte depletion can be so profound that it impairs the child’s ability to eradicate infection, despite the normal neutrophil counts seen in these patients. Third, prolonged hypoprolactinemia may be a previously unrecognized risk factor contributing to the pathophysiology of lymphoid depletion syndrome. Prolonged lymphopenia, lymphoid depletion, and death from nosocomial sepsis may occur, in part, due to absence of the counterregulatory hormone that ameliorates stress-mediated immune cell apoptosis. Absolute neutropenia has long been recognized as a risk factor for nosocomial sepsis in children and adults. Patients with absolute neutrophil counts Ͻ 500 and fever are treated empirically with broad spectrum antibiotics. If fever and neutropenia persists for Ͼ5 days, then antifungal therapy is added to the empiric regimen. Patients with prolonged neutropenia are also treated with growth factors or white blood cell transfusions to prevent or cure nosocomial sepsis. Persistent neutropenia, unresponsive to these ther- apies, is associated with death from nosocomial sepsis. Although

absolute lymphopenia has long been taken for granted as an inno- Downloaded from cent bystander in patients with critical illness, the preponderance of clinical data suggests otherwise (4, 5, 20–22). The HIV and cancer chemotherapy literature has identiﬁed CD4 counts Ͻ 1500 in infants, 1000 in 1- to 5-year-olds, 500 in 6- to 12-year-olds, and 200 in older children as a risk factor for nosocomial infection with

viruses, fungus, and protozoa (23). Prophylaxis and empiric anti- http://www.jimmunol.org/ microbial strategies are used as the standard of care in these populations. The primary immune deﬁciency and bone marrow transplantation literature recognize the importance of decreased CD20 counts to the development of hypogammaglobulinemia and an increased risk of nosocomial sepsis (24). These children are treated with i.v. Ig (IVIG) prophylaxis on a 3- to 4-wk basis to maintain IVIG levels Ͼ 500 at all times. In our patient population, children with lymphopenia commonly had depressed CD4 and/or CD20 cell counts. Similar to clinical experience with neutropenia, tran- by guest on September 29, 2021 sient lymphopenia is a risk factor for nosocomial infection. Pro- longed lymphopenia is a risk factor for uneradicable nosocomial sepsis, MOF, and death. Gurevitch et al. (25) previously reported lymphoid depletion at an autopsy in low birth weight infants who died with systemic Ag-related disease or sepsis. Hotchkiss et al. (7) similarly found lymphoid depletion in adults dying from sepsis. These patients had reduced lymphoid CD4 and CD20 cell numbers with the degree of reduction in cell numbers related to the duration of sepsis before death. Patients with sepsis Ͻ 7 days had less depletion than patients with sepsis Ͼ 7 days. These investigators also found Ͼ3% apoptotic cells in specimens with lymphoid depletion (7). In our study, all autopsy specimens were obtained from children who had sepsis-associated MOF for Ͼ7 days. The patterns of lymphoid depletion were similar to those reported by Gurevitch et al. (25) and Hotchkiss et al (7). There was a reduction in CD4, CD20, and CD21 cells with Ͼ3% of cells demonstrating apoptosis. It is pos- sible that this profound depletion of immune cells needed for cell- mediated immunity, humoral immunity, and Ag presentation and

MOF and two patients without. Patients without lymphoid depletion had more numerous and robust lymphoid follicles than those without. B, Im- munohistochemical staining for T cells (CD4) in the periarteriolar zone of the spleens of two patients with lymphoid depletion and MOF and two patients without, showing a decrease in this cell population in patients with lymphoid depletion. Magniﬁcation is ϫ200. C, Immunohistochemical FIGURE 5. Immunohistochemical staining for B cells, T cells, and fol- staining for mature dendritic cells (CD21) in the spleen of one patient with licular dendritic cells. A, Immunohistochemical staining for B cells (CD20) lymphoid depletion and MOF (patient 44) and one patient without (patient in splenic lymphoid follicles of two patients with lymphoid depletion and 12), showing decreased numbers in the lymphoid-depleted patient. 3770 LYMPHOPENIA AND LYMPHOCYTE APOPTOSIS AND HYPOPROLACTINEMIA Downloaded from

FIGURE 6. TUNEL staining for apoptosis. A representative field of splenocytes stained with a blue nuclear stain and a green TUNEL stain in a patient with lymphoid depletion and MOF (patient 53) and one without (patient 12). TUNEL-positive apoptotic cells (arrows) are those in which the green and blue colocalize to the same cell. All images are at ϫ600 magnification. http://www.jimmunol.org/ processing contributed to uneradicable infection in these patients. are used in the intensive care unit, prolactin release is inhibited Experimental evidence supporting the role of lymphocytes in bac- strongly by dopamine at dosage levels as low as 0.1 ␮g/kg/min terial killing has been provided by Hotchkiss et al. (8), who dem- (38). We found that dopamine infusion use was associated with onstrated that administration of a polycaspase inhibitor to a rodent hypoprolactinemia in our patients; however, hypoprolactinemia model of experimental sepsis increased lymphocyte counts, re- was also observed in patients who did not receive dopamine infu- duced bacterial counts, and improved survival. sions. Loss of normal patterns of secretion of other pituitary hor- The causes of prolonged lymphopenia and lymphoid depletion mones have been demonstrated in patients with prolonged critical are likely multifactorial. Deficiencies in factors necessary for lym- by guest on September 29, 2021 phocyte growth and proliferation, particularly zinc, may contribute to lymphopenia in patients with prolonged marginal nutrition (26). Iatrogenic immune suppression also plays a role as demonstrated by the contribution of steroids and immune suppressants to the development of lymphopenia in our patients. However, our study suggests that previously unappreciated prolonged hypoprolactinemia may be another important contributor to lymphopenia and lymphocyte apoptosis. As both a growth factor and an antiapoptotic hormone, prolactin may support circulating lymphocyte numbers in humans as it does in animal models of stress. Withdrawal of the proliferation cofactor IL-2 has been shown to induce apoptosis-by-neglect (27). The absence of prolactin, which is a necessary cofactor for IL-2-mediated lymphocyte proliferation, may have a similar effect (28). Prolactin is known to protect against glucocorticoid-induced lymphocyte apoptosis in vitro and in animal models (29, 30), and levels of glucocorticoids commonly seen in stressed patients have been shown to cause lymphocyte apoptosis (31, 32). Because prolactin release occurs simultaneously with stress-induced hypothalamic/pituitary/adrenal axis activation, it may protect the organism from the immune-suppressive effects of stress cortisol. Prolactin can mediate these effects, in part, through regulation of expression of antiapoptotic genes (particularly BcL-2) by lymphocytes in vitro (33, 34). Overexpression of BcL-2 in transgenic mice has been shown to decrease lymphocyte apoptosis and improve survival from sepsis (35). Patients undergoing stress from uncomplicated surgery or mod- erate infections have increased prolactin levels (36, 37); however, our critically ill population did not show this stress response. Pi- FIGURE 7. Lymphocyte counts (ALC) in patients with and without tuitary prolactin release is inhibited tonically by endogenous do- prolonged hypoprolactinemia. Patients with prolonged hypoprolactinemia pamine release by the hypothalamus. When dopamine infusions had lower lymphocyte counts (p Ͻ 0.05). The Journal of Immunology 3771 illness (12). Whether hypoprolactinemia in some critically ill pa- age; therefore, the ALC Ͻ 1000 cutoff may or may not be predic- tients is caused by increased endogenous hypothalamic dopamine tive in adults. Also, the role of different prolactin secretalogues production or other types of pituitary dysfunction remains to be will likely be important. For example, metoclopramide is used studied. commonly in critically ill children, whereas haloperidol (another Chaudry and colleagues (13, 14, 39) have addressed the rela- central dopamine antagonist) is used commonly in critically ill tionship of prolactin, reduced lymphocyte proliferation, and sus- adults. ceptibility to death from sepsis in the experimental setting. These In summary, prolonged lymphopenia identifies critically ill in- investigators induced hemorrhagic shock and caused reduced lym- fants and children at high risk for nosocomial infection, lymphoid phocyte proliferation in mice. When subsequently challenged with depletion, and death from nosocomial sepsis-induced MOF. Occult sepsis using the cecal-ligation puncture model, survival was im- hypoprolactinemia likely contributes to the pathogenesis of this paired; however, treatment with prolactin at the time of hemor- condition. What prolactin level is associated with the best outcome rhage restored lymphocyte and macrophage function and improved from critical illness has yet to be determined. Investigations of survival from sepsis (14). This protection was also achieved with novel strategies that 1) substitute for lymphocyte function (e.g., treatment with the prolactin secretalogue, metoclopramide, which antibiotic prophylaxis against infection for low CD4 counts and acts as a partial dopamine antagonist. Metoclopramide increased IVIG supplementation for hypogammaglobulinemia), 2) improve prolactin levels, increased splenocyte proliferation, reduced sys- lymphocyte function (e.g., zinc supplementation), 3) remove iat- temic IL-6 production, and improved survival (13, 39). These find- rogenic sources of lymphopenia (e.g., rapid tapering of steroids, ings provide biologic plausibility for the potential of prolactin and lympholytics), and 4) reverse hypoprolactinemia (e.g., dopamine prolactin secretalogues to help reverse susceptibility to lymphoid infusion withdrawal, administration of prolactin secretalogues, or Downloaded from depletion and nosocomial infection in critically ill patients. How- careful titration of prolactin infusion to target levels associated ever, it is important to note that in vitro and animal studies suggest with immune preservation) are warranted in this easily identified a biphasic effect of prolactin on the immune system similar to that critically ill population. seen in other target organs (40, 41). Hypoprolactinemia (in humans, Ͻ2.5 ng/ml; Ref. 17) or extreme hyperprolactinemia (in Acknowledgments humans, approximately Ͼ200 ng/ml) are associated with immune- We thank Christine Marco and Steve Tomarello for their technical support. http://www.jimmunol.org/ suppressive effects. Oberbeck et al. (42) recently demonstrated the immune-suppressive effects of high-dose prolactin. In this study, Disclosures exogenous prolactin administered at levels 60-fold higher than The authors have no financial conflict of interest. those seen during stress-induced release of the endogenous hormone lead to increased sepsis-induced mortality and impaired lym- References phocyte function (42). 1. Watson, R. S., J. A. Carcillo, W. T. Linde-Zwirble, G. Clermont, J. Lidicker, and In contrast, prolactin levels in the normal (2.5–20 ng/ml; Ref. D. C. Angus. 2003. The epidemiology of severe sepsis in children in the United States. Am. J. Respir. Crit. Care Med. 167:695. 17) to stress range (10–100 ng/ml; Ref. 43) are associated with 2. Meakins, J. L., J. B. Pietsch, O. Bubenick, R. Kelly, H. Rode, J. Gordon, and by guest on September 29, 2021 immune preservation, particularly in the setting of an apoptotic L. D. MacLean. 1977. Delayed hypersensitivity: indicator of acquired failure of stimulus, such as stress cortisol release. In this regard, our findings host defenses in sepsis and trauma. Ann. Surg. 186:241. 3. Pellegrini, J. D., A. K. De, K. Kodys, J. C. Puyana, R. K. Furse, and support the animal data documenting the immune-suppressive ef- C. Miller-Graziano. 2000. Relationships between T lymphocyte apoptosis and fects of hypoprolactinemia in states of stress. Administration of anergy following trauma. J. Surg. Res. 88:20. prolactin secretalogues such as metoclopramide and haloperidol to 4. O’Sullivan, S. T., J. A. Lederer, A. F. Horgan, D. H. Chin, J. A. Mannick, and M. L. Rodrick. 1995. Major injury leads to predominance of the T helper-2 human subjects have been shown to raise prolactin into the stress lymphocyte phenotype and diminished interleukin-12 production associated with range (44, 45). These drugs may deserve investigation for use in decreased resistance to infection. Ann. Surg. 222:482. critically ill populations who are at risk for lymphopenia lymphoid 5. Menges, T., J. Engel, I. Welters, R. M. Wagner, S. Little, R. Ruwoldt, M. Wollbrueck, and G. Hempelmann. 1999. Changes in blood lymphocyte pop- depletion and nosocomial sepsis. ulations after multiple trauma: association with posttraumatic complications. Crit. There are limitations to consider in our study. A prolactin-bind- Care Med. 27:733. 6. Gogos, C. A., E. Drosou, H. P. Bassaris, and A. Skoutelis. 2000. Pro- versus ing protein that interferes with biologic activity is known to exist. anti-inflammatory cytokine profile in patients with severe sepsis: a marker for Its binding characteristics in pathologic states have not been de- prognosis and future therapeutic options. J. Infect. Dis. 181:176. scribed, so it is unclear how protein binding might affect the pro- 7. Hotchkiss, R. S., K. W. Tinsley, P. E. Swanson, R. E. Schmeig, J. J. Hui, K. C. Chang, D. F. Osborne, B. D. Freeman, J. P. Cobb, T. G. Buchman, and lactin levels measured in this study (46). The biologic activity of I. E. Karl. 2001. Sepsis induced apoptosis a progressive and profound depletion immunoreactive prolactin in this patient population may be con- of B and CD4ϩ lymphocytes in humans. J. Immunol. 166:6952. firmed in a future study. Because the two patient cohorts were 8. Hotchkiss, R. S., K. C. Chang, P. E. Swanson, K. W. Tinsley, J. J. Hui, P. Klender, S. Xanthoudakis, S. Roy, C. Black, G. Grimm, et al. 2000. Caspase collected at different times, the possibility that changes in clinical inhibitors improve survival in sepsis: a critical role of the lymphocyte. Nat. Im- practice over time influenced patient outcomes cannot be ruled out. munol. 1:496. 9. Yu-Lee, L. Y. 1997. Molecular actions of prolactin in the immune system. Proc. The TUNEL method used in autopsy specimens only identified the Soc. Exp. Biol. Med. 215:35. group of cells that were in the process of apoptotic death. Cells that 10. Freeman, M. E., B. Kanyicska, A. Lerant, and G. Nagy. 2000. Prolactin: struc- are identifiable as apoptotic are phagocytosed within a few hours ture, function, and regulation of secretion. Physiol. Rev. 80:1523. 11. Buckley, A., and D. Buckley. 2000. Prolactin regulation of apoptosis-associated (47). Although a delay in tissue fixation has been reported to in- gene expression in T cells. Ann. NY Acad. Sci. 917:522. crease the percentage of cells that nick end labeling in situ, the 12. Van den Berghe, G., and F. de Zegher. 1996. Anterior pituitary function during delay was similar for the groups with and without TUNEL-positive critical illness and dopamine treatment. Crit. Care Med. 24:1580. 13. Zellweger, R., M. W. Wichmann, A. Ayala, and I. H. Chaudry. 1998. Metoclo- tissues. We documented lymphocyte depletion and apoptosis in pramide: a novel and safe immunomodulating agent for restoring the depressed patients who died; however, we can only speculate on the causes macrophage immune function after hemorrhage. J. Trauma 44:70. 14. Zellweger, R., X. H. Zhu, M. W. Wichmann, A. Ayala, C. M. DeMaso, and of prolonged lymphopenia in patients who recovered. Recent pub- I. H. Chaudry. 1996. Prolactin administration following hemorrhagic shock im- lications have reported the presence of early indicators of apopto- proves macrophage cytokine release capacity and decreases mortality from sub- sis in the circulating lymphocytes of moderately critically ill pa- sequent sepsis. J. Immunol. 157:5748. 15. Wilkinson, J. D., M. M. Pollack, U. E. Ruttimann, N. L. Glass, and T. S. Yeh. tients (48). Extrapolation of these findings directly to adults will 1986. Outcome of pediatric patients with multiple organ system failure. Crit. also require further study. Lymphocyte numbers decrease with Care Med. 14:271. 3772 LYMPHOPENIA AND LYMPHOCYTE APOPTOSIS AND HYPOPROLACTINEMIA

16. Vankrieken, L. 2000 Immulite reproductive hormone assays: multicenter refer- 33. Leff, M. A., D. J. Buckley, J. S. Krumenacker, J. C. Reed, T. Miyashita, and ence range data. Diagnostic Products Corporation Document No. ZB157-d. Di- A. R. Buckley. 1996. Rapid modulation of the apoptosis regulatory genes, bcl-2 agnostic Product Corporation, Los Angeles, p. 3. and bax by prolactin in rat Nb2 lymphoma cells. Endocrinology 137:5456. 17. Friesen, H. G. 1978. Human prolactin. Ann. R. Coll. Phys. Surg. Can. 11:275. 34. Buckley, A. R., D. J. Buckley, M. A. Leff, D. S. Hoover, and N. S. Magnuson. 18. Dallman, P. R. 1987. White blood cells: developmental changes in numbers. In 1995. Rapid induction of pim-1 expression by prolactin and interleukin-2 in rat Pediatrics, 19th Ed. A. M. Rudolph, J. I. E. Hoffman, and C. D. Rudolph, eds. Nb2 lymphoma cells. Endocrinology 136:5252. Appleton and Lange, Norwalk, p. 1061. 35. Hotchkiss, R. S., P. E. Swanson, C. M. Knudson, K. C. Chang, J. P. Cobb, 19. Goetz, A. M., C. Squier, M. M. Wagener, and R. R. Muder. 1994. Nosocomial D. F. Osborne, K. M. Zollner, T. G. Buchman, S. J. Korsmeyer, and I. E. Karl. infections in the human immunodeficiency virus-infected patient: a two-year sur- 1999. Overexpression of Bcl-2 in transgenic mice decreases apoptosis and im- vey. Am. J. Infect. Control 22:334. proves survival in sepsis. J. Immunol. 162:4148. 20. Stiehm, E. R. 1989. Immunologic Disorders in Infants and Children. W. B. Saunders, Philadelphia, p. 171. 36. Noel, G. L., H. K. Suh, J. G. Stone, and A. G. Frantz. 1972. Human prolactin and 21. Rajan, G., and J. W. Sleigh. 1997. Lymphocyte counts and the development of growth hormone release during surgery and other conditions of stress. J. Clin. subsequent sepsis. Intensive Care Med. 23:1187. Endocrinol. Metab. 35:840. 22. Laupland, K. B., D. B. Gregson, D. A. Zygun, C. J. Doig, G. Mortis, and 37. Chikanza, I. C. 1999. Prolactin and neuroimmunomodulation: in vitro and in vivo D. L. Church. 2004. Severe bloodstream infections: a population-based assess- observations. Ann. NY Acad. Sci. 876:119. ment. Crit. Care Med. 32:992. 38. Van den Berghe, G., F. de Zegher, and P. Lauwers. 1994. Dopamine suppresses 23. Working Group on Antiretroviral Therapy and Medical Management of Infants, pituitary function in infants and children. Crit. Care Med. 2:1747. Children, and Adolescents with HIV Infection. 1998. Antiretroviral therapy and 39. Knoferl, M. W., M. K. Angele, A. Ayla, W. G. Cioffi, K. I. Bland, and medical management of pediatric HIV infection. Pediatrics 102(Suppl. 4):1005. I. H. Chaudry. 2000. Insight in to the mechanism by which metoclopramide 24. Stiehm, E. R. 1997. Human intravenous immunoglobulin in primary and second- improves immune function after trauma-hemorrhage. Am. J. Physiol. 279:c72. ary antibody deficiencies. Pediatr. Infect. Dis. J. 16:696. 40. Spangelo, B. L., N. R. Hall, P. C. Ross, and A. L. Goldstein. 1987. Stimulation 25. Gurevitch, P., H. Ben-Hur, and B. Czernobilsky. 1999. Pathology of lymphoid of in vivo antibody production and concanavalin-A-induced mouse spleen mito- organs in low birth weight infants subjected to antigen related diseases: a mor- genesis by prolactin. Immunopharmacology 14:11. phologic and morphometric study. Pathology 27:121. 26. Fraker, P. J., L. E. King, T. Laakko, and T. L. Vollmer. 2000. The dynamic link 41. Prins, G. S., and C. Lee. 1983 Biphasic response of the rat lateral prostate to Downloaded from between the integrity of the immune system and zinc status. J. Nutr. 130(Suppl. increasing levels of serum prolactin. Biol. Reprod. 29:938. 5S):1399S. 42. Oberbeck, R., D. Schmitz, K. Wilsenack, M. Schuler, C. Biskup, 27. Rathmell, J. C., and C. B. Thompson. 2002. Pathways of apoptosis in lymphocyte M. Schedlowski, D. Nast-Kolb, and M. S. Exton. 2003. Prolactin modulates development, homeostasis and disease. Cell 109:s97. survival and cellular immune functions in septic mice. J. Surg. Res. 113:248. 28. Clevenger, C. V., S. W. Altmann, and M. B. Prystowsky. 1991. Requirement of 43. Devins, S. S., A. Miller, B. L. Herndon, L. Otoole, and G. Reisz. 1992. Effects nuclear prolactin for interleukin-2-stimulated proliferation of T lymphocytes. Sci- of dopamine on T-lymphocyte proliferative responses and serum prolactin con- ence 253:77. centrations in critically ill patients. Crit. Care Med. 20:1644.

29. Fletcher-Chiappini, S. E., M. M. Compton, H. A. La Voie, E. B. Day, 44. Ijaiya, K., B. Roth, and A. Schwenk. 1980. The effects of arginine, insulin, and http://www.jimmunol.org/ R. J. Witorsch, and M. M. Comptom. 1993. Glucocorticoid-prolactin interactions metaclopramide on growth hormone, prolactin and cortisol release in children. in Nb2 lymphoma cells: antiproliferative versus anticytolytic effects. Proc. Soc. Clin. Endocrinol. 12:589. Exp. Biol. Med. 202:345. 45. Van Putten, T., S. R. Marder, and J. Mintz. 1991. Serum prolactin as a correlate 30. Krishnan, N., O. Thellin, D. J. Buckley, N. D. Horseman, and A. R. Buckley. of clinical response to haloperidol. J. Clin. Psychopharmacol. 11:357. 2003. Prolactin suppresses glucocorticoid-induced thymocyte apoptosis in vivo. Endocrinology 44:2102. 46. Kline, J. B., and C. V. Clevenger. 2001. Identiﬁcation and characterization of the 31. Ayala, A., C. D. Herdon, D. L. Lehman, C. M. DeMaso, C. A. Ayala, and prolactin-binding protein in human serum and milk. J. Biol. Chem. 276:24760. I. H. Chaudry. 1995. The induction of accelerated thymic programmed cell death 47. McCarthy, N. J., and G. I. Evan. 1998. Methods for detecting and quantifying during polymicrobial sepsis: control by corticosteroids but not tumor necrosis apoptosis. Curr. Top. Dev. Biol. 36:259. factor. Shock 3:259. 48. Schroeder, S., C. Lindemann, D. Decker, S. Klaschik, R. Hering, C. Putensen, 32. Tarcic, N., H. Ovadia, D. W. Weiss, and J. Weidenfeld. 1998. Restraint stress- A. Hoeft, A. von Ruecker, and F. Stuber. 2001. Increased susceptibility to apo-

induced thymic involution and cell apoptosis are dependent on endogenous glu- ptosis in circulating lymphocytes of critically ill patients. Langenbecks Arch. by guest on September 29, 2021 cocorticoids. J. Neuroimmunol. 82:40. Surg. 386:42.